Anti-IL13Rα2 antibody, its antigen-binding fragment and use

Abstract

The present invention relates to an antibody, or an antigen-binding fragment thereof, capable of binding to IL13Rα2. The antibody and its antigen-binding fragment are particularly useful in the construction of chimeric antigen receptors (CARs) and CAR-based immunotherapy for the treatment of IL13Rα2-expressing cancer diseases.

Description

[Technical Field]

[0001] This embodiment generally relates to an anti-IL13Rα2 antibody and its antigen-binding fragment, and more particularly to its use in cancer treatment. [Background technology]

[0002] Immunotherapy has revolutionized cancer treatment. However, glioblastoma, a high-grade brain cancer, has yet to benefit from the breakthroughs of immunotherapy, and the disease remains lethal. Adoptive transfer of patient-derived T cells, ex vivo modified to express chimeric antigen receptors (CARs) against antigens expressed on the surface of cancer cells, is one form of immunotherapy currently being intensively studied. Artificial transmembrane CAR molecules consist of an extracellular antigen-binding moiety, usually derived from antibody-derived single-chain variable fragments (scfv), hinge regions, transmembrane domains, and T cell receptor complexes (CD3ζ), and an intracellular signaling domain derived from one or more T cell costimulatory molecules, e.g., CD28, 4-1BB. Thus, CAR T cell therapy combines the specificity of antibodies with the killing efficacy of T lymphocytes. CD19-targeted CAR T cells are highly effective in treating refractory B-cell malignancies and are approved for use in acute lymphoblastic leukemia and non-Hodgkin lymphoma in both the United States and Europe. CAR T cells targeting solid tumors are not yet approved. The idea of ​​modifying NK cells and macrophages using CAR molecules, which have been named CAR NK and CAR macrophages, has also been explored.

[0003] Interleukin-13 receptor subunit alpha-2 (IL13Rα2), also known as surface antigen classification 213A2 (CD213A2), is a membrane protein that binds interleukin-13 (IL-13). IL13Rα2 is closely related to IL13Rα1 (CD213A1), which forms a receptor complex with the interleukin-4 receptor alpha (IL4Rα), a subunit shared by the IL-13 and IL-4 receptors. When IL-13 binds to the IL13Rα1 / IL4Rα receptor complex, a signaling process that results in the activation of JAK1, STAT3, and STAT6 is initiated. In clear contrast, IL13Rα'2 binds IL-13 with high affinity as a monomer and lacks a functional cytoplasmic domain, and thus does not appear to function as a signal mediator. IL13Rα2 is expressed at low levels in healthy human cells and tissues, except for some expression in the testis and pituitary gland. However, IL13Rα2 has been shown to be selectively overexpressed in various cancers that are targeted for cancer treatment, including glioblastoma.

[0004] CAR T cells against IL13Rα2 have been evaluated in a small clinical trial for patients with glioblastoma (Clin Cancer Res 2015, 21:4062-4072). In one patient, the treatment resulted in durable regression over a period of time until IL13Rα2-negative tumor clones grew back (N Engl J Med 2016,375:2561-2569).

[0005] U.S. Patent No. 9,914,909 discloses T cells expressing a chimeric antigen receptor (CAR) that includes an extracellular domain that binds IL-13 or a variant thereof to IL13Rα2, a transmembrane region, and an intracellular signaling domain. The CAR T cells are said to be useful for the treatment of glioblastoma.

[0006] U.S. Patent No. 9,868,788 discloses an antibody that specifically binds to a linear epitope spanning the extracellular portion of human IL13Rα2 and an extracellular portion having at least 90% sequence identity with canine IL13Rα2. The antibody conjugated to a chemotherapeutic agent is said to be useful for the treatment of glioblastoma.

[0007] U.S. Patent No. 10,308,719 discloses an antibody that binds to a CAR construct comprising IL13Rα2 and this antibody fragment, and its fragment, and their use in the treatment of glioblastoma. The antibody inhibits the interaction between IL-13 and IL13Rα2. N-linked glycosylation of IL13Rα2 contributes to the interaction of the antibody with IL13Rα2.

[0008] Mol Cancer Ther 2008,7(6):1579-1587 discloses a fusion of a single-chain Fv (scFv) obtained from a human scFv antibody phage library and Pseudomonas aeruginosa exotoxin (PE) to obtain an anti-IL13Rα2 (scFv)-PE38 immunotoxin. However, the resulting immunotoxin did not mediate a higher antitumor effect compared to an immunotoxin previously developed in the form of a fusion between IL-13 and PE38 (IL-13-PE38). This was due to the low affinity of the scFv portion of the immunotoxin for the target antigen. There is still a need for improvement in the treatment of glioblastoma and other cancers characterized by overexpression of IL13Rα2. SUMMARY OF THE INVENTION

[0009] It is a general object to provide an anti-IL13Rα2 antibody and its antigen-binding fragment. It is a specific object to provide such an anti-IL13Rα2 antibody and its antigen-binding fragment useful in CAR-based immunotherapy. This and other objects are achieved by the embodiments disclosed herein. The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0010] The embodiments of this model relate to an antibody capable of binding to IL13Rα2, or an antigen-binding fragment thereof. In one embodiment, the antibody, or its antigen-binding fragment, is specific to an epitope within the β-sheet region of IL13Rα2, including the first β-chain at amino acid numbers 68-75 in IL13Rα2, the loop after the first β-chain, the second β-chain at amino acid numbers 101-109 in IL13Rα2, the loop before the second β-chain, and the third β-chain at amino acid numbers 124-128 in IL13Rα2.

[0011] In another embodiment, the antibody, or its antigen-binding fragment, comprises a variable heavy chain (VH) domain complementarity-determining region 1 (CDR1) containing the amino acid sequence GFTFX1X2X3X4, each X n n=1-4 are independently selected from the groups G, A, S, and Y. The antibody, or its antigen-binding fragment, also contains the VH domain CDR2 containing the amino acid sequence IB1B2B3B4B5B6T, each B m m=1~6 is independently selected from the group consisting of G, S, and Y. The antibody, or its antigen-binding fragment, has the amino acid sequence AR-Z H - Further comprising a VH domain CDR3 containing Z1DY, where Z1 is selected from the group consisting of F, M, I and L, Z HThis is an amino acid sequence selected from the group consisting of VVRSTYGY (SEQ ID NO: 15), YGHYAYGSY (SEQ ID NO: 16), YSSSGWYYGF (SEQ ID NO: 17), TPYSAY (SEQ ID NO: 18), RYRSHRPGLS (SEQ ID NO: 19), FHPRYGY (SEQ ID NO: 20), GSYSHYGAHY (SEQ ID NO: 21), YYHYDYGYYY (SEQ ID NO: 22), YSPFY (SEQ ID NO: 3), RNYWEHGGGS (SEQ ID NO: 24), HHYGYYPPGSVYY (SEQ ID NO: 25), and VEYTYYGSEGSPV (SEQ ID NO: 26). The antibody, or its antigen-binding fragment, further comprises a variable light chain (VL) domain CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 12) and a VL domain CDR2 containing the amino acid sequence AAS. The antibody, or its antigen-binding fragment, contains the amino acid sequence QQ-Z L - Further includes VL domain CDR3 containing T, Z L This is an amino acid sequence selected from the group consisting of TYYSPH (SEQ ID NO: 28), DYYLF (SEQ ID NO: 29), SYSTPY (SEQ ID NO: 30), FYSYPL (SEQ ID NO: 31), AFSPS (SEQ ID NO: 32), SYDTLL (SEQ ID NO: 33), ALSSLP (SEQ ID NO: 34), FSTRLS (SEQ ID NO: 35), GYSFPP (SEQ ID NO: 4), STYPF (SEQ ID NO: 37), YGSNPL (SEQ ID NO: 38), and RYNGLF (SEQ ID NO: 39).

[0012] The present invention also relates to an antibody according to the present invention, or a CAR comprising an antigen-recognition domain including its antigen-binding fragment, a transmembrane domain, and an intracellular signaling domain; a T cell receptor (TCR) complex comprising an antibody according to the present invention, or an antigen-recognition domain comprising its antigen-binding fragment; and a conjugate comprising an antibody according to the present invention, or its antigen-binding fragment and an effector molecule.

[0013] The present invention further relates to an epitope of IL13Rα2. The epitope is located within the β-sheet region of IL13Rα2, which includes a first β-chain of amino acids 68-75, a loop following the first β-chain, a second β-chain of amino acids 101-109, a loop preceding the second β-chain, and a third β-chain of amino acids 124-128.

[0014] Further embodiments also relate to a pharmaceutically acceptable carrier comprising an antibody according to the present invention, or a nucleic acid molecule encoding its antigen-binding fragment, CAR, and / or TCR complex; a vector comprising a nucleic acid molecule; a cell comprising an antibody according to the present invention, or its antigen-binding fragment, CAR, TCR complex, nucleic acid, and / or vector; and a pharmaceutical composition comprising an antibody according to the present invention, or its antigen-binding fragment, CAR, TCR complex, conjugate, nucleic acid molecule, vector, and / or cell.

[0015] The present invention also relates to antibodies, or their antigen-binding fragments, CARs, TCR complexes, conjugates, nucleic acid molecules, vectors, cells, and / or pharmaceutical compositions, for use as drugs, particularly for use in treating or delaying the onset of IL13Rα2-expressing cancer diseases.

[0016] The present invention also relates to a method for identifying IL13Rα2-positive cells. The method comprises contacting a biological sample with an antibody or its antigen-binding fragment in accordance with the present invention, and measuring the amount of the antibody or its antigen-binding fragment bound to at least one cell in the biological sample, thereby identifying at least one cell as an IL13Rα2-positive cell.

[0017] The antibody of the embodiment, and its antigen-binding fragment, even when converted to a single-chain variable fragment (scFv) format, also specifically binds to the epitope on L13Rα2 and retains highly specific and strong binding to IL13Rα2. CAR constructs based on the antigen-binding fragment of the antibody of the embodiment exhibit high cytotoxicity when used in CAR T-cell immunotherapy, thereby enabling their use in the treatment of IL13Rα2-expressing cancers. Along with their further objectives and advantages, some embodiments can be best understood by referring to the following description, along with the accompanying drawings. [Brief explanation of the drawing]

[0018] [Figure 1] The results of enzyme-linked immunosorbent assay (ELISA) are shown. Bacterial supernatants of 1E10B9 scFv (B9 scFv) and mAb47 scFv (47 scFv) were screened by ELISA at three different concentrations (2, 20, and 200-fold dilutions) for binding to human IL13Rα2 and an unrelated protein (streptavidin). Two colonies (clone_1 and clone_2) were evaluated for each of the two scFv. Streptavidin-specific scFv G-strep-1 was included as a reference. Binding was detected using HRP-labeled anti-FLAG antibody, and absorbance values ​​were measured at 450 nm (y-axis). Reported values ​​are the average of two measurements. Blank values ​​(signals obtained from samples with only culture medium added, without scFv) were subtracted. [Figure 2]The results of homogeneous time-resolved fluorescence (HTRF) are shown. Bacterial supernatants of 1E10B9 scFv (X-ME107-B9) and mAb47 scFv (X-ME107-47) were screened for binding to IL13Rα2 by HTRF. Irrelevant scFvs were included as negative controls, predicted to bind to unrelated proteins. Binding signals (665 nm) and background / noise signals (615 nm) were measured, and the R value, the signal ratio, was calculated for each sample (y-axis). Reported values ​​are the average of two measurements. Blank values ​​(signals obtained from samples with only culture medium added, without scFvs) were also subtracted. [Figure 3-1] Gel electrophoresis of 44 types of purified W-ME107 scFv and reference clone mAb47 scFv is shown. [Figure 3-2] Gel electrophoresis of 44 types of purified W-ME107 scFv and reference clone mAb47 scFv is shown. [Figure 4] The ELISA results are shown. Binding signals are measured as absorbance (y-axis) at 450 nm for 44 W-ME107 scFv clones, reference clone mAb47 scFv, and G-strep-1 scFv against human IL13Rα2-avi (hollow bar), human IL13Rα2-Fc (black bar), mouse IL13Rα2-Fc (dark gray bar), human IL13Rα1-Fc (upward oblique stripe bar), unrelated protein (solid dot bar), and streptavidin (light gray bar). [Figure 5] The results of scFv binding to cell lines are shown. Selected scFv cells were incubated with either human glioblastoma cell line U-87MG (endogenously expressing high levels of hIL13Rα2) or human non-small cell lung cancer cells A549 (as a negative control). The cells were further stained with anti-FLAG-PE antibody and analyzed by flow cytometry. Binding of scFv to cells is shown as mean fluorescence intensity (MFI, y-axis). [Figure 6-1]This document shows surface plasmon resonance (SPR) sensorgrams of 12 W-ME107 scFv clones that bind to IL13Rα2 in the presence and absence of IL-13. It also includes a sensorgram of streptavidin scFv G-strep-1 (negative control, predicted to bind to streptavidin). Activity of purified clone 47 scFv was not detected. [Figure 6-2] This document shows surface plasmon resonance (SPR) sensorgrams of 12 W-ME107 scFv clones that bind to IL13Rα2 in the presence and absence of IL-13. It also includes a sensorgram of streptavidin scFv G-strep-1 (negative control, predicted to bind to streptavidin). Activity of purified clone 47 scFv was not detected. [Figure 6-3] This document shows surface plasmon resonance (SPR) sensorgrams of 12 W-ME107 scFv clones that bind to IL13Rα2 in the presence and absence of IL-13. It also includes a sensorgram of streptavidin scFv G-strep-1 (negative control, predicted to bind to streptavidin). Activity of purified clone 47 scFv was not detected. [Figure 6-4] This document shows surface plasmon resonance (SPR) sensorgrams of 12 W-ME107 scFv clones that bind to IL13Rα2 in the presence and absence of IL-13. It also includes a sensorgram of streptavidin scFv G-strep-1 (negative control, predicted to bind to streptavidin). Activity of purified clone 47 scFv was not detected. [Figure 6-5] This document shows surface plasmon resonance (SPR) sensorgrams of 12 W-ME107 scFv clones that bind to IL13Rα2 in the presence and absence of IL-13. It also includes a sensorgram of streptavidin scFv G-strep-1 (negative control, predicted to bind to streptavidin). Activity of purified clone 47 scFv was not detected. [Figure 6-6]This document shows surface plasmon resonance (SPR) sensorgrams of 12 W-ME107 scFv clones that bind to IL13Rα2 in the presence and absence of IL-13. It also includes a sensorgram of streptavidin scFv G-strep-1 (negative control, predicted to bind to streptavidin). Activity of purified clone 47 scFv was not detected. [Figure 6-7] This document shows surface plasmon resonance (SPR) sensorgrams of 12 W-ME107 scFv clones that bind to IL13Rα2 in the presence and absence of IL-13. It also includes a sensorgram of streptavidin scFv G-strep-1 (negative control, predicted to bind to streptavidin). Activity of purified clone 47 scFv was not detected. [Figure 7-1] The sensorgrams from the epitope binning experiment are shown. 10 nM hIL13Rα2-avi was injected into a clone mAb47 mIgG1-immobilized surface and pre-incubated with a 10-fold molar excess (100 nM) of human IL-13, W-ME107-10, W-ME107-27, W-ME107-75, or W-ME107-117. As control samples, 10 nM hIL13Rα2-avi pre-incubated with a 10-fold molar excess of BI-8 scFv (negative control) or clone 47 mIgG1 (positive control) was also evaluated. [Figure 7-2] The sensorgrams from the epitope binning experiment are shown. 10 nM hIL13Rα2-avi was injected into a clone mAb47 mIgG1-immobilized surface and pre-incubated with a 10-fold molar excess (100 nM) of human IL-13, W-ME107-10, W-ME107-27, W-ME107-75, or W-ME107-117. As control samples, 10 nM hIL13Rα2-avi pre-incubated with a 10-fold molar excess of BI-8 scFv (negative control) or clone 47 mIgG1 (positive control) was also evaluated. [Figure 8]The residual plot from HDX-MS mapping of W-ME107-117 against IL13αR2 is shown, with the output figures representing different time points and the vertical lines representing the sum of differences per peptide. The measurements between two vertical lines in the compartmentalized region are not statistically significant (differences between antigens alone or in the presence of scFv did not exceed repeated variances at a 95% confidence level). [Figure 9] The ELISA results are shown. Binding signals and absorbance at 450 nm (y-axis) were plotted against the peptide and hIL13Rα2 antigen (x-axis). Binding of W-ME107-117 scFv (black bar) and W-ME107-75 (gray bar) was the only one detected against hIL13Rα2. [Figure 10] The structure of IL13Rα2 (light gray, residues 31-328) in complex with IL-13 (black) (PDB code 3LB6) is shown, highlighting the W-ME107-117 epitope. The epitope sequence, determined by HDX-MS, is colored dark gray and located on domain 1 of the receptor. The left panel shows the structure in a schematic representation, with β-chains represented by arrows and helices as spirals. The epitope sequence is found on one of two β-sheets (chains 4, 3, 6, 7) and includes residues in chains 3, 6, 7, and in the loop regions between chains 3 and 4 and between chains 5 and 6, both loops facing domain 2. The right panel shows the structure in a surface representation, with IL-13 removed to highlight that the epitope region does not interact with the ligand. The IL-13 binding site is represented as a black circle. [Figure 11] A schematic diagram of the lentiviral construct is shown. The EF1α promoter is used to drive the expression of a chimeric antigen receptor (CAR) containing a selected scFv and an intracellular T cell activation domain. The CAR construct and the green fluorescent protein (GFP) used for detection are isolated using the T2A autocleavage peptide. [Figure 12]This report shows the results of CAR T cell cytotoxicity. Luciferase-expressing U-87MG cells (expressing IL13Rα2) or Mel526 cells (not expressing IL13Rα2) were co-cultured for 24 hours with different CAR T cell constructs at effector ratios ranging from 0:1 to 25:1 for the target. Target cell viability was evaluated by measuring luciferase activity. The relative viability of target cells in the co-culture (y-axis) was measured compared to an untreated control (tumor cells only). The assay was performed by biological and experimental replication. Values ​​are shown as mean ± SEM. [Figure 13] This shows the proliferation of different CAR T cell constructs at target recognition. CAR T cells were stained with blue-violet dye and co-cultured with U-87MG for 4 days. Co-culture with lovastatin served as a non-replication control. CAR T cells replicated at target recognition were classified according to the number of cell divisions (0, 1, 2, 3, and 4 or more divisions). The figure shows the percentage of CD3+GFP+ cells during division. The percentage was assessed by measuring the percentage of the total CD3+GFP+ population within each divisional peak. Representative data are shown. [Figure 14A]This figure highlights the epitope sequences of W-ME107-10, W-ME107-27, and W-ME107-75 as revealed by HDX-MS. (A) The structure of IL13Rα2 (light gray) in complex with IL-13 (black) (PDB code 3LB6) is rotated 180 degrees compared to the field of view in Figure 10. The epitope sequences are colored dark gray along with numbered residue boundaries, and all are located on domain 3 of the receptor, with the exception of one sequence beginning in domain 2. The structure is shown in a schematic representation. (B-E) show IL13Rα2 in surface representation, highlighting the differences in epitopes predicted for W-ME107-10, W-ME107-27, and W-ME107-75 based on HDX-MS data along with binding data. Epitope regions are colored dark gray. IL-13 (black) is left as a schematic representation for clarity. (B) and (C) show the predicted binding regions for W-ME107-10 and W-ME107-27, respectively. In (D), the binding region for W-ME107-75 is shown in dark gray. W-ME107-75 behaves differently from W-ME107-10 and W-ME107-27 in many experiments and is therefore predicted to have a different binding site. The epitope is located in the very C-terminal region of the receptor, including residues 329-337. These residues are not visible in the reported structure but are included as dotted lines in the figure. They are presumed to be important in W-ME107-75 binding because they are non-conserved in mouse IL13Rα2. (E) Here, the protein structure is rotated 180 degrees, and the beginning of peptides 228-245, which are part of the epitope in W-ME107-10 and W-ME107-27 but not in W-ME107-75, are seen as dark gray. [Figure 14B]This figure highlights the epitope sequences of W-ME107-10, W-ME107-27, and W-ME107-75 as revealed by HDX-MS. (A) The structure of IL13Rα2 (light gray) in complex with IL-13 (black) (PDB code 3LB6) is rotated 180 degrees compared to the field of view in Figure 10. The epitope sequences are colored dark gray along with numbered residue boundaries, and all are located on domain 3 of the receptor, with the exception of one sequence beginning in domain 2. The structure is shown in a schematic representation. (B-E) show IL13Rα2 in surface representation, highlighting the differences in epitopes predicted for W-ME107-10, W-ME107-27, and W-ME107-75 based on HDX-MS data along with binding data. Epitope regions are colored dark gray. IL-13 (black) is left as a schematic representation for clarity. (B) and (C) show the predicted binding regions for W-ME107-10 and W-ME107-27, respectively. In (D), the binding region for W-ME107-75 is shown in dark gray. W-ME107-75 behaves differently from W-ME107-10 and W-ME107-27 in many experiments and is therefore predicted to have a different binding site. The epitope is located in the very C-terminal region of the receptor, including residues 329-337. These residues are not visible in the reported structure but are included as dotted lines in the figure. They are presumed to be important in W-ME107-75 binding because they are non-conserved in mouse IL13Rα2. (E) Here, the protein structure is rotated 180 degrees, and the beginning of peptides 228-245, which are part of the epitope in W-ME107-10 and W-ME107-27 but not in W-ME107-75, are seen as dark gray. [Figure 14C]This figure highlights the epitope sequences of W-ME107-10, W-ME107-27, and W-ME107-75 as revealed by HDX-MS. (A) The structure of IL13Rα2 (light gray) in complex with IL-13 (black) (PDB code 3LB6) is rotated 180 degrees compared to the field of view in Figure 10. The epitope sequences are colored dark gray along with numbered residue boundaries, and all are located on domain 3 of the receptor, with the exception of one sequence beginning in domain 2. The structure is shown in a schematic representation. (B-E) show IL13Rα2 in surface representation, highlighting the differences in epitopes predicted for W-ME107-10, W-ME107-27, and W-ME107-75 based on HDX-MS data along with binding data. Epitope regions are colored dark gray. IL-13 (black) is left as a schematic representation for clarity. (B) and (C) show the predicted binding regions for W-ME107-10 and W-ME107-27, respectively. In (D), the binding region for W-ME107-75 is shown in dark gray. W-ME107-75 behaves differently from W-ME107-10 and W-ME107-27 in many experiments and is therefore predicted to have a different binding site. The epitope is located in the very C-terminal region of the receptor, including residues 329-337. These residues are not visible in the reported structure but are included as dotted lines in the figure. They are presumed to be important in W-ME107-75 binding because they are non-conserved in mouse IL13Rα2. (E) Here, the protein structure is rotated 180 degrees, and the beginning of peptides 228-245, which are part of the epitope in W-ME107-10 and W-ME107-27 but not in W-ME107-75, are seen as dark gray. [Figure 14D]This figure highlights the epitope sequences of W-ME107-10, W-ME107-27, and W-ME107-75 as revealed by HDX-MS. (A) The structure of IL13Rα2 (light gray) in complex with IL-13 (black) (PDB code 3LB6) is rotated 180 degrees compared to the field of view in Figure 10. The epitope sequences are colored dark gray along with numbered residue boundaries, and all are located on domain 3 of the receptor, with the exception of one sequence beginning in domain 2. The structure is shown in a schematic representation. (B-E) show IL13Rα2 in surface representation, highlighting the differences in epitopes predicted for W-ME107-10, W-ME107-27, and W-ME107-75 based on HDX-MS data along with binding data. Epitope regions are colored dark gray. IL-13 (black) is left as a schematic representation for clarity. (B) and (C) show the predicted binding regions for W-ME107-10 and W-ME107-27, respectively. In (D), the binding region for W-ME107-75 is shown in dark gray. W-ME107-75 behaves differently from W-ME107-10 and W-ME107-27 in many experiments and is therefore predicted to have a different binding site. The epitope is located in the very C-terminal region of the receptor, including residues 329-337. These residues are not visible in the reported structure but are included as dotted lines in the figure. They are presumed to be important in W-ME107-75 binding because they are non-conserved in mouse IL13Rα2. (E) Here, the protein structure is rotated 180 degrees, and the beginning of peptides 228-245, which are part of the epitope in W-ME107-10 and W-ME107-27 but not in W-ME107-75, are seen as dark gray. [Figure 14E]This figure highlights the epitope sequences of W-ME107-10, W-ME107-27, and W-ME107-75 as revealed by HDX-MS. (A) The structure of IL13Rα2 (light gray) in complex with IL-13 (black) (PDB code 3LB6) is rotated 180 degrees compared to the field of view in Figure 10. The epitope sequences are colored dark gray along with numbered residue boundaries, and all are located on domain 3 of the receptor, with the exception of one sequence beginning in domain 2. The structure is shown in a schematic representation. (B-E) show IL13Rα2 in surface representation, highlighting the differences in epitopes predicted for W-ME107-10, W-ME107-27, and W-ME107-75 based on HDX-MS data along with binding data. Epitope regions are colored dark gray. IL-13 (black) is left as a schematic representation for clarity. (B) and (C) show the predicted binding regions for W-ME107-10 and W-ME107-27, respectively. In (D), the binding region for W-ME107-75 is shown in dark gray. W-ME107-75 behaves differently from W-ME107-10 and W-ME107-27 in many experiments and is therefore predicted to have a different binding site. The epitope is located in the very C-terminal region of the receptor, including residues 329-337. These residues are not visible in the reported structure but are included as dotted lines in the figure. They are presumed to be important in W-ME107-75 binding because they are non-conserved in mouse IL13Rα2. (E) Here, the protein structure is rotated 180 degrees, and the beginning of peptides 228-245, which are part of the epitope in W-ME107-10 and W-ME107-27 but not in W-ME107-75, are seen as dark gray. [Figure 15A] This shows the profiling and characterization of modified CAR T cells. (A) shows IFNγ secretion into culture medium from unstimulated control (mock) CAR T cells, W-ME107-10 CAR T cells, W-ME107-27 CAR T cells, W-ME107-55 CAR T cells, W-ME107-75 CAR T cells, and W-ME107-117 CAR T cells. [Figure 15B] This shows the profiling and characterization of modified CAR T cells. (B) shows the IFNγ secretion into the culture medium of control (mock) CAR T cells, W-ME107-10 CAR T cells, W-ME107-27 CAR T cells, W-ME107-55 CAR T cells, W-ME107-75 CAR T cells, and W-ME107-117 CAR T cells co-cultured with U87UU or U343MG tumor cells. [Figure 15C] Profiling and characterization of modified CAR T cells are shown. (C) shows CAR expression over time in control (mock) CAR T cells, W-ME107-10 CAR T cells, W-ME107-27 CAR T cells, W-ME107-55 CAR T cells, W-ME107-75 CAR T cells, and W-ME107-117 CAR T cells. [Figure 15D] Profiling and characterization of modified CAR T cells are shown. (D) Surface activation markers (PD1, TIM-3, LAG-3, CD69, and CD25) on control (mock) CAR T cells, W-ME107-27 CAR T cells, and W-ME107-117 CAR T cells in the presence or absence of tumor cell stimulation are shown. [Figure 16A] This study demonstrates that modified CAR T cells regulate glioblastoma tumor growth in vivo. (A) Schematic diagram of the experimental procedure. [Figure 16B] This shows that modified CAR T cells regulate glioblastoma tumor growth in vivo. (B) shows tumor growth at various days after tumor transplantation for control (mock) CAR T cells, W-ME107-10 CAR T cells, W-ME107-75 CAR T cells, and W-ME107-117 CAR T cells. [Figure 16C] This shows that modified CAR T cells regulate glioblastoma tumor growth in vivo. (C) shows the survival percentage of mice at various days after tumor transplantation for control (mock) CAR T cells, W-ME107-10 CAR T cells, W-ME107-75 CAR T cells, and W-ME107-117 CAR T cells. [Figure 17A] This study demonstrates that different complementarity-determining regions (CDRs) in the heavy and light chains of scFv influence CAR expression in CAR-T cells. (A) CAR expression on human Jurcut cells in each construct. (ns: no statistical significance; *: P<0.05; **: P<0.01). [Figure 17B] This shows that different complementarity-determining regions (CDRs) of the heavy and light chains of scFv influence CAR expression in CAR-T cells. (B) Representative plots showing GFP signaling as an indicator of transdextrins and CAR surface staining signals in each construct. [Figure 18A] The basal-level activation of modified CAR T cells is reduced when the intracellular signaling domain is removed. (A) Schematic diagram of a lentiviral construct used to express a CAR or decoy CAR (a CAR molecule that does not contain an intracellular signaling domain, designated as dCAR). [Figure 18B] The basal level activation of modified CAR T cells is reduced when the intracellular signaling domain is removed. (B) The schematic diagram shows CAR molecules and decoy CAR molecules on the cell membrane. [Figure 18C] The basal level activation of modified CAR T cells is reduced when the intracellular signaling domain is removed. (C) IFNγ secretion from different CAR-T cell constructs 7 days after transduction, without stimulation. [Modes for carrying out the invention]

[0019] This embodiment generally relates to an anti-IL13Rα2 antibody and its antigen-binding fragment, and more particularly to its use in cancer treatment. This embodiment relates to an antibody specific to interleukin-13 receptor subunit α-2 (IL13Rα2) and its antigen-binding fragment. The antibody and antigen-binding fragment of this embodiment are particularly suitable for use in chimeric antigen receptor (CAR) and CAR-based immunotherapy for various diseases, including cancers, characterized by the expression and presentation of IL13Rα2.

[0020] Antibody CAR constructs against IL-13Rα2 are known in the art, as exemplified in the literature cited in the background section. However, the prior art solutions have various drawbacks that limit their use in cancer treatment. For example, the monoclonal anti-IL13Rα2 antibody 1E10B9 (U.S. Patent No. 9,868,788; Debinski, et al., New agents for targeting of IL-13RA2 expressed in primary human and canine brain tumors, PLoS One 2013, 8(10):e77719) loses its ability to specifically bind to human IL13Rα2 when converted from IgG antibody to single-chain variable fragment (scFv) format, as shown in the examples section. Loss of antigen binding during switching between antibody formats is not uncommon, especially when transitioning from IgG to scFv. Modified protein folding that affects the structure of the antigen-binding site can best explain this. Therefore, while the prior art monoclonal anti-IL13Rα2 antibody 1E10B9 can be used in IgG form, it is not suitable for conversion to scFv format and is therefore unsuitable for use in CAR immunotherapy.

[0021] The monoclonal anti-IL13Rα2 antibody mAb47 (U.S. Patent No. 10,308,719; Balyasnikova et al., Characterization and immunotherapeutic implications for a novel antibody targeting interleukin (IL)-13 receptor α2, J Biol Chem 2012, 287(36):30215-30227; Kim et al., A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors, Sci Rep 2015, 5:18133) showed retention of binding to human IL13Rα2 upon conversion to scFv format. However, in comparative examples using the scFv of the present invention in CAR immunotherapy, CAR T cells generated based on mAb47 scFv showed only minimal target cell killing, whereas CAR T cells generated by this embodiment already showed sufficient cytotoxicity even with a low effector:target cell ratio. Therefore, the antigen-binding fragments of the antibodies according to the embodiments are superior to the antigen-binding fragments of antibody mAb47 when used in CAR immunotherapy. Furthermore, antibody mAb47 competes with ligand interleukin-13 (IL-13) for binding to the receptor IL13Rα2, whereas some antibodies of the embodiments and their antigen-binding fragments do not compete with IL-13 for binding to IL13Rα2.

[0022] In this specification, all amino acids in the variable region of an antibody or its antigen-binding fragment, including the complementarity-determining region (CDR) described herein, are therefore numbered and identified in accordance with the International ImMunoGeneTics (IMGT) information system and nomenclature (Lefranc et al., Dev Comp Immunol. (2003) 1:55-77).

[0023] The amino acid numbers in the human IL13Rα2 protein sequence follow the accession number NP_000631 and the NCBI reference sequence NP_000631.1 dated April 26, 2021, as well as the sequence presented below in this specification (SEQ ID NO: 107). MAFVCLAIGC LYTFLISTTF GCTSSSDTEI KVNPPQDFEI VDPGYLGYLY LQWQPPLSLD HFKECTVEYE LKYRNIGSET WKTIITKNLH YKDGFDLNKG IEAKIHTLLP WQCTNGSEVQ SSWAETTYWI SPQGIPETKV QDMDCVYYNW QYLLCSWKPG IGVLLDTNYN LFYWYEGLDH ALQCVDYIKA DGQNIGCRFP YLEASDYKDF YICVNGSSEN KPIRSSYFTF QLQNIVKPLP PVYLTFTRES SCEIKLKWSI PLGPIPARCF DYEIEIREDD TTLVTATVEN ETYTLKTTNE TRQLCFVVRS KVNIYCSDDG IWSEWSDKQC WEGEDLSKKT LLRFWLPFGF ILILVIFVTG LLLRKPNTYP KMIPEFFCDT

[0024] The specificity of an antibody, or an antigen-binding fragment thereof, can be determined based on, and / or, its binding affinity. Affinity is the equilibrium constant for the dissociation of an antigen from an antibody, or an antigen-binding fragment thereof, (K D ), and is a measure of the strength of binding between an antigen determinant, i.e., an epitope, and an antigen-binding on an antibody, or an antigen-binding fragment thereof. The smaller the value of K D , the stronger the binding strength between the antigen determinant and the antibody, or an antigen-binding fragment thereof. Alternatively, affinity can also be expressed as an affinity constant (K D ) which is 1 / K A . As will be apparent to those skilled in the art, affinity can be determined by methods known per se, depending on the specific antigen of interest.

[0025] Binding strength is a measure of the binding strength between an antibody, or its antigen-binding fragment, and the associated antigen. Binding strength is related to both the antigenic determinant and its affinity for the antigen-binding site on the antibody, or its antigen-binding fragment, and the number of associated binding sites present on the antibody, or its antigen-binding fragment.

[0026] Typically, an antibody or its antigen-binding fragment is 10 -5 ~10 -12 moles / liter (M) or less, and preferably 10 -7 ~10 -12 M or less, and more preferably 10 -8 ~10 -12 The equilibrium dissociation constant (K) of M D ) and, in other words, 10 5 ~10 12 M -1 The above, and preferably 10 7 ~10 12 M -1 More preferably 10 8 ~10 12 M -1 affinity constant (K A ) binds to those antigens. Usually 10 -4 Any K greater than M D Value (or 10) 4 M -1 any K smaller than A The value is considered to indicate nonspecific binding.

[0027] Preferably, the antibody of the embodiment, or its antigen-binding fragment, binds to IL13Rα2 with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example less than 5 nM. The specific binding of an antibody, or its antigen-binding fragment, to an antigen or antigenic determinant can be determined by any preferred method known in itself, for example, scatchard analysis and / or competitive binding assays such as radioimmunoassays (RIAs), enzyme immunoassays (EIAs), and sandwich competitive assays; Luminex® multiplex assays and various variations thereof known in the art.

[0028] The inventors have identified a novel epitope or antigenic determinant region in IL13Rα2, which is highly suitable for targeting with antibodies and their antigen-binding fragments. In particular, antibodies and their antigen-binding fragments that target this novel epitope are useful in CAR immunotherapy. This epitope corresponds to the second and largest β-sheet in domain 1 of IL13Rα2, as shown in Figure 10. This is the N-terminal portion of the receptor, and domain 1 consists of a β-sandwich folded between two β-sheets on each other. The epitope is located in three of the four chains in the largest β-sheet and is located in a region away from the IL-13 binding site relative to IL13Rα2. See Figure 10.

[0029] The antibody that specifically binds to the epitopes in this β-sheet, and its antigen-binding fragment, exhibited high specificity for IL13Rα2 and did not bind to IL13Rα1. The antigen-binding fragment showed excellent cytotoxic activity against cancer cells expressing IL13Rα2 in CAR T-cell immunotherapy.

[0030] The embodiments of this model relate to an antibody or antigen-binding fragment thereof that can bind to IL13Rα2. The antibody or antigen-binding fragment is specific to an epitope within the β-sheet region or domain of IL13Rα2, including the first β-chain at amino acid numbers 68-75, the loop following the first β-chain, the second β-chain at amino acid numbers 101-109, the loop preceding the second β-chain, and the third β-chain at amino acid numbers 124-128.

[0031] The amino acid sequence of IL13Rα2 is shown in SEQ ID NO: 107. Amino acid numbers 68-75 correspond to the amino acid sequence EYELKYRN (SEQ ID NO: 108), amino acid numbers 101-109 correspond to the amino acid sequence IEAKIHTLL (SEQ ID NO: 109), and amino acid numbers 124-128 correspond to the amino acid sequence AETTY (SEQ ID NO: 110).

[0032] Figure 10 shows the largest β-sheet of domain 1, which has a hatched ellipse around three β-chains numbered 3, 6, and 7 that form an epitope. β-chains 6 and 7 are interconnected by a loop region containing an alpha-helix and a bend. β-chains 3 and 6 are interconnected by an amino acid sequence containing two folds and two β-chains (numbered 4 and 5; β-chain 4 belongs to the largest β-sheet together with chains 3, 6, and 7, and β-chain 5 belongs to the first smaller β-sheet of domain 1).

[0033] In one embodiment, the antibody, or its antigen-binding fragment, has specificity for an epitope comprising at least one peptide, also called an epitope region, selected from the group consisting of amino acid numbers 67-81, 96-106, and 123-128 in IL13Rα2. In one embodiment, the first peptide or epitope region (VEYELKYRNIGSETW, SEQ ID NO: 44) substantially corresponds to β-chain 3 and the subsequent folding portion of β-chain 3 in IL13Rα2. The second peptide or epitope region (DLNKGIEAKIH, SEQ ID NO: 45) substantially corresponds to the short stretch of β-chain 6 and the preceding loop, while the third peptide or epitope region (WAETTY, SEQ ID NO: 46) substantially corresponds to β-chain 7.

[0034] The antibody, or its antigen-binding fragment, has specificity for at least one of these three peptide or epitope regions in the β-sheet region of IL13Rα2. Therefore, the antibody, or its antigen-binding fragment, has specificity for the first peptide or epitope (VEYELKYRNIGSETW, SEQ ID NO: 44), the antibody, or its antigen-binding fragment, has specificity for the second peptide or epitope region (DLNKGIEAKIH, SEQ ID NO: 45), or the antibody, or its antigen-binding fragment, has specificity for the third peptide or epitope region (WAETTY, SEQ ID NO: 46).

[0035] In one embodiment, the antibody, or its antigen-binding fragment, has specificity for at least two of these three peptide or epitope regions in the β-sheet region of IL13Rα2. Thus, the antibody, or its antigen-binding fragment, has specificity for the first peptide or epitope (VEYELKYRNIGSETW, SEQ ID NO: 44) and the second peptide or epitope region (DLNKGIEAKIH, SEQ ID NO: 45), the antibody, or its antigen-binding fragment, has specificity for the first peptide or epitope region (VEYELKYRNIGSETW, SEQ ID NO: 44) and the third peptide or epitope region (WAETTY, SEQ ID NO: 46), or the antibody, or its antigen-binding fragment, has specificity for the second peptide or epitope region (DLNKGIEAKIH, SEQ ID NO: 45) and the third peptide or epitope region (WAETTY, SEQ ID NO: 46).

[0036] In a preferred embodiment, the antibody, or its antigen-binding fragment, has specificity for all three peptide or epitope regions in the β-sheet region of IL13Rα2, namely, the first peptide or epitope region (VEYELKYRNIGSETW, SEQ ID NO: 44), the second peptide or epitope region (DLNKGIEAKIH, SEQ ID NO: 45), and the third peptide or epitope region (WAETTY, SEQ ID NO: 46).

[0037] The present invention also relates to an epitope of IL13Rα2. This epitope is located within the β-sheet region of IL13Rα2, which includes a first β-chain of amino acids 68-75, a loop following the first β-chain, a second β-chain of amino acids 101-109, a loop preceding the second β-chain, and a third β-chain of amino acids 124-128.

[0038] In one embodiment, the epitope comprises at least one peptide, preferably at least two peptides, and more preferably all three peptides, selected from the group consisting of amino acid numbers 67-81, i.e., VEYELKYRNIGSETW (SEQ ID NO: 44), amino acid numbers 96-106, i.e., DLNKGIEAKIH (SEQ ID NO: 45), and amino acid numbers 123-128, i.e., WAETTY (SEQ ID NO: 46) in IL13Rα2.

[0039] In one embodiment, the antibody, or its antigen-binding fragment, includes a variable heavy chain (VH) domain complementarity-determining region 3 (CDR3) containing the amino acid sequence YSPFY (SEQ ID NO: 3). In one embodiment, the antibody, or its antigen-binding fragment, comprises a VH domain CDR3 containing the amino acid sequence YSPFYM (SEQ ID NO: 9). In a specific embodiment, the antibody, or its antigen-binding fragment, comprises a VH domain CDR3 containing, preferably, the amino acid sequence ARYSPFYMDY (SEQ ID NO: 10). In one embodiment, the antibody, or its antigen-binding fragment, comprises a variable light chain (VL) domain CDR3 containing the amino acid sequence GYSFPP (SEQ ID NO: 4). In certain embodiments, the antibody, or its antigen-binding fragment, comprises a VL domain CDR3 comprising, preferably, the amino acid sequence QQGYSFPPT (SEQ ID NO: 11). In one embodiment, the antibody, or its antigen-binding fragment, comprises a VH domain CDR1 containing the amino acid sequence SGSY (SEQ ID NO: 1). In one embodiment, the antibody, or its antigen-binding fragment, comprises a VH domain CDR1 comprising the amino acid sequence GFTFSGSY (SEQ ID NO: 106), preferably consisting of the same. In one embodiment, the antibody, or its antigen-binding fragment, comprises the amino acid sequence SGSYMS (SEQ ID NO: 5), preferably the amino acid sequence GFTFSGSYMS (SEQ ID NO: 6), and preferably an extended VH domain CDR1 comprising the same. In one embodiment, the antibody, or its antigen-binding fragment, comprises a VH domain CDR2 containing the amino acid sequence YGSGGY (SEQ ID NO: 2). In certain embodiments, the antibody, or its antigen-binding fragment, comprises a VH domain CDR2 comprising the amino acid sequence IYGSGGYT (SEQ ID NO: 7), preferably consisting of the same. In one embodiment, the antibody, or its antigen-binding fragment, comprises an extended VH domain CDR2 comprising, preferably, the amino acid sequence SIYGSGGYTY (SEQ ID NO: 8). As used herein, extended CDR refers to an amino acid sequence comprising at least one additional amino acid residue beyond the amino acid of a CDR as defined according to IMGT nomenclature. In one embodiment, the antibody, or its antigen-binding fragment, comprises a VL domain CDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 12), preferably consisting thereof. In one embodiment, the antibody, or its antigen-binding fragment, comprises an amino acid sequence AAS, preferably a VL domain CDR2 comprising the same. The antibody, or its antigen-binding fragment, may include the VH domain and / or VL domain CDR regions of at least one of the above embodiments, preferably at least two, more preferably at least three, and even more preferably at least four, at least five, or all of the CDR regions.

[0040] In one embodiment, the antibody, or its antigen-binding fragment, comprises at least two of the above-described VH domains and / or VL domain CDR regions. Such embodiments include: an antibody, or its antigen-binding fragment, comprising VH domain CDR1 and VH domain CDR2 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1 and VH domain CDR3 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1 and VL domain CDR1 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1 and VL domain CDR2 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1 and VL domain CDR3 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR2 and VH domain CDR1 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR2 and VL domain CDR1 as defined above; and VH domain CDR2 and VL domain CDR3 as defined above. An antibody containing CDR2 or its antigen-binding fragment; an antibody containing the VH domain CDR2 and VL domain CDR3 as defined above, or its antigen-binding fragment; an antibody containing the VH domain CDR3 and VL domain CDR1 as defined above, or its antigen-binding fragment; an antibody containing the VH domain CDR3 and VL domain CDR2 as defined above, or its antigen-binding fragment; an antibody containing the VH domain CDR3 and VL domain CDR3 as defined above, or its antigen-binding fragment; an antibody containing the VL domain CDR1 and VL domain CDR2 as defined above, or its antigen-binding fragment; an antibody containing the VL domain CDR1 and VL domain CDR3 as defined above, or its antigen-binding fragment; and an antibody containing the VL domain CDR2 and VL domain CDR3 as defined above, or its antigen-binding fragment.

[0041] In one embodiment, the antibody, or its antigen-binding fragment, comprises at least three of the above-described VH domains and / or VL domain CDR regions. Such embodiments include: an antibody, or its antigen-binding fragment, comprising VH domain CDR1, VH domain CDR2, and VH domain CDR3 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1, VH domain CDR2, and VL domain CDR1 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1, VH domain CDR2, and VL domain CDR2 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1, VH domain CDR2, and VL domain CDR3 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1, VH domain CDR3, and VL domain CDR2 as defined above; an antibody, or its antigen-binding fragment, comprising VH domain CDR1, VH domain CDR3, and VL domain CDR3 as defined above; An antibody comprising the VH domain CDR1, VL domain CDR1, and VL domain CDR2 as defined above, or its antigen-binding fragment; an antibody comprising the VH domain CDR1, VL domain CDR1, and VL domain CDR3 as defined above, or its antigen-binding fragment; an antibody comprising the VH domain CDR1, VL domain CDR2, and VL domain CDR3 as defined above, or its antigen-binding fragment; such embodiments include an antibody comprising the VH domain CDR2, VH domain CDR3, and VL domain CDR1 as defined above, or its antigen-binding fragment; an antibody comprising the VH domain CDR2, VH domain CDR3, and VL domain CDR3 as defined above, or its antigen-binding fragment; an antibody comprising the VH domain CDR2, VL domain CDR1, and VL domain CDR2 as defined above, or its antigen-binding fragment;An antibody containing the VH domain CDR2, VL domain CDR1, and VL domain CDR3 as defined above, or its antigen-binding fragment; an antibody containing the VH domain CDR2, VL domain CDR2, and VL domain CDR3 as defined above, or its antigen-binding fragment; an antibody containing the VH domain CDR3, VL domain CDR1, and VL domain CDR2 as defined above, or its antigen-binding fragment; an antibody containing the VH domain CDR3, VL domain CDR1, and VL domain CDR3 as defined above, or its antigen-binding fragment; and an antibody containing the VL domain CDR1, VL domain CDR2, and VL domain CDR3 as defined above, or its antigen-binding fragment.

[0042] In one embodiment, the antibody, or its antigen-binding fragment, comprises at least four of the above-described VH domains and / or VL domain CDR regions. Such embodiments include an antibody or antigen-binding fragment thereof comprising VH domain CDR1, VH domain CDR2, VH domain CDR3 and VL domain CDR1 as defined above; an antibody or antigen-binding fragment thereof comprising VH domain CDR1, VH domain CDR2, VH domain CDR3 and VL domain CDR2 as defined above; an antibody or antigen-binding fragment thereof comprising VH domain CDR1, VH domain CDR2, VH domain CDR3 and VL domain CDR3 as defined above; an antibody or antigen-binding fragment thereof comprising VH domain CDR1, VH domain CDR2, VL domain CDR1 and VL domain CDR2 as defined above; an antibody or antigen-binding fragment thereof comprising VH domain CDR1, VH domain CDR2, VL domain CDR2 and VL domain CDR3 as defined above Fragments; antibodies containing VH domain CDR1, VH domain CDR3, VL domain CDR1 and VL domain CDR2 as defined above, or their antigen-binding fragments; antibodies containing VH domain CDR1, VH domain CDR3, VL domain CDR1 and VL domain CDR3 as defined above, or their antigen-binding fragments; antibodies containing VH domain CDR1, VH domain CDR3, VL domain CDR2 and VL domain CDR3 as defined above, or their antigen-binding fragments; antibodies containing VH domain CDR1, VL domain CDR1, VL domain CDR2 and VL domain CDR3 as defined above, or their antigen-binding fragments; antibodies containing VH domain CDR2, VH domain CDR3, VL domain CDR1 and VL domain CDR2 as defined above, or their antigen-binding fragments; antibodies containing VH domain CDR2, VH domain CDR3, VL domain CDR1 and VL domain CDR3 as defined above, or their antigen-binding fragments;An antibody or its antigen-binding fragment comprising the VH domain CDR2, VH domain CDR3, VL domain CDR2, and VL domain CDR3 as defined above; an antibody or its antigen-binding fragment comprising the VH domain CDR2, VL domain CDR1, VL domain CDR2, and VL domain CDR3 as defined above; and an antibody or its antigen-binding fragment comprising the VH domain CDR3, VL domain CDR1, VL domain CDR2, and VL domain CDR3 as defined above.

[0043] In one embodiment, the antibody, or its antigen-binding fragment, comprises at least five of the above-described VH domains and / or VL domain CDR regions. Such embodiments include: an antibody, or its antigen-binding fragment, comprising the above-described VH domain CDR1, VH domain CDR2, VH domain CDR3, VL domain CDR1 and VL domain CDR2; an antibody, or its antigen-binding fragment, comprising the above-described VH domain CDR1, VH domain CDR2, VH domain CDR3, VL domain CDR1 and VL domain CDR3; an antibody, or its antigen-binding fragment, comprising the above-described VH domain CDR1, VH domain CDR2, VH domain CDR3, VL domain CDR2 and VL domain CDR3 The following are included: an antibody or antigen-binding fragment thereof comprising VH domain CDR1, VH domain CDR2, VL domain CDR1, VL domain CDR2 and VL domain CDR3 as defined above; an antibody or antigen-binding fragment thereof comprising VH domain CDR1, VH domain CDR3, VL domain CDR1, VL domain CDR2 and VL domain CDR3 as defined above; and an antibody or antigen-binding fragment thereof comprising VH domain CDR2, VH domain CDR3, VL domain CDR1, VL domain CDR2 and VL domain CDR3 as defined above. In one embodiment, the antibody, or its antigen-binding fragment, includes all six of the above-described VH domains and VL domain CDR regions. Such embodiments include an antibody, or its antigen-binding fragment, that includes the VH domain CDR1, VH domain CDR2, VH domain CDR3, VL domain CDR1, VL domain CDR2, and VL domain CDR3 as defined above.

[0044] In one embodiment, the antibody, or its antigen-binding fragment, comprises, preferably, a VH domain comprising the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSGSYMSWVRQAPGKGLEWVSSIYGSGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSPFYMDYWGQGTLVTVSS (SEQ ID NO: 13).

[0045] In one embodiment, the antibody, or its antigen-binding fragment, comprises a VL domain, preferably consisting of the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSFPPTFGQGTKLEIK (SEQ ID NO: 14). In certain embodiments, the antibody, or its antigen-binding fragment, comprises a VH domain, preferably comprising the amino acid sequence of SEQ ID NO: 13, and a VL domain, preferably comprising the amino acid sequence of SEQ ID NO: 14.

[0046] A particularly preferred antibody of the embodiment is referred to herein as ME107-117 or W-ME107-117, and its corresponding antigen-binding fragment is referred to as ME107-117 scFv or W-ME107-117 scFv. This antibody and its antigen-binding fragment specifically bind to an epitope in the β-sheet region of IL13Rα2 (Figure 10) and include the VH and VL domain CDR regions described above.

[0047] Another embodiment relates to an antibody or antigen-binding fragment thereof that can bind to IL13Rα2. The antibody or antigen-binding fragment thereof comprises a VH domain CDR1 having the amino acid sequence GFTFX1X2X3X4, preferably consisting of each X n n=1-4 are independently selected from the group consisting of G, A, S, and Y. The antibody, or its antigen-binding fragment, also comprises a VH domain CDR2, preferably consisting of the amino acid sequence IB1B2B3B4B5B6T, and each B m m=1~6 is independently selected from the group consisting of G, S, and Y. The antibody, or its antigen-binding fragment, has the amino acid sequence AR-Z H - Further comprising a VH domain CDR3 containing, preferably consisting of Z1DY, where Z1 is selected from the group consisting of F, M, I and L, Z H This is an amino acid sequence selected from the group consisting of VVRSTYGY (SEQ ID NO: 15), YGHYAYGSY (SEQ ID NO: 16), YSSSGWYYGF (SEQ ID NO: 17), TPYSAY (SEQ ID NO: 18), RYRSHRPGLS (SEQ ID NO: 19), FHPRYGY (SEQ ID NO: 20), GSYSHYGAHY (SEQ ID NO: 21), YYHYDYGYYY (SEQ ID NO: 22), YSPFY (SEQ ID NO: 3), RNYWEHGGGS (SEQ ID NO: 24), HHYGYYPPGSVYY (SEQ ID NO: 25), and VEYTYYGSEGSPV (SEQ ID NO: 26). The antibody, or its antigen-binding fragment, contains the amino acid sequence QSISSY (SEQ ID NO: 12), preferably comprising a VL domain CDR1. The antibody, or its antigen-binding fragment, also contains the amino acid sequence AAS, preferably comprising a VL domain CDR2, and the amino acid sequence QQ-Z L -Includes, preferably consisting of, a VL domain CDR3 containing T, Z LThis is an amino acid sequence selected from the group consisting of TYYSPH (SEQ ID NO: 28), DYYLF (SEQ ID NO: 29), SYSTPY (SEQ ID NO: 30), FYSYPL (SEQ ID NO: 31), AFSPS (SEQ ID NO: 32), SYDTLL (SEQ ID NO: 33), ALSSLP (SEQ ID NO: 34), FSTRLS (SEQ ID NO: 35), GYSFPP (SEQ ID NO: 4), STYPF (SEQ ID NO: 37), YGSNPL (SEQ ID NO: 38), and RYNGLF (SEQ ID NO: 39). In one embodiment, the antibody, or its antigen-binding fragment, comprises an extended VH CDR1 comprising, preferably, the amino acid sequence GFTFX1X2X3X4MX5. In this embodiment, each X n n=1 to 5 are independently selected from the groups G, A, S, and Y. In one embodiment, X5 is S or G. In one embodiment, the antibody, or its antigen-binding fragment, comprises an extended VH CDR2 having the amino acid sequence JIB1B2B3B4B5B6TY, preferably consisting of the same. In this embodiment, each B m m=1~6 are independently selected from the group consisting of G, S, and Y, and J is selected from the group consisting of A, Y, G, and S. In one embodiment, J is selected from the group consisting of A, Y, and S. In one embodiment, X1 is S or Y, X2 is S or G, X3 is S or Y, and X4 is A, Y, or G. In one embodiment, B1 is S or Y, B2 is G, B3 is S, G or Y, B4 is G, B5 is S or G, and B6 is S or Y. In one embodiment, Z H -Z1 is an amino acid sequence selected from the group consisting of YGHYAYGSYF (SEQ ID NO: 40), TPYSAYI (SEQ ID NO: 41), GSYSHYGAHYL (SEQ ID NO: 42), and YSPFYM (SEQ ID NO: 9). In one embodiment, Z LThis is an amino acid sequence selected from the group consisting of DYYLF (SEQ ID NO: 29), FYSYPL (SEQ ID NO: 31), ALSSLP (SEQ ID NO: 34), and GYSFPP (SEQ ID NO: 4).

[0048] The preferred embodiments of the antigen-binding fragments at present are referred to herein as W-ME107-7, W-ME107-10, W-ME107-16, W-ME107-27, W-ME107-55, W-ME107-67, W-ME107-75, W-ME107-112, W-ME107-117, W-ME107-128, W-ME107-150, and W-ME107-156. Accordingly, preferred antibodies have CDR regions and VH and VL domains corresponding to these antigen-binding fragments. Table 1 shows VH domain CDR1, Table 2 shows VH domain extension CDR1, Table 3 shows VH domain CDR2, and Table 4 shows VH domain extension CDR2. Table 5 shows VH domain CDR3, and Table 6 shows VL domain CDR3. W-ME107-7, W-ME107-10, W-ME107-16, W-ME107-27, W-ME107-55, W-ME107-67, W-ME107-75, W-ME107-112, W-ME107-117, W-ME107-128, W-ME107-150, and W-ME107-156 all have a common VL domain CDR1 QSISSY (sequence number 12) and a common VL domain CDR2 AAS. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0049] Particularly preferred antigen-binding fragments of the embodiment are W-ME107-10, W-ME107-27, W-ME107-75, and W-ME107-117, and particularly preferred antibodies are antibodies having the VH and VL CDR regions of W-ME107-10, W-ME107-27, W-ME107-75, and W-ME107-117.

[0050] In one embodiment, the antibody, or its antigen-binding fragment, comprises the VH CDR region of clone W-ME107-10, as shown in Tables 1, 3, and 5, and preferably as identified in Tables 2, 4, and 5. In one embodiment, the antibody, or its antigen-binding fragment, includes the VL CDR3 of clone W-ME107-10, as identified in Table 6, along with common VL domains CDR1 and CDR2 regions.

[0051] In certain embodiments, the antibody, or its antigen-binding fragment, includes the VH CDR region of clone W-ME107-10, as shown in Tables 1, 3, and 5, preferably identified in Tables 2, 4, and 5, and the VL CDR3 region of clone W-ME107-10, as identified in Table 6, along with common VL domain CDR1 and CDR2 regions.

[0052] In one embodiment, the antibody, or its antigen-binding fragment, comprises the VH CDR region of clone W-ME107-27, as shown in Tables 1, 3, and 5, and preferably as identified in Tables 2, 4, and 5. In one embodiment, the antibody, or its antigen-binding fragment, includes the VL CDR3 of clone W-ME107-27, as identified in Table 6, along with common VL domains CDR1 and CDR2 regions.

[0053] In certain embodiments, the antibody, or its antigen-binding fragment, comprises the VH CDR region of clone W-ME107-27, as identified in Tables 1, 3, and 5, preferably as identified in Tables 2, 4, and 5, and the VL CDR3 region of clone W-ME107-27, as identified in Table 6, along with common VL domain CDR1 and CDR2 regions.

[0054] In one embodiment, the antibody, or its antigen-binding fragment, comprises the VH CDR region of clone W-ME107-75, as identified in Tables 1, 3, and 5, preferably as identified in Tables 2, 4, and 5. In one embodiment, the antibody, or its antigen-binding fragment, includes the VL CDR3 of clone W-ME107-75, identified in Table 6, along with common VL domains CDR1 and CDR2 regions.

[0055] In certain embodiments, the antibody, or its antigen-binding fragment, includes the VH CDR region of clone W-ME107-75, as identified in Tables 1, 3, and 5, preferably as identified in Tables 2, 4, and 5, and the VL CDR3 region of clone W-ME107-75, as identified in Table 6, along with common VL domain CDR1 and CDR2 regions.

[0056] In one embodiment, the antibody, or its antigen-binding fragment, comprises the VH CDR region of clone W-ME107-117, as identified in Tables 1, 3, and 5, preferably as identified in Tables 2, 4, and 5. In one embodiment, the antibody, or its antigen-binding fragment, includes the VL CDR3 of clone W-ME107-117, as identified in Table 6, along with the common VL domains CDR1 and CDR2 regions.

[0057] In certain embodiments, the antibody, or its antigen-binding fragment, includes the VH CDR region of clone W-ME107-117, as identified in Tables 1, 3, and 5, preferably as identified in Tables 2, 4, and 5, and the VL CDR3 region of clone W-ME107-117, as identified in Table 6, along with common VL domain CDR1 and CDR2 regions.

[0058] W-ME107-10 is (Sequence ID 86):EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGHYAYGSYFDYWGQGTLVTVSS The VH domain and (SEQ ID NO: 87): Includes the VL domain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYYLFTFGQGTKLEIK.

[0059] W-ME107-27 is (Sequence ID 88): EVQLLESGGGLVQPGGSLRLSCAASGFTFYGSYMGWVRQAPGKGLEWVSYISGYGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTPYSAYIDYWGQGTLVTVSS The VH domain and (SEQ ID NO: 89): Includes the VL domain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYSYPLTFGQGTKLEIK.

[0060] W-ME107-75 is (Sequence ID 90): EVQLLESGGGLVQPGGSLRLSCAASGFTFYSYGMSWVRQAPGKGLEWVSYISGGGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYSHYGAHYLDYWGQGTLVTVSS The VH domain and (SEQ ID NO: 91): Includes the VL domain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALSSLPTFGQGTKLEIK.

[0061] W-ME107-117 is (Sequence ID 13): EVQLLESGGGLVQPGGSLRLSCAASGFTFSGSYMSWVRQAPGKGLEWVSSIYGSGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSPFYMDYWGQGTLVTVSS The VH domain and (SEQ ID NO: 14): Includes the VL domain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSFPPTFGQGTKLEIK.

[0062] In some embodiments, the VH domain of an antibody, or its antigen-binding fragment, is fused to the VL domain via a linker. Various such linkers interconnecting the VH and VL domains in the antibody and its antigen-binding fragment may be used depending on the embodiment. In certain embodiments, the linker is a peptide linker. For example, a peptide linker may consist of, for example, the amino acids glycine (G) and / or serine (S). A non-limiting example of such a peptide linker includes, preferably consists of, the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 27).

[0063] Those skilled in the art will understand that minor changes, such as the substitution of one, two, three, four or more amino acid residues in an amino acid sequence, including the deletion or addition of amino acids, can occur without affecting the functional properties of the antibody or its antigen-binding fragment, such as its ability to bind to IL13Rα2. These changes may be present in the amino acid sequence of the CDR in the heavy and / or light chain variable region, in the amino acid sequence outside the CDR region, i.e., in the framework region, or in both the CDR and the CDR regions. Accordingly, some embodiments also include antibodies or their antigen-binding fragments having at least 75%, preferably at least 80%, for example, at least 85%, and more preferably at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences presented herein or in the sequence listings.

[0064] As used herein, sequence identity refers to the sequence similarity between two amino acid sequences, such as peptide or protein sequences. Similarity is determined by sequence alignment to determine the structural and / or functional relationship between sequences. Sequence identity between amino acid sequences can be determined by comparing the alignment of sequences using the Needleman-Wunsch Global Sequence Alignment Tool, available from the National Center for Biotechnology Information (NCBI), Bethesda, Md., USA, for example via http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, with default parameter settings (for protein alignment, Gap costs Existence: 11, Extension: 1). The sequence comparisons and percentages mentioned herein were determined using this software. For example, when comparing the level of sequence identity for a given amino acid sequence, this should preferably be performed over the entire length of the amino acid sequence; that is, the global alignment method is used to avoid short regions of high identity overlap that would result in a high overall identity rating. For example, a short polypeptide fragment containing five amino acids may have 100% identical sequences to the five amino acid regions within the overall amino acid sequence. However, this does not constitute 100% amino acid identity unless the fragment forms part of a longer sequence that also contains identical amino acids at other equivalent positions in the amino acid sequence. Molecules are identical at positions where equivalent positions in the sequences being compared are occupied by identical amino acids. The scoring of alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the sequences being compared. When comparing sequences, optimal alignment may require gaps introduced into one or more sequences to account for possible insertions and deletions.

[0065] In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. In some embodiments, the antibody is a genetically engineered antibody, such as a single-chain antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is a chimeric antibody. As used herein, a chimeric antibody refers to an antibody that contains a constant domain from one species and a variable domain from a second species. In some embodiments, the antibody is a humanized antibody. As used herein, humanization refers to an antibody having at least one CDR region of non-human origin that is modified to have a structure and immunological function that is more similar to a true human antibody than to the antibody of its original origin. An example of a humanized antibody is an antibody having a CDR region of non-human antibody grafted onto a human antibody. Humanization may also include, in addition to or instead of, selective amino acid substitutions to make the non-human amino acid sequence more similar to a human sequence.

[0066] Suitable antibody production methods are known in the art. For example, the standard hybridoma method is described in Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001).

[0067] Monoclonal antibodies can be prepared by any technique that enables the production of antibody molecules by serially passaged cell lines in culture. These include, but are not limited to, the hybridoma technique first described in Nature 256:495-497, 1975, the human B-cell hybridoma technique (Immunol Today 4:72, 1983; Proc Natl Acad Sci 80:2026-2030, 1983), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York NY, pp 77-96, (1985)). Polyclonal antibodies can be prepared by immunizing animals with an immunogen containing the IL13Rα2 antigen and collecting antiserum from the immunized animals. A wide range of animal species, including but not limited to rabbits, mice, rats, hamsters, goats, sheep, pigs, or horses, can be used for antiserum production.

[0068] Alternatively, antibodies and their antigen-binding fragments may be selectively produced using phage display selection as described herein. As used herein, antibody antigen-binding fragments can be selected from the group consisting of single-chain antibodies, Fv fragments, scFv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, single-domain antibodies (sdAb), scFv-Fc fragments, di-scFv fragments, and CDR regions. Currently, the preferred embodiment of an antigen-binding fragment is the single-chain variable fragment (scFv).

[0069] In some embodiments, the antibody, or its antigen-binding fragment, specifically binds to IL13Rα2, preferably human IL13Rα2. In some embodiments, the antibody, or its antigen-binding fragment, specifically binds to non-human IL13Rα2, either additionally or instead. Non-limiting examples of such non-human IL13Rα2 include canine IL13Rα2, feline IL13Rα2, bovine IL13Rα2, horse IL13Rα2, sheep IL13Rα2, rat IL13Rα2, and / or mouse IL13Rα2. In one embodiment, the antibody, or its antigen-binding fragment, does not bind to human IL13Rα1.

[0070] One embodiment relates to a chimeric antigen receptor (CAR). The CAR comprises an antigen recognition domain containing an antibody or its antigen-binding fragment according to the embodiment. The CAR also comprises a transmembrane domain and an intracellular signaling domain. Generally, a CAR includes an external domain, a transmembrane domain, and an endodomain. The external domain of a CAR may contain an antigen recognition region, which may be an scFv. The external domain may also contain a signal tag or a peptide that guides the CAR into the endoplasmic reticulum.

[0071] The transmembrane domain is the portion of the CAR that crosses the cell membrane. Generally, the transmembrane domain can be derived from any transmembrane protein. In some embodiments, the transmembrane domain includes a hydrophobic alpha-helix. Non-limiting examples of transmembrane domains that may be contained in a CAR include all or part of the transmembrane domain of surface antigen classification 28 (CD28), all or part of the transmembrane domain of CD8α, all or part of the transmembrane domain of CD27, all or part of the transmembrane domain of CD137(4-1BB), all or part of the transmembrane domain of CD134(OX40), all or part of the transmembrane domain of CD3ε, all or part of the transmembrane domain of CD3ζ, all or part of the transmembrane domain of CD3γ, all or part of the transmembrane domain of CD3δ, all or part of the transmembrane domain of TCRα, and all or part of the transmembrane domain of TCRβ, preferably including all or part of the transmembrane domain of CD28 or all or part of the transmembrane domain of CD8α.

[0072] The endodomain of a CAR contains at least one signaling domain. Non-limiting examples of such signaling domains that may be contained in a CAR include the zeta chain of CD3 (CD3ζ), CD28, CD137 (4-1BB), ICOS, CD27, CD40, OX40 (CD134), or Myd88, preferably CD3ζ and / or CD137. In one embodiment, the CAR includes a hinge domain or spacer that interconnects the antigen recognition domain and the transmembrane domain.

[0073] Relevant aspects of the embodiment define a T cell receptor (TCR) complex comprising an antigen recognition domain containing an antibody or its antigen-binding fragment according to the embodiment. The TCR is a molecule found on the surface of T cells that is involved in the recognition of antigen fragments as a peptide that binds to major histocompatibility complex (MHC) molecules. When the TCR engages with the antigenic peptide and MHC, the T cell is activated through a series of biochemical events mediated by signaling, i.e., related enzymes, co-receptors, differentiation adapter molecules, and activated or released transcription factors. The TCR complex is typically the TCR molecule bound to the CD3γ chain, CD3δ chain, and two CD3ε chains. These chains bind to the TCR and ζ chain (zeta chain) to generate an activation signal in the T cell. Together, the TCR, ζ chain, and CD3 molecules constitute the TCR complex. Non-exclusive examples of usable TCRs include the CMVpp65 TCR and the TARP TCR.

[0074] Another aspect of the embodiment relates to a conjugate comprising an antibody, or its antigen-binding fragment, and an effector molecule, according to the embodiment. The conjugate of the embodiment includes an antibody, or its antigen-binding fragment, as a targeting domain or molecule and an effector domain or molecule. The antibody, or its antigen-binding fragment, directs the conjugate to IL13Rα2-expressing cells, including tumor cells.

[0075] The antibody of the embodiment, or its antigen-binding fragment, may be used in conjunction with various so-called effector domains or molecules. In some embodiments, the effector molecule is selected from the group consisting of a detectable label, a cytotoxin, a metal, another antibody, or its antigen-binding fragment, a nucleic acid sequence, and a lipid bilayer docking moiety.

[0076] In one embodiment, the effector domain may be used for detection and diagnostic purposes. In such cases, the effector domain is a detectable label, such as an irradiative label, a fluorescent label, or a chemiluminescent label. The conjugate can then be used for the diagnosis and imaging of IL13Rα2, for example, in IL13Rα2-expressing cells in the body of a subject. Depending on the specific detectable label, various imaging techniques, such as X-ray imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), or single-photon emission computed tomography (SPECT), may then be used.

[0077] Instead of using detectable labels, the antibody of the embodiment, or its antigen-binding fragment, has an element such as hydrogen or carbon in at least one amino acid residue of the antibody, or its antigen-binding fragment, or its isotope, for example, deuterium instead of hydrogen and 12 Instead of C 13 It can be detected by substituting with C.

[0078] Paramagnetic metals and metals containing radioactive isotopes can also be used as effector domains for imaging purposes. Cytotoxins, or cytotoxic agents, can be used as effector domains to exert cytotoxicity and kill IL13Rα2-expressing cells when the conjugate targets cells using an antibody or its antigen-binding fragment. Typical examples of cytotoxins are chemotherapeutic agents. Any chemotherapeutic agent, including but not limited to alkylating agents, antimetabolites, antimicrotubule inhibitors, topoisomerase inhibitors, and cytotoxic antibiotics, can be used according to the embodiment. In another embodiment, the effector domain is an apoptosis tag, which induces apoptosis in cells expressing IL13Rα2 and targeted by a conjugate. An example of such an apoptosis tag is the TRAIL protein. In yet another embodiment, the effector domain is a T cell or B cell epitope, or a nucleic acid sequence encoding a T cell or B cell epitope, which causes specific induction of T cell or B cell immunity against the epitope. The effector domain may be another antibody or its antigen-binding fragment. For example, the effector domain could be the Fc domain of IgG or other immunoglobulin. The Fc domain is then used to enable purification via a protein A affinity column. Additionally, or instead, the Fc domain may improve the in vivo half-life of the conjugate. Furthermore, the Fc region enables dimerization / multimerization of the conjugate.

[0079] Further embodiments of the embodiments include nucleic acid molecules encoding antibodies, or their antigen-binding fragments, CARs, and / or TCR complexes, according to the embodiments. When used herein, nucleic acid molecules include polynucleotides, oligonucleotides, and nucleic acid sequences, typically meaning macromolecules of DNA or RNA, which may be single-stranded or double-stranded, and which may include natural, unnatural, or modified nucleotides, and which may include natural, unnatural, or modified nucleotide interbondings, such as phosphoramidate bonds or phosphorothioate bonds rather than phosphate diesters found between nucleotides in unmodified oligonucleotides. Nucleic acid molecules also include complementary DNA (cDNA) and messenger RNA (mRNA).

[0080] Nucleic acid molecules can also encode molecules other than antibodies or their antigen-binding fragments, depending on the embodiment. An example of such another molecule is Helicobacter pylori (HP) neutrophil-effective activating protein (NAP), HP-NAP is a dodecamer protein that acts as a pathogenic factor in H. pylori bacterial infections. It is made up of 12 monomer subunits, each subunit consisting of four alpha helices. The surface of HP-NAP is strongly positively charged and has the ability to interact with and activate human leukocytes (WBCs) (also denoted as leukocytes).

[0081] Another aspect of the embodiment relates to a vector containing nucleic acid molecules according to the embodiment. The vector is preferably an expression vector, i.e., a vector containing an expression vector which includes at least one nucleic acid molecule containing a coding sequence that can be expressed in a host cell, such as a host T cell, by being transcribed and translated. In some embodiments, the expression vector is selected from DNA molecules, RNA molecules, plasmids, episomal plasmids, and viral vectors. In one embodiment, the vector is a viral vector. In a particular embodiment, the viral vector is selected from the group consisting of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, Semryki forest virus, poliovirus, and hybrid vectors.

[0082] Lentiviruses are a subclass of retroviruses. They are adapted as gene delivery vehicles (vectors) due to their ability to integrate into the genomes of non-dividing cells. This is a unique characteristic of lentiviruses, as other retroviruses can only infect dividing cells. The RNA genome of the virus is reverse transcribed when the virus enters a cell, such as a T cell, producing DNA, which is then inserted into the genome at a specific location by the viral integrase enzyme. The vector (here called a provirus) remains in the genome and is passed on to the cell's offspring when the cell divides. For safety reasons, lentiviral vectors typically never contain the genes necessary for their replication. To create lentiviruses, several plasmids are introduced into a so-called packaging cell line, usually HEK293. One or more plasmids, commonly called packaging plasmids, encode virion proteins, such as the capsid and reverse transcriptase. Another plasmid contains the genetic material delivered by the vector. It is transcribed to produce a single-stranded RNA viral genome, marked by the presence of a Ψ (psi) sequence. This sequence is used to package the genome within the virion.

[0083] Retroviruses play a central role in current gene therapy techniques. Recombinant retroviruses, such as Moloney's mouse leukemia virus, have the ability to stably integrate into the host genome. They contain reverse transcriptase, which enables integration into the host genome. Retroviral vectors can be either replication-capable or replication-deficient. Replication-deficient vectors are the most common choice. This is because the virus has coding regions for genes that are substituted or deleted with other genes necessary for additional rounds of virion replication and packaging. These viruses can infect T cells and deliver their viral load, but cannot subsequently follow the typical lytic pathway that leads to cell lysis and cell death. If the vector is a lentiviral or retroviral vector, the nucleic acid sequences encoding the CAR and / or TCR complex and HP-NAP and / or immunologically equivalent HP-NAP fragments are preferably RNA sequences.

[0084] As used herein, adenovirus vectors include adenovirus vectors and adenovirus-derived virus vectors. Adenovirus DNA is not integrated into the genome and does not replicate during cell division. Adenovirus-derived viral vectors are adenovirus-based but have undergone various modifications, such as modifications related to the nucleotide sequences encoding replication proteins, i.e., regulatory proteins and surface proteins.

[0085] Adeno-associated viruses (AAVs) are small viruses that infect humans and some primate species. AAVs can infect both dividing and non-dividing cells and can integrate their genome into the host cell's genome. Furthermore, AAVs mostly exist as episomes and undergo long-term stable expression. AAVs package single-stranded DNA and require the process of second-strand synthesis, while self-complementary adeno-associated viruses (scAAVs) package both strands, which anneal together to form double-stranded DNA. By skipping second-strand synthesis, scAAVs enable rapid expression in cells. If the vector is an adenovirus vector, the nucleic acid sequences encoding the CAR and / or TCR complex and HP-NAP and / or immunologically equivalent HP-NAP fragments are preferably DNA sequences.

[0086] Semliki Forest Virus is a positive-strand RNA virus with a genome of approximately 13,000 base pairs encoding nine proteins. The 5' two-thirds of the genome encode four non-structural proteins related to RNA synthesis, while structural proteins are encoded in the 3' one-third. Of the structural proteins, the C protein constitutes an icosahedral capsid covered by a lipid bilayer derived from the host cell. The outermost surface of the virus is almost completely covered by glycoprotein E1 and E2 heterodimers arranged in interconnected trimers that form the outer shell. The trimers are fixed in the membrane by an E2 cytoplasmic domain that binds to the nucleocapsid. Due to its broad host range and efficient replication, it has also been developed as a vector.

[0087] A hybrid vector is a vector virus that is genetically engineered to possess the properties of two or more vectors. For example, a hybrid vector could be a combination of an adenovirus and a lentivirus.

[0088] In some embodiments, the antibody, or its antigen-binding fragment, or nucleic acid molecule encoding the CAR and / or TCR complex, is under the transcriptional control of a promoter. In some embodiments, the promoter is selected from the group consisting of the human EF1α promoter, the CMV promoter, and the CAG promoter, preferably from the EF1α promoter. In one embodiment, the vector comprises a nucleic acid molecule encoding a CAR and / or TCR complex under the transcriptional control of a promoter such as the EF1α promoter. The vector also preferably has a signal peptide for secretion and comprises a nucleic acid molecule encoding HP-NAP under the transcriptional control of an inducible promoter such as the inducible NFAT-IL-2 promoter. Further embodiments of the embodiment relate to cells comprising antibodies, or their antigen-binding fragments, CARs, TCR complexes, nucleic acid molecules, and / or vectors according to the embodiment. The nucleic acid or vector can then be transcribed in cells to produce antibodies, or their antigen-binding fragments, CARs, and / or TCR complexes, within the cells. In some embodiments, the cells are selected from a group consisting of T cells, natural killer (NK) cells, B cells, monocytes, and macrophages. In certain embodiments, the cells are T cells.

[0089] The embodiments also relate to pharmaceutical compositions comprising antibodies, or their antigen-binding fragments, CARs, TCR complexes, cells, conjugates, nucleic acids, and / or vectors, and pharmaceutically acceptable carriers according to the embodiments.

[0090] A pharmaceutically acceptable carrier includes a pharmaceutically acceptable carrier, vehicle and / or excipient, or a combination thereof, that is compatible with the other components of the pharmaceutical composition. Non-limiting examples of such a pharmaceutically acceptable carrier include injectable solutions such as physiological saline or buffered injectable solution.

[0091] The embodiments also relate to antibodies or their antigen-binding fragments, CARs, TCR complexes, and conjugates, where the effector molecule is a cytotoxin, nucleic acid molecule, vector, cell, and / or pharmaceutical composition, according to embodiments for use as a drug. In certain embodiments, the effector molecule is a cytotoxin, nucleic acid molecule, vector, cell, and / or pharmaceutical composition according to the embodiment, an antibody or its antigen-binding fragment, CAR, TCR complex, or conjugate can be used in the treatment or delay of the onset of cancerous diseases characterized by the expression of IL13Rα2 on cancer cells, i.e., IL13Rα2-expressing cancers.

[0092] In some embodiments, IL13Rα2-expressing cancers are selected from the group consisting of glioblastoma, medulloblastoma, breast cancer, head and neck cancer, pancreatic cancer, kidney cancer, ovarian cancer, colon cancer, liver cancer, lung cancer, urothelial carcinoma, melanoma, and Kaposi's sarcoma.

[0093] Further embodiments relate to methods for treating, reducing, and / or preventing IL13Rα2-expressing cancer in patients. The method comprises administering an effective amount of an antibody, or its antigen-binding fragment, CAR, TCR complex, or conjugate, to a patient, where the effector molecule is a cytotoxin, nucleic acid molecule, vector, cell, and / or pharmaceutical composition according to the embodiments.

[0094] As used herein, effective dose refers to the effective amount for administration and time required to obtain a desired outcome. For example, in inhibiting tumor growth, effective dose is the amount that induces, for example, remission, a reduction in tumor burden, and / or prevents tumor propagation or growth, compared to the response obtained without administration of cells. Effective dose may vary depending on factors such as the patient's disease state, age, sex, and / or weight. As used herein, treating or therapy means a method for obtaining a beneficial or desired outcome, including clinical outcomes, which is well understood in the art. Beneficial or desired clinical outcomes may include reduction or improvement of one or more symptoms or conditions, reduction of disease severity, stabilization of the disease state, i.e., prevention of exacerbation, prevention of disease spread, delay or slowing of disease progression, improvement or mitigation of disease state, reduction of disease recurrence, and remission. Treating or therapy may also include an extension of survival compared to the predicted survival time without treatment.

[0095] As used herein, prevention or mitigation means methods of reducing or preventing the risk of developing a disease or condition, which are well understood in the art and may include delaying or prolonging the onset of the disease. For example, patients who are predisposed to developing a disease, such as due to genetics or a genetic predisposition, may benefit from the administration of antibodies, or their antigen-binding fragments, cells, conjugates and / or pharmaceutical compositions, according to embodiments for preventing the onset of the disease, reducing its risk, delaying and / or slowing it.

[0096] The patient is preferably human. However, the embodiments may also be applied to non-human patients, i.e., non-human mammals including, for example, primates, monkeys, apes, cattle, sheep, pigs, goats, horses, cattle, dogs, mice, rats, and guinea pigs. Antibodies, or their antigen-binding fragments, cells, conjugates, and / or pharmaceutical compositions according to the embodiments, may be administered to the patient by various routes, e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, or intratumor administration.

[0097] The present invention also relates to a method for identifying IL13Rα2-positive cells. The method includes contacting a biological sample with an antibody or its antigen-binding fragment according to the embodiments, and measuring the amount of the antibody or its antigen-binding fragment bound to at least one cell in the biological sample, thereby identifying at least one cell as an IL13Rα2-positive cell.

[0098] In some embodiments, the antibody, or its antigen-binding fragment, is fused to or bound to a detectable label, such as one containing an isotope or using a biotin-avidin or biotin-streptavidin bond, as previously described herein. Alternatively, IL13Rα2-positive cells can be detected using flow cytometry (FCM) or enzyme-linked immunosorbent assay (ELISA).

[0099] Examples The examples herein describe the development of IL13Rα2-targeted human single-chain antibody variable fragments (scFv) for use in chimeric antigen receptor (CAR) cell therapy for the treatment of cancer.

[0100] Example 1 - Conversion of 1E10B9 and mAb47 IgG to scFv and binding characterization by ELISA and HTRF A literature review identified two existing monoclonal anti-IL13Rα2 agents: 1E10B9 (US Patent No. 9,868,788; Debinski, et al., New agents for targeting of IL-13RA2 expressed in primary human and canine brain tumors, PLoS One 2013, 8(10):e77719) and mAb clone 47 (mAb47) (US Patent No. 10,308,719; Balyasnikova et al., Characterization and immunotherapeutic implications for a novel antibody targeting interleukin(IL)-13 receptor α2, J Biol Chem 2012, 287(36):30215-30227; Kim et al., A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors, Sci Rep 2015, 5:18133). These are both mouse antibodies produced by hybridoma technology. In the case of 1E10B9, a peptide fragment encoding a portion of the extracellular domain of IL13Rα2, which has 100% homology between human and canine sequences, was synthesized and used as an immunogen. The resulting antibody 1E10B9 therefore binds both human and canine homologous species of the receptor. mAb47 was obtained by using the complete extracellular region of the receptor (IL13Rα2-Fc) as an immunogen. The reported affinity for human IL13Rα2 is K DThe antibody has a molecular weight of 1.4 nM and was found not to bind to human IL13Rα1 or mouse IL13Rα2 (Balyasnikova et al., Characterization and immunotherapeutic implications for a novel antibody targeting interleukin(IL)-13 receptor α2, J Biol Chem 2012, 287(36):30215-30227). Furthermore, this antibody competes with IL-13 for binding to IL13Rα2.

[0101] The genes encoding the scFv proteins of 1E10B9 and mAb47 were synthesized, the proteins were expressed in bacteria, and their binding to human IL13Rα2 was tested by ELISA and homogeneous time-resolved fluorescence (HTRF). The results of these assays showed that only mAb47 scFv retained the ability to bind its full-length parental IgG counterpart.

[0102] Materials and methods gene synthesis The sequences of the variable domains of the heavy chain (VH) and light chain (VL) of 1E10B9 and mAb47 were obtained from U.S. Patent No. 9,868,788 and No. 10,308,719, respectively. The first seven amino acids of the VH of 1E10B9 were not included in the patent. To generate the complete VH gene, the first seven amino acids of the most homologous mouse VH gene were included based on a search of the IMGT database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi). By fusing VH to VL via a glycine-serine linker ((Gly4Ser)3), genes encoding the corresponding scFv constructs 1E10B9 scFv (also known as X-ME107-B9) (SEQ ID NO: 92) and mAb47 scFv (also known as X-ME107-47) (SEQ ID NO: 93) were formed. 1E10B9 scFv(VH+linker+VL+3xFLAG+Hisx6)(Sequence ID 92) [ka] mAb47 scFv(VH+linker+VL+3xFLAG+Hisx6)(SEQ ID NO: 93) [ka]

[0103] The 1E10B9 scFv and mAb47 scFv were synthesized and subcloned using GenScript (Piscataway, NJ, USA). Codon optimization was performed based on Escherichia coli expression. After synthesis, the scFv gene was inserted into the pHAT-6 vector (SciLifeLab, Stockholm, Sweden) using restriction enzymes SfiI and NotI. The pHAT-6 vector yields secreted scFv with a triple FLAG tag and a hexahistidine (Hisx6) tag at the C-terminus.

[0104] The vector is transformed into Top10 E.coli, and the desired sequence is obtained. This was confirmed by sequencing (GATC, Germany).

[0105] Protein expression Small-scale expression of 1E10B9 scFv and mAb47 scFv, with two colonies per scFv, was performed in 96-deep-well plates. After overnight incubation, the bacteria were centrifuged and the supernatant was used for functional evaluation by both ELISA and HTRF.

[0106] ELISA Human IL13Rα2-avi (see Example A1) and the negative control protein streptavidin were coated in PBS at a concentration of 1 μg / ml in 384-well plates overnight at 4°C.

[0107] The plates were washed twice with Milli-Q water and blocked for 2 hours in blocking buffer (phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) + 0.05% Tween 20®). Triple-FLAG-tagged scFv 1E10B9 and mAb47 present in the bacterial supernatant were diluted 1:2, 1:20, and 1:200 in blocking buffer and conjugated. A streptavidin-specific assay control scFv (G-strep-1) was also included. Binding detection was enabled by HRP-labeled anti-FLAG M2 antibody (Sigma-Aldrich #A8592), followed by incubation with 1-step Ultra TMB ELISA substrate (Thermofisher Scientific #34029). Colorimetric analysis signal generation was stopped by adding 1M sulfuric acid, and the plates were read at 450 nm. All samples were assayed in duplicate.

[0108] Uniform time-resolved fluorescence (HTRF) 1E10B9, mAb47, and positive assay control scFv were diluted 1:5 in assay buffer (PBS supplemented with 0.1% BSA) and conjugated to human IL13Rα2-avi and unrelated proteins (both diluted to 200 nM in assay buffer). Binding detection was enabled using a terbium-labeled anti-FLAG antibody (Cisbio #611FG2TL) for the donor molecule and streptavidin-labeled XL665 (Cisbio #610SAXL) for the receptor molecule. Plates were incubated in the dark at room temperature for 2 hours and then analyzed at 615 nm (background / noise signal) and 665 nm (binding signal) using an Envision spectrometer (Perkin Elmer). All samples were assayed in duplicate.

[0109] result 4 μg vectors encoding 1E10B9 scFv and mAb47 scFv were obtained from GenScript. DNA sequencing confirmed the correct sequences. Small-scale expression was performed, and the binding of these small sets to antigens was analyzed using ELISA and HTRF.

[0110] The results of these analyses showed that mAb47 scFv recognizes human IL13Rα2 (Figures 1 and 2). 1E10B9 scFv did not produce a detectable signal for any of its contained antigens in either ELISA or HTRF.

[0111] conclusion The scFv gene encoding VH and VL of IL13Rα2, specific to mouse IgG antibodies 1E10B9 and mAb47, was successfully synthesized, inserted into pHAT6, expressed, and analyzed for binding by ELISA and HTRF.

[0112] mAb47 scFv showed retained binding to human IL13Rα2, while no binding to the negative control protein was detected. 1E10B9 scFv, however, did not show binding to human IL13Rα2. This is in contrast to what has been reported for its full-length IgG counterpart (Kim et al., A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors, Sci Rep 2015, 5:18133). Loss of antigen binding during alternation between antibody formats is not uncommon, especially when transitioning from IgG to scFv. Modified protein folding affecting the structure of the antigen-binding site best explains this.

[0113] Example 2 - Phage display selection for human and mouse IL13Rα2 using a phage library Phage display selection was performed to enable the isolation of scFv fragments specific to human and mouse IL13Rα2. scFv fragments were selected from two different human phage libraries (SciLifeLib1 and 2). Primary screening of a total of 920 clones by ELISA yielded 673 positive clones, which were sent for sequencing. Of these, 304 were found to have unique sequences.

[0114] Materials and methods antigen Table 7 lists the human and mouse versions of IL13Rα2 used as antigens for phage display selection. [Table 7]

[0115] Phage display selection Biopanning was performed using two human synthetic scFv phage libraries, SciLifeLib1 and SciLifeLib2 (SciLifeLab, Stockholm, Sweden), with four selection rounds of enrichment. SciLifeLib1 and 2 are unsensitized human synthetic scFv libraries similar in design and composition to those previously reported (Sall, et al., Protein Eng Des Sel (2016) 29:427-437). Briefly, human germline genes IGHV3-23 and IGKV1-39 were used as the library backbone, and Kunkel mutagenesis was used to introduce diversity into four of the six complementarity-determining regions (CDRs): CDR-H1, CDR-H2, CDR-H3, and CDR-L3. Selection of biotinylated samples (hIL13Rα2-avi) was performed using streptavidin-coated magnetic beads (Dynabeads M-280, ThermoFisher Scientific, #11206D). Similarly, Fc-fused mIL13Rα2-Fc were captured using protein G-binding magnetic beads (Dynabeads ThermoFisher Scientific, #10004D). In two tracks, the antigen was alternated between human and mouse IL13Rα2 in different rounds to preferentially select interspecies reactive scFv. Furthermore, in two other tracks, human IL-13 (Prospec #cyt-446) was included in the selection buffer. Selection pressure was increased by gradually decreasing the amount of antigen and by increasing the number and intensity of washes between different rounds. Elution of antigen-binding phages was performed using the trypsin-aprotinin method. The entire selection process, excluding the phage target protein incubation step, was automated and performed using a Kingfisher Flex robot. Different parameter combinations resulted in a scheme encompassing six distinct selection tracks.

[0116] Recloning and expression of scFv To produce soluble scFv, phagemide DNA was isolated from the third and fourth rounds of each selected track. In a pool, the gene encoding the scFv fragment was digested with restriction enzymes and subcloned into the screening vector pHAT-6, resulting in a signal for scFv secretion along with a triple FLAG tag and a hexahistidine (His6) tag at the C-terminus. The construct was then transformed into TOP10 E. coli. Single colonies were harvested, cultured, and IPTG-induced soluble scFv expression in a 96-well format. In total, 920 scFv clones present in the bacterial supernatant were prepared for primary ELISA screening.

[0117] ELISA sorting Mouse IL13Rα2-Fc was directly coated overnight at 4°C in 384-well ELISA plates with 1 μg / ml of PBS, while human IL13Rα2-avi was indirectly coated overnight at 4°C via streptavidin, similarly with 1 μg / ml of PBS. Two negative control proteins, streptavidin and BSA, were also coated.

[0118] FLAG-tagged W-ME107 scFv clones present in bacterial supernatant were diluted 1:3 with blocking buffer (PBS supplemented with 0.5% BSA + 0.05% Tween 20®) and conjugated to coated proteins. Binding was detected using HRP-labeled anti-FLAG M2 antibody (Sigma-Aldrich #A8592), followed by incubation with TMB ELISA substrate (Thermofisher Scientific #34029). Colorimetric analysis signal generation was stopped by adding 1M sulfuric acid, and the plates were read at 450 nm. All samples were assayed in duplicate. Two reference scFvs, mAb47 scFv and G-strep-1 scFv, were also included.

[0119] DNA sequencing method 673 positive scFv clones showing binding to human and / or mouse IL13Rα2 were sent to GATC Biotech (Ebersberg, Germany) for Sanger DNA sequencing.

[0120] result A total of six selection tracks were performed in parallel on human and / or mouse IL13Rα2 using SciLifeLib1 and 2. After recloning of the selected scFv clones, 92 clones (colonies) were taken from selection rounds 3 and 4, resulting in a total of 920 selected clones.

[0121] ELISA screening identified 673 potential hits with distinct binding characteristics. The majority of hits showed binding only to human IL13Rα2, while the remaining hits showed binding to both human and mouse IL13Rα2. DNA sequencing of 673 hits identified 304 unique sequences in the W-ME107 clone.

[0122] conclusion scFv clones that bind to human and / or mouse IL13Rα2 were successfully isolated by phage display selection. After initial ELISA screening of 920 clones and DNA sequencing of 673 positive hits, a total of 304 unique sequence scFv clones were identified. The majority of clones bound only to human IL13Rα2, but one set showed binding to both human and mouse IL13Rα2.

[0123] Example 3 - Secondary sorting of 304 sequence-unique scFv clones by ELISA and HTRF To facilitate further ranking of the 304 sequence-unique scFv clones, secondary ELISA and homogeneous time-resolved fluorescence (HTRF) screening were performed on all sequence-unique clones. These analyses confirmed the results of the initial ELISA screening (Example 2) and identified two major clonal groups based on binding specificity. One group bound only to human IL13Rα2, while the other group included clones that bound to both human and mouse IL13Rα2. Using the generated ELISA and HTRF data, the number of clones was further reduced to produce a list of 160 scFvs. These 160 clones were selected based on two main criteria: 1) showing high signal to both mouse and human IL13Rα2 (external reactivity) or 2) showing high signal to human IL13Rα2 and low background signal to unrelated targets.

[0124] Materials and methods ELISA Human IL13Rα2-avi was indirectly coated via streptavidin into a 384-well ELISA plate, while mouse IL13Rα2-Fc and negative control proteins (streptavidin and unrelated proteins) were directly coated into the wells. Details of the IL13Rα2 proteins used in this example are listed in Table 7. After coating the plates overnight at 4°C, the plates were washed twice with Milli-Q water and blocked for 2 hours in blocking buffer (PBS supplemented with 0.5% BSA + 0.05% Tween 20®). Triple-FLAG tagged W-ME107 scFv clones present in the bacterial supernatant were diluted 1:10 in blocking buffer and conjugated. Binding was detected using HRP-labeled anti-FLAG M2 antibody (Sigma-Aldrich #A8592), followed by incubation with 1-step Ultra TMB ELISA substrate (Thermofisher Scientific #34029). Colorimetric analysis signal generation was stopped by adding 1M sulfuric acid, and the plates were analyzed at 450 nm. All samples were assayed in duplicate. Two positive controls, mAb47 scFv and G-strep-1, were also included.

[0125] HTRF W-ME107 scFv clones, as well as reference mAb47 scFv and G-strep-1 scFv, were diluted 1:5 in assay buffer (PBS supplemented with 0.1% BSA) and conjugated to hIL13Rα2-avi or an unrelated antigen diluted to 200 nM in assay buffer. Binding detection was enabled using the donor molecule terbium-labeled anti-FLAG antibody (Cisbio #611FG2TL) and the receptor molecule streptavidin-labeled XL665 (Cisbio #610SAXL). Plates were incubated in the dark at room temperature for 2 hours and then analyzed using an Envision spectrometer (Perkin Elmer) at 615 nm (background / noise signal) and 665 nm (binding signal). The R-value for each sample was obtained by dividing the 665 nm value by the 615 nm value. All samples were assayed in duplicate.

[0126] result ELISA The scFv clones can be divided into two main groups based on binding specificity. The first group, which includes the majority of scFv, bound only to human IL13Rα2, while clones belonging to the second group bound to both human and mouse IL13Rα2. The reference clone mAb47 scFv showed binding only to human IL13Rα2. HTRF 304 scFv clones were also analyzed for binding to human IL13Rα2 using a FRET-based homogeneous solution assay. In this assay as well, a far greater number of clones showed clear binding to human IL13Rα2 and did not show significant binding to the negative control included in this assay. Surprisingly, mAb47 scFv showed an extremely weak binding signal.

[0127] conclusion ELISA and HTRF sorting were successfully performed on 304 sequence-unique scFv clones isolated by phage display selection. Based on these results, along with the results from the Luminex assay performed on the same sample in Example 4, 160 clones were selected for further screening by SPR. These 160 clones could be divided into two subsets based on their binding characteristics: i) clones that bind only to human IL13Rα2, and ii) clones that bind to both human and mouse IL13Rα2.

[0128] Example 4 - Evaluation of binding specificity of 304 scFv clones using Luminex In this embodiment, all 304 unique scFv clones, as well as the reference clone mAb47 scFv, were further evaluated for specificity using a Luminex-based method. This analysis showed that 304 clones of the eight strains exhibited nonspecific binding to several unrelated proteins. The remaining 296 clones, as well as the reference clone mAb47, showed specific binding to their target antigen, human IL13Rα2.

[0129] Along with the Luminex® data shown in Example 4, the number of clones was further reduced using ELISA and HTRF data generated from the same sample in Example 3 to produce a list of 160 scFv. In addition to good performance with Luminex®, these 160 clones were selected based on two main criteria: 1) showing a high signal to both mouse and human IL13Rα2 (external reactivity) or 2) showing a high signal to human IL13Rα2 and a low background signal to unrelated targets.

[0130] Materials and methods Biotinylated human IL13Rα2-avi and 31 biotinylated unrelated proteins were individually conjugated to specific neutraavidin-conjugated Luminex bead IDs. After conjugation, all bead IDs were mixed and incubated with scFv clones present in bacterial supernatant, diluted 1:10 in assay buffer (PBS supplemented with 3% BSA, 0.05% Tween 20®, and 10 μg / ml neutraavidin). Each of the 31 unrelated proteins contained at least one positive control scFv. Binding of clones to specific protein-conjugated beads was enabled by R-PE-labeled anti-FLAG M2 (Prozyme #PJ315) antibody and subsequently analyzed using a FlexMAP 3D analyzer. All samples were assayed in duplicate, and the average of the resulting binding signals, corresponding to the median intensity fluorescence (MFI), was calculated for each clone for each bead ID.

[0131] result Of the 304 scFv clones, 296 showed specific binding to human IL13Rα2. Eight clones, however, showed binding to several unrelated proteins in addition to binding to human IL13Rα2. Notably, for each of the 31 unrelated proteins, at least one positive control scFv was assayed. All of these interacted specifically with their respective homologous antigens, as predicted. This ensures that all antigens functionally bound to their respective beads.

[0132] conclusion The 32Plex Luminex®-based assay was successfully performed on 304 sequence-unique scFv clones isolated by phage display selection. Only 8 scFv clones showed nonspecific binding to unrelated proteins included in the assay. The remaining 296 clones, and the reference clone mAb47, showed specific binding to human IL13Rα2 of their target antigens and showed no significant binding to any of the 31 unrelated proteins.

[0133] Example 5 - Dynamic sorting of 160 array-unique scFv using SPR 160 W-ME107 scFv clones from Example 4, as well as a reference clone mAb47 scFv, were selected for further characterization by surface plasmon resonance (SPR) using a dynamical sorting-based method that allows for efficient ranking of different clones.

[0134] Materials and methods Dynamic sorting was performed using a BIACORE® T200 analyzer (GE Healthcare). Anti-FLAG M2 antibody (Sigma-Aldrich #F1804), which functions as a capture ligand, was immobilized on all four surfaces of the CM5-S amine sensor tip according to the manufacturer's recommended conditions.

[0135] 160 FLAG-tagged W-ME107 clones present in the bacterial supernatant were injected and captured on the chip surface, followed by injection of hIL13Rα2-avi, hIL13Rα2-Fc, or mIL13Rα2-Fc at 50 nM. Details of the IL13Rα2 proteins used in this study are described in Table 8. The surface was regenerated with 10 mM glycine-HCl pH 2.2. All experiments were performed at 25°C in running buffer (HBS supplemented with 0.05% Tween 20®, pH 7.5).

[0136] Response curve sensograms were obtained for all scFv clones by subtracting the response curve of the reference surface from the response curve of the surface immobilized with anti-FLAG M2 antibody. The data were analyzed using BIACORE® T200 Evaluation 3.1 software. [Table 8]

[0137] result Anti-FLAG M2 antibody was immobilized on all four surfaces of the CM5 S tip, yielding captured scFv clones with similar RU levels. After injection of IL13Rα2 at 50 nM, each surface was successfully regenerated using a low pH acid solution.

[0138] Data analysis was performed by visual inspection (not shown) of sensorgrams. From the sensorgrams, it was clear that the clones could be divided into three groups based on their binding characteristics: (i) scFv clones that bind to both human IL13Rα2-avi and human IL13Rα2-Fc, as well as mouse IL13Rα2-Fc; (ii) scFv clones that bind to both human IL13Rα2 variants but show no measurable binding to mouse IL13Rα2-Fc; and (iii) scFv that bind only to human IL13Rα2-avi but not to human IL13Rα2-Fc, with a small number of these also showing binding to mouse IL13Rα2-Fc.

[0139] Table 9 describes 44 binding patterns for 160 scFvs. This set was considered most promising based on binding to human receptors. More specifically, high binding response and a favorable slow off-rate were considered. In addition, clones exhibiting heterogeneous reactivity were included. [Table 9] TIFF0007844012000012.tif145162

[0140] conclusion Dynamic sorting yielded clones exhibiting different binding characteristics: (i) clones that bind to both human IL13Rα2-avi and Fc-fused human IL13Rα2 (IL13Rα2-Fc), as well as mouse IL13Rα2-Fc; (ii) clones that bind to both human IL13Rα2 variants but show no measurable binding to mouse IL13Rα2-Fc; and (iii) clones that bind only to human IL13Rα2-avi but show no measurable binding to human IL13Rα2-Fc, some of which also show binding to mouse IL13Rα2-Fc. The generated data allowed for efficient ranking of the different clones and a further reduction in the number of clones from 160 to 44.

[0141] Examples 6-44: Small-scale protein purification and ELISA analysis of scFv clones Forty-four clones and a reference clone, mAb47 scFv, were selected for small-scale protein production and purification based on the results shown in Examples 3–5.

[0142] Protein purification was performed using Protein A or nickel-bonded magnetic beads in a Kingfisher Flex analyzer in a 96-well format. All 44 W-ME107 scFv clones could be purified to sufficient purity and protein concentration. The reference clone mAb47 scFv, however, was insufficiently expressed, resulting in lower purity and concentration. When analyzed by ELISA, most purified clones showed binding to their predicted antigens, human and / or mouse IL13Rα2.

[0143] Materials and methods Production and Purification Each of the 44+1 scFv clones was produced in 15 ml of E. coli culture. After 18 hours of protein production at 30°C, the cells were lysed with B-PER reagent (Thermofisher Scientific #78248). The clarified bacterial lysates of the W-ME107 scFv clone and the reference clone mAb47 scFv were mixed with protein A-binding magnetic beads (Thermofisher Scientific #88846) or MagneHis Ni particle magnetic beads (Promega #V8548), respectively, to enable purification using a Kingfisher Flex instrument. The buffer of the eluted scFv clones was replaced with PBS using a Zeba 96-well spin desalting plate (Thermofisher Scientific #89807).

[0144] Purified scFv clones were analyzed by gel electrophoresis under reducing conditions to measure purity and integrity, and protein concentration was measured by standard BCA (bicinchoninic acid) assay (Thermofisher Scientific #23227) according to manufacturer-recommended conditions.

[0145] ELISA Human IL13Rα2-avi, human IL13Rα2-Fc, and mouse IL13Rα2-Fc were coated in PBS at a concentration of 1 μg / ml in 384-well ELISA plates at 37°C for 45 minutes. Details of the IL13Rα2 proteins used in this example are listed in Table 8. Three negative control proteins (streptavidin, BSA, and an unrelated protein) were also coated. FLAG-tagged scFv was diluted to 1 μg / ml in blocking buffer (PBS supplemented with 0.5% BSA + 0.05% Tween 20®) and conjugated to the coated proteins. Binding was detected using HRP-labeled anti-FLAG M2 antibody (Sigma-Aldrich #A8592), followed by incubation with TMB ELISA substrate (Thermofisher Scientific #34029). The colorimetric analysis signal generation was stopped by adding 1M sulfuric acid, and the plate was read at 450nm. All samples, including the reference clone mAb47 scFv and the positive assay control G-strep-1 scFv (specific to streptavidin), were dual-assayed.

[0146] result Production and Purification For 44 W-ME107 scFv samples, SDS-PAGE showed sufficient sample purity, i.e., one major band correlated with the scFv predicted molecular weight (approximately 30 kDa) and either absent or only faintly weak E. coli-derived protein bands (Figure 3). The reference clone mAb47, however, exhibited low sample purity and contained multiple E. coli-derived protein bands. Furthermore, the protein band corresponding to its predicted molecular weight was considerably weaker than all other bands on the gel, indicating a low sample concentration for this clone. The protein concentration of the purified scFv samples was measured by a standard BCA assay. All 44 W-ME107 scFv clones were purified to a sufficient protein concentration in the range of 0.1–0.6 mg / ml.

[0147] ELISA Most purified W-ME107 scFv clones showed binding to their target antigen, human and / or mouse IL13Rα2, and non-binding or very low binding to coated negative control proteins. However, some clones exhibited low or no antigen binding.

[0148] conclusion Forty-four W-ME107 scFv clones and reference clone mAb47 were purified on a small scale using a Kingfisher Flex instrument. All scFv clones, except for reference clone mAb47, showed sufficient sample purity and sufficient protein concentration. When assayed using ELISA-based methods, the majority of clones showed binding to their target antigen, human and / or mouse IL13Rα2. However, some clones did not function as expected, exhibiting low binding or no antigenic binding.

[0149] Example 7 - Investigation of binding of 44 scFv clones to human IL13Rα1 by ELISA. In Example 6, 44 selected clones for small-scale protein purification and the reference clone mAb47 scFv were tested for binding to human IL13Rα1 using an ELISA-based method. The results showed that all W-ME107 scFv clones showed binding to their target antigens, human and / or mouse IL13Rα2, but no significant binding to human IL13Rα1. The same results were obtained for the reference clone mAb47 scFv.

[0150] Materials and methods The coating proteins hIL13Rα2-avi (Example A1), hIL13Rα2-Fc (RnD Systems #7147-IR), mIl13Rα2-Fc (RnD Systems #539-IR), and hIL13Rα1-Fc (RnD Systems #146-IR) were diluted to 1 μg / ml in PBS and directly coated onto 384-well ELISA plates. Two negative control proteins, streptavidin, and an unrelated protein were also coated. After incubating the plates with the coating proteins overnight at 4°C, the plates were washed twice with Milli-Q water and blocked for 2 hours in blocking buffer (PBS supplemented with 0.5% BSA + 0.05% Tween 20®). Triple-flag tagged W-ME107 scFv clones present in the bacterial supernatant were diluted 1:10 in blocking buffer and conjugated. Binding was detected using an HRP-labeled anti-FLAG M2 antibody (Sigma-Aldrich #A8592), followed by incubation with a 1-step Ultra TMB ELISA substrate (Thermofisher Scientific #34029). Colorimetric analysis signal generation was stopped by adding 1M sulfuric acid, and the plate was analyzed at 450 nm. All samples, including the reference clone mAb47 scFv and the positive ELISA control G-strep-1 scFv (specific to streptavidin), were dual-assayed.

[0151] result All W-ME107 scFv clones showed binding to their target antigens, human and / or mouse IL13Rα2, as previously reported (Examples 3 and 5) (Figure 4). Significant binding to human IL13Rα1 or to negative control proteins was not detected in any of the clones. The reference clone mAb47 scFv showed binding only to human IL13Rα2 and not to mouse IL13Rα2 or human IL13Rα1, while the positive assay control G-strep-1 scFv bound to the streptavidin coat well.

[0152] conclusion Significant binding to human IL13Rα1 was not detected in any of the 44 W-ME107 scFv clones or the reference clone mAb47 scFv when assayed from bacterial supernatant (Figure 4). In contrast to the results of kinetic sorting (Example 5), all clones here showed binding to the human IL13Rα2 protein in both avi-tagged and Fc-fused forms. In contrast, in Example 5, 12 of the 44 clones did not show measurable binding to the Fc-fused variant. This discrepancy can likely be explained by differences in the sensitivity of the two methods, but it could also be due to differences in experimental setup. In this example, the receptor was attached to a surface, while in Examples 5 and 9, the receptor was in solution. Differences in receptor tagging or fusion proteins can significantly affect how the antigen behaves and how different epitopes are presented differently in the two experimental setups. Binding to mouse IL13Rα2 correlates well with previous SPR data (Example 5), meaning that scFv that do not show measurable binding to the target in affinity screening are also considered "non-binders" in this experimental configuration. Several scFv showed low signals for all analyzed proteins (represented by W-ME107-97, W-ME107-101, W-ME107-129, W-ME107-130, W-ME107-137, W-ME107-141, W-ME107-151, and W-ME107-157) (Figure 4).

[0153] Example 8 - Cell Binding We evaluated the binding of selected scFv to IL13Rα2-expressing cells and confirmed its binding to receptors on the surface of target cells.

[0154] Materials and methods To enable scFv binding to IL13Rα2-expressing cells, 100 ng of purified scFv (Example 6) was used to bind 1 x 10⁶ human glioblastoma cell line U-87MG (original Uppsala University clone with confirmed IL13Rα2 expression, PMID: 27582061) or human non-small cell lung cancer cells (A549). 5 Individual cells were evaluated by incubation at room temperature (RT) (approximately 20-25°C) for 20 minutes. Human glioblastoma cell line U-87MG endogenously expressed high levels of human IL13Rα2 (hIL13Rα2), while human non-small cell lung cancer cells did not express hIL13Rα2. After washing (1x PBS, 0.1% BSA, 3 mM EDTA), the FLAG tags were stained with PE-labeled anti-FLAG antibody at room temperature for 20 minutes. Reading was performed using a CytoFLEX flow cytometer (Beckman Coulter, CA).

[0155] result Selected scFvs were incubated with IL13Rα2-expressing cells to confirm their binding to receptors on the surface of target cells. All scFvs were able to specifically bind to the human glioblastoma cell line U-87MG, which endogenously expresses high levels of hIL13Rα2, but did not bind to negative control human non-small cell lung cancer cells (A549) (Figure 5).

[0156] conclusion Flow cytometry successfully assessed the cell binding ability of selected W-ME107 clones, which exhibited different binding affinities to target cell lines. Off-target binding, i.e., binding to non-glioma cells, was not observed in this binding assay.

[0157] Examples 9-11: Single-cycle dynamics of W-ME107 scFv clones using SPR Surface plasmon resonance (SPR) analysis using a single-cycle dynamical approach was performed on 11 of the most promising W-ME107 clones selected based on the results of Examples 5, 7, and 8, and on the reference clone mAb47 scFv, to determine their dynamical parameters relative to human IL13Rα2-avi, human IL13Rα2-Fc, and mouse IL13Rα2-Fc, respectively. Analysis of the obtained data showed that low nanomolar range K was applied to both human and mouse IL13Rα2. D The highest affinity binding clones that exhibit the specified values ​​are shown.

[0158] Materials and methods The anti-FLAG M2 antibody (Sigma-Aldrich #F1804), which functions as a capture ligand, was immobilized on all four surfaces of the CM5-S amine sensor tip of the BIACORE® T200 analyzer (GE Healthcare) according to the manufacturer's recommended conditions. Protein A-purified FLAG-tagged W-ME107 clones were injected and captured on the chip surface so that the response units (RUs) between clones were equal. The reference clone mAb47 was captured unpurified from the bacterial supernatant, however, because the yield of protein purification was too low (Example 6). Human IL13Rα2-avi, human IL13Rα2-Fc, and mouse IL13Rα2-Fc, each consisting of five concentration ranges between 1.2 and 100 nM in a 3-fold dilution series, were prepared in running buffer (HBS supplemented with 0.05% Tween 20®, pH 7.5) and sequentially injected onto the chip. After the dissociation phase, the chip surface was regenerated with 10 mM glycine-HCl pH 2.1. Response curve sensograms for all scFv clones were obtained by subtracting the response curve of the reference surface from the response curve of the surface immobilized with anti-FLAG M2 antibody. The data were analyzed using BIAeval version 3.1 (GE Healthcare), and dynamic parameters were calculated assuming a 1:1 Langmuir-coupled model.

[0159] result The data showed that all W-ME107 clones exhibited low-nanomolar to nanomolar affinity for human hIL13Rα2-avi, with the highest affinity clone showing 3.1 nM K D It was shown that the value was W-ME107-27 (Table 10). All but four clones (W-ME107-112, W-ME107-117, W-ME107-128, and W-ME107-156) also bound to the mouse version of the receptor. This correlated well with the data shown in Example 5. Surprisingly, while all analyzed clones clearly showed binding to hIL13Rα2-avi, only about half showed binding to the Fc-fusion human construct (hIL13Rα2-Fc). This was demonstrated by W-ME107-10, W-ME107-27, W-ME107-55, W-ME107-112, W-ME107-143, and W-ME107-150, which showed a high response to hIL13Rα2-avi (>50RU) but almost no response to the Fc-fusion human construct. This was also confirmed during dynamic sorting analysis (Example 5).

[0160] Binding affinity of the reference clone mAb47 scFv (present in bacterial supernatant) to human IL13Rα2-avi, K D (M) was identified as 1.1nM. [Table 10] TIFF0007844012000014.tif22162 All scFv samples are purified samples except for the clone mAb47 scFv present in the bacterial supernatant. Values ​​reported as "--" indicate that the binding parameter could not be determined. Values ​​reported as "**" indicate that the data is indeterminate due to a low binding response (RU).

[0161] conclusion Single-cycle reaction kinetics analysis for 11 W-ME107 scFv clones and reference clone mAb47 was successfully performed using a BIACORE® T200 instrument. For human IL13Rα2-avi, human IL13Rα2-Fc, and mouse IL13Rα2-Fc, the obtained kinetic parameters for each clone correlated well with the estimated affinity obtained during kinetic sorting analysis (Example 5). The best affinity-binding clone, W-ME107-27, had a K2 of 3.1 nM. D It had a value. W-ME107-27 also had the best affinity (K) for mouse IL13Rα2-Fc. D It was a clone that exhibited a molecular weight of 2.4 nM.

[0162] Surprisingly, in this example, several clones showed good binding to hL13Rα2-avi, but not to hIL13R2α2-Fc (W-ME107-10, W-ME107-27, W-ME107-55, W-ME107-112, W-ME107-143, and W-ME107-150). This is despite the fact that the amino acids contained in these protein constructs are almost identical (Table 8). However, different epitopes may be presented differently in the two constructs. For example, dimerization resulting from Fc fusion may cause steric hindrance to some epitopes.

[0163] Example 10 - Tm measurement of 11 W-ME107 scFv clones using nanoDSF Eleven purified W-ME107 scFv clones were subjected to melting temperature (Tm) measurement using nanoDSF on a Prometheus NT.48 instrument to evaluate their stability.

[0164] Materials and methods NanoDSF technology measures the intrinsic fluorescence of a protein while it is undergoing thermal denaturation, thereby revealing the characteristics of protein unfolding under natural conditions. The protein A purified W-ME107 scFv clone (Example 6) was diluted to 0.1 mg / ml with PBS and loaded into a "high-sensitivity" capillary (NanoTemper #PR-C006) by capillary action. This was then attached to a Prometheus NT.48 instrument. The melting temperature gradient was set to 20°C to 95°C, with a heating rate of 1°C / min. Tryptophan emissions were measured at 330 nm and 350 nm, and the calculated ratios were plotted against temperature to obtain melting curves for each clone. From these, the Tm value was estimated using the software PR.ThermoControl (NanoTemper Inc).

[0165] result The melting temperature was determined by nano-DSF. Table 11 shows the Tm values ​​(inflection points) for each of the 11 scFv clones. [Table 11]

[0166] conclusion The melting temperature, Tm (°C), was determined for all 11 W-ME107 scFv clones. The Tm values ​​were within the predicted range, with the exception of W-ME107-27 and W-ME107-143, which showed somewhat lower Tm values ​​of 55°C and 57°C respectively. All clones were approximately 60°C. <Tm<70℃であった。 Furthermore, all clones showed only one thawing event, with the exception of W-ME107-7, which showed two thawing events. This is likely due to tryptophan-containing contaminants or the heterogeneous mixture of W-ME107-7 (the present population of folded and misfolded cells). The determined Tm values ​​can be used as a measure of protein stability, and the obtained data show that clones W-ME107-27 and W-ME107-143 exhibit lower stability compared to other scFv clones included in this analysis.

[0167] Example 11 - Analysis of IL13 inhibition by binding of W-ME107 scFv to IL13Rα2 by SPR Twelve W-ME107 scFv clones and a reference clone, mAb47 scFv, were analyzed for their ability to compete with IL13 for receptor binding using SPR-based methods. Each W-ME107 clone was captured on an anti-FLAG M2 antibody immobilized on the surface of an SPR chip and bound to the IL13α2 receptor itself, as well as to IL13Rα2 pre-incubated with IL-13. Seven of the twelve clones, W-ME107-7, W-ME107-10, W-ME107-27, W-ME107-55, W-ME107-67, W-ME107-112, and W-ME107-150, were found to exhibit IL-13 competition for receptor binding. Some clones showed almost complete loss of binding to present IL-13, while others showed only partial loss of receptor binding. Binding of the clones to IL13Rα2 pre-incubated with a negative control protein failed to block receptor binding. These findings indicate that these seven clones possess a binding epitope that overlaps with, or is very close to, the ligand-binding site of IL-13 on the receptor.

[0168] Five clones unaffected by the presence of IL-13—namely W-ME107-16, W-ME107-75, W-ME107-117, W-ME107-128, and W-ME107-156—bound to an epitope different from the IL-13 binding site of IL13Rα2. The reference clone mAb47 scFv did not show binding activity in this experiment, and thus, no data could be obtained. However, in the literature, it has been reported that this antibody competes with IL-13 for binding to IL13Rα2 (Balyasnikova et al., Characterization and immunotherapeutic implications for a novel antibody targeting interleukin(IL)-13 receptor α2, J Biol Chem 2012, 287(36):30215-30227).

[0169] Materials and Methods The anti-FLAG M2 antibody (Sigma-Aldrich #F1804), which functions as a capture ligand, was immobilized on all four surfaces of the CM5-S amine sensor chip by ECD / NHS chemistry using a BIACORE® T200 instrument (GE Healthcare) according to the manufacturer's recommended conditions. Twelve protein A-purified FLAG-tagged W-ME107 clones (Example 6), the positive reference clone mAb47, and the negative control G-strep-1 scFv (specific for streptavidin) were injected and captured on the chip surface, followed by injection of 100 nM hIL13Rα2-avi (Example A1), 100 nM hIL13Rα2-avi + 200 nM IL13 (Prospec #cyt-446), 100 nM IL13Rα2-avi + 200 nM streptavidin (Sigma-Aldrich #SA4762), or 200 nM streptavidin. The surface was regenerated with 10 mM glycine-HCl pH 2.1. All experiments were performed at 25 °C in HBS, pH 7.5 supplemented with 0.05% Tween 20®. Response curve sensorgrams for all scFv clones were obtained by subtracting the response curve of the reference surface from the surface on which the anti-FLAG M2 antibody was immobilized.

[0170] Results The anti-FLAG M2 antibody was successfully immobilized on all four surfaces of the CM5 series S tip. After scFv clone capture and antigen injection, the tip surface was successfully regenerated at a low pH. All 12 W-ME107 scFv clones showed binding to hIL13Rα2, as well as to hIL13Rα2 pre-incubated with negative control streptavidin. None of the clones showed binding to streptavidin alone. When the clones were tested for binding to IL-13 and pre-incubated hIL13Rα2, only five clones (W-ME107-16, W-ME107-75, W-ME107-117, W-ME107-128, and W-ME107-156) showed retained binding to the receptor-ligand complex. For the remaining seven clones (W-ME107-7, W-ME107-10, W-ME107-27, W-ME107-55, W-ME107-67, W-ME107-112, and W-ME107-150), binding was almost completely blocked in the presence of IL-13, as indicated by a decrease in response units (RUs) (Figure 6). No binding activity was observed with the reference clone mAb47 scFv. As expected, the negative control G-strep-1 scFv showed binding only to streptavidin and pre-incubated hIL13Rα2, as well as to streptavidin alone.

[0171] conclusion The results showed that seven of the twelve W-ME107 scFv clones were unable to bind to the hIL13Rα2 receptor in the presence of IL-13. These results indicate that these clones possessed binding epitopes that overlapped with, or were in close proximity to, the ligand-binding site of IL-13 on the receptor. The remaining five clones were able to bind to hIL13Rα2 even when IL-13 was bound to the receptor. All clones exhibiting competition for binding to IL-13 also showed no detectable binding at all, or only very slight binding, to the Fc-fused human IL13Rα2 construct (hIL13Rα2-Fc, RnD systems #7147-IR) in previously performed kinetic measurements (Examples 5 and 9).

[0172] Example 12 - Epitope binning experiment using clone mAb47 mIgG1 We purchased the full-length antibody clone mAb47 mIgG1 and analyzed it using an SPR-based epitope binning method. We also measured its reaction rate constant with respect to human IL13Rα2. Clone mAb47 mIgG1 exhibits the following equilibrium dissociation constant (K) for human IL13Rα2. D Affinity defined as (M), K D It showed =0.9nM. Furthermore, epitope binning data showed that clone mAb47 mIgG1 competed with or interfered with IL-13, W-ME107-10 scFv and W-ME107-27 scFv for binding to human IL13Rα2. This indicates that they all have overlapping or adjacent epitopes on the receptor. The data also showed that W-ME107-75 scFv and W-ME107-117 scFv did not interfere with clone mAb47 mIgG1 for binding to human IL13Rα2, thus indicating that they have detached, non-overlapping epitopes.

[0173] Materials and methods Single-cycle dynamics, SCK Reference clone mAb47 mIgG1 (Creative Biolabs #NEUT-1190QC) was diluted to 50 μg / ml in 10 nM NaAc pH 4.0 and immobilized on CM5 Series S tips using NHS / EDC chemistry according to manufacturer's recommended conditions. A 4-fold dilution series of five concentrations of human IL13Rα2-avi, ranging from 50 nM to 0.2 nM, was sequentially injected onto the tip surface. The coupling sensorgram is subtracted from the blank reference surface sensorgram (without ligand fixation), and then applied to a 1:1 Langmuir coupling model to determine the dynamic parameters, K a (M -1 s -1 ), K d (s -1 ) and K D I obtained (M).

[0174] Epitope Binning 10 nM hIL13Rα2-avi was injected onto the surface of immobilized cloned mAb47 mIgG1, either alone or pre-incubated with a 10x molar excess (100 nM) of human IL-13 (Prospec #cyt-446) or W-ME107-10, W-ME107-27, W-ME107-75, or W-ME107-117 scFv clones. As control samples, 10 nM hIL13Rα2-avi pre-incubated with a 10x molar excess of BI-8 scFv (negative control) or cloned mAb47 mIgG1 (positive control) was also evaluated. After the association phase, the chip surface was regenerated using 10 nM glycine-HCl, pH 2.1. Binding sensorgrams were subtracted from blank reference surface sensorgrams (without immobilized antibody) to obtain the binding level and response units (RU) for each sample.

[0175] result Clone mAb47 mIgG1 was successfully immobilized on CM5 Series S chips using NHS / EDC chemistry, and its activity was maintained after regeneration in 10 nM glycine-HCl, pH 2.1. The affinity of clone mAb47 mIgG1 for hIL13Rα2 is 0.9 nM in the sub-nanomole range. D It was identified as the value. The observed bond rate constant (k a ) and dissociation rate constant (k d ) are, respectively, 5.9 x 10 5 M -1 s -1 and 5.4x10 -4 s -1It was identified as such. Epitope binning showed that when hIL13Rα2 was pre-incubated with a 10-fold molar excess of human IL-13, W-ME107-10 scFv, W-ME107-27 scFv, or positive control clone mAb47 mIgG1, respectively, and subsequently injected onto a clone mAb47 mIgG1-immobilized surface, the observed binding response (RU, y-axis) was reduced compared to injection of hIL13Rα2 alone (Figure 7). This was not observed for W-ME107-75 scFv, W-ME107-117 scFv, and negative control BI-8 scFv, in which case the binding response was almost the same as that of injection of hIL13Rα2 alone (Figure 7).

[0176] conclusion The affinity of reference clone mAb47 mIgG1 for human IL13Rα2 was identified as being in the sub-nanomole range (0.9 nM K). D value). Epitope binning results showed that W-ME107-10 and W-ME107-27, as well as human IL-13, competed with reference clone mAb47 mIgG1 for binding to the hIL13Rα2 receptor. The results from epitope binning analysis correlated well with the SPR analysis previously performed on the W-ME107 scFv clone (Example 11). This example showed that W-ME107-10 scFv and W-ME107-27 scFv competed with human IL-13 for binding to hIL13Rα2, while W-ME107-75 and W-ME107-117 did not.

[0177] In summary, the results of Examples 11 and 12 showed that W-ME107-10 and W-ME107-27, as well as reference clone mAb47, bound to or were very close to the human IL-13 binding site on the human IL13α2 receptor, whereas W-ME107-75 and W-ME107-117 did not. The overlapping epitopes of IL-13 and clone mAb47 are also suggested by Balyasnikova et al., Characterization and immunotherapeutic implications for a novel antibody targeting interleukin(IL)-13 receptor α2, J Biol Chem 2012, 287(36):30215-30227. In this literature, they demonstrated significant inhibition of the interaction between human soluble IL-13 and hIL13Rα2 by this antibody using a plate-based competitive assay.

[0178] Example 13 - Epitope mapping experiment using HDX-MS When a protein is dissolved in a buffer containing heavy water (D2O), the hydrogens attached to heteroatoms (e.g., -OH, -NH, -SH) are replaced by deuterium. In hydrogen-deuterium exchange mass spectrometry (HDX-MS), the rate of deuterium uptake correlates with the degree of intramolecular hydrogen bonding of the peptide backbone amide group (-NHCO-). Backbone amide-NH hydrogens already involved in hydrogen bonding, e.g., -NHCO- hydrogens located in the α-helix or β-sheet, exchange more slowly than their NH hydrogens available to share with the acceptor. Consequently, backbone amide H located in disordered regions exchange more quickly than their hydrogens involved in the β-sheet or α-helix.

[0179] Nowadays, ligand binding to protein targets can lead to local changes during hydrogen bonding, such as structural stabilization or destabilization, allowing this technique to identify the binding interface of protein complexes. For most hydrogens, H / D exchange is short-lived, and as soon as the polypeptide chain comes into contact with a water-containing solution, the deuterium incorporated as -OD, -SD is replaced again by hydrogen. This decrease is called "reverse exchange," and it is very rapid for deuterium bonded to heteroatoms located in the side chains of amino acid residues. Fortunately, under acidic conditions (pH around 2.3) and temperatures near 0°C, reverse exchange at peptide backbone amide groups (-NDCO-) is slowed down to a timeframe suitable for analysis by liquid chromatography and mass spectrometry (LC-MS). In this way, each D atom becomes one mass unit heavier than the H atom, so the degree of incorporation can be monitored by MS. Furthermore, when deuterium labeling is combined with enzymatic proteolysis, the deuteration profiles of different regions within the protein can be monitored.

[0180] Materials and methods A control sample (IL13Rα2 alone) was prepared by mixing 3 μL of human IL13Rα2-avi (see Example A1) (1.6 mg / mL) with 24 μL of deuterated PBS. Antigen / scFv complexes were prepared as follows: 40 μL of hIL13Rα2-avi (1.6 mg / mL) was mixed with 34.5 μL of W-ME107-117 scFv (1.46 mg / mL), 74 μL of W-ME107-10 scFv (0.68 mg / mL), 57.2 μL of W-ME107-27 scFv (0.88 mg / mL), and 122.8 μL of W-ME107-75 scFv (0.41 mg / mL) using an average molecular weight of 30 kDa for each scFv, in a 1:1 antigen / scFv molar ratio. The complex was concentrated to an initial volume of 40 μL using a 0.5 mL Amicon Ultra 10 kDa protein concentrator. The labeling reaction was carried out by mixing 3 μL of antigen / scFv complex with 24 μL of deuterated PBS for 4 minutes, 10 minutes, and 60 minutes (triple incubation was performed except for the 4-minute incubation for W-ME107-27 and W-ME107-75, which was performed in double incubation). After incubation, the reaction was quenched by adding 25 μL of a solution containing 6 M urea, 100 mM TCEP, and 0.5% TFA, lowering the pH to approximately 2.3 and the temperature to approximately 4°C.

[0181] The samples were analyzed using an automated HDX-MS system (CTC PAL / Biomotif HDX). In this system, samples were automatically labeled, quenched, digested, washed, and isolated at 2°C. The samples were digested at 60 μL / min for 2 minutes using an immobilized pepsin column (2.1 x 30 mm), followed by an online desalting step using a 2 mm ID x 10 mm length C-18 pre-column (ACE HPLC Columns, Aberdeen, UK) with 0.1% formic acid at 400 μL / min for 1 minute. The digestible peptides were then separated by a linear gradient of 8 / 18 to 55% of ACN in 0.1% formic acid using a 2 mm ID x 50 mm length HALO C18 / 1.8 μm analytical column operated at 60 μL / min. All experiments were conducted using an LTQ Elite Orbitrap mass spectrometer (Thermo Fisher Scientific) operating at m / z 400 with a resolution of 120,000. All HDX-MS data were processed using HDExaminer version 2.5.1.Mascot, which was used for peptide identification in a dedicated database, with a precursor mass tolerance of 10 ppm and a mass error of 0.05 Da MS / MS.

[0182] result In this HDX-MS experiment, IL13Rα2 was labeled with deuterium using deuterated buffer under two different experimental conditions. One set of experiments used IL13Rα2 alone, while in the other, IL13Rα2 was labeled in the presence of equimolar concentrations of W-ME107-117 scFv. After labeling, the reaction was stopped and the protein was digested with proteolytic enzymes. In a subsequent step, the individual masses of each deuterated peptide derived from digested IL13Rα2 were measured by liquid chromatography-mass spectrometry (LC-MS). Under these experimental conditions, the difference in deuterium incorporation between IL13Rα2 alone and IL13Rα2 in the presence of W-ME107-117 scFv can be attributed solely to the change in deuterium incorporation due to the binding of W-ME107-117 scFv. Since it is well known in the art that antibodies cause a decrease in deuterium incorporation at the antigen / antibody interface (epitope), the difference in deuterium incorporation between these two experimental conditions was identified and mapped into the three-dimensional (3D) structure of IL13Rα2 containing IL-13 (PDB 3LB6, Lupardus, et al., Structure (2010) 18:332-342). Peptides derived from IL13Rα2 that have a statistically significant difference (Student's t-test, three replicate experiments per experimental condition, and 99% confidence interval, p<0.01) between IL13Rα2 alone and IL13Rα2 in the presence of W-ME107-117 scFv were identified, and if they clustered in a region between 15 and 20 angstroms and showed sufficient solvent exposure to allow for precise binding of the scFv, they were considered to belong to that epitope.

[0183] The following peptides were identified as epitopes for W-ME107-117 scFv: amino acid sequence VEYELKYRNIGSETW (SEQ ID NO: 44) corresponding to positions 67-81; amino acid sequence DLNKGIEAKIH (SEQ ID NO: 45) corresponding to positions 96-106; and amino acid sequence WAETTY (SEQ ID NO: 46) corresponding to positions 123-128 (Figure 8 and Tables 12 and 13). These three sequences map to domain 1 of IL13Rα2 and are part of the second β-sheet of this β-sandwich domain. The epitope region more specifically consists of adjacent β-chains 3, 6, and 7, the loop after β-chain 3, and the loop before β-chain 6 (Figure 10). Both of these loops point toward domain 2 of the receptor. The epitope region forms a continuous surface region, which coincided with that typically observed at antigen / antibody boundaries. The amino acids protruding from the β-sheet were primarily large amino acids (Trp, Lys, His, and Glu), providing several charged groups for potential hydrogen bonding to the antibody. These residues had a distinct electron density in the reported structure and were considered to form stacking interactions with each other. The epitope region was located on the side of domain 1, which did not interact with IL-13. [Table 12] [Table 13] TIFF0007844012000018.tif10162

[0184] Peptides mapped to clones W-ME107-10, W-ME107-27, and W-ME107-75 overlapped in different combinations and were all located in domain 3, with the exception of one peptide that opened in domain 2. All peptides are highlighted in Figure 14, and the start and end amino acids are marked with numbers (Figure 14A). HDX-MS of W-ME107-10 gave the following two peptides: FTFQLQNIVKPLPPVYLT (SEQ ID NO: 43) corresponding to amino acid numbers 228-245 and EIKLKWSIPLGPIPARCFD (SEQ ID NO: 36) corresponding to amino acid numbers 253-271. The results for W-ME107-27 gave the same two peptides with an additional peptide SEWSDKQCWGLNDIF (SEQ ID NO: 23) corresponding to amino acid numbers 323-337, where residues 332-337 are derived from the recombinant protein affinity tag. The epitope mapped to W-ME107-75 consisted of two peptides having the same amino acid numbers 253-271 and 323-337 as described above.

[0185] Mapping of these three different peptides to their three-dimensional structures showed that they all cluster toward the C-terminal region of IL13Rα2. Both clones W-ME107-10 and W-ME107-27 contained peptides 228–245. This peptide began in domain 2 and continued to domain 3. Close to the beginning of the peptide, Gln231, Gln233, and Asn234 were extremely close to the IL-13 binding site, although none of the residues appeared to interact structurally (Figure 14E). Structural analysis of IL13Rα2 suggests that it would be difficult for scFv to interact with these residues while simultaneously interacting with the other peptides mentioned above. Conformational changes in IL13Rα2 may explain the inclusion of these residues in the HDX-MS results.

[0186] Clone W-ME107-75 lacked peptides 228-245 in its epitope and was found not to compete for binding to IL-13, whereas the other two clones did compete. Peptides 253-271 are adjacent to 228-245 and located on the IL-13 binding site side, possessing Arg268 which forms binding to the ligand. All three clones shared this peptide in epitope mapping. However, due to the different binding profile of W-ME107-75, this scFv does not appear to bind to peptides 253-271 in the same manner as W-ME107-10 and W-ME107-27, but rather appears to bind more to the leading edge of peptides 253-271 (Figure 14D).

[0187] Peptides 323–337 are precisely the C-terminal portion of the receptor, located opposite 228–245. The structure reported in the Protein Data Bank (PDB) ends at residue 329, and it is not possible to state where the C-terminal portion continues. This is likely a flexible region of this structure. Considering the data from Examples 5 and 9 on how different scFvs bound to mouse IL13Rα2, and then investigating where the conserved residues are located in the structure, provided further insights into why W-ME107-75 differs from W-ME107-10 and W-ME107-27. There is a band of conserved residues in mouse and human IL13Rα2 that extends along the tip of the domain and consists of residues 237–241, 263–269, and 323–326. W-ME107-27 is thought to bind to this region like W-ME107-10, but it lacks interaction with peptide residues 323-326 (Figures 14B and 14C). On the other hand, W-ME107-75 did not bind to this conserved region as much, but rather to amino acid residues 253-266 and precisely the C-terminal residues 332-337. This means a region further down from domain 3 compared to W-ME107-10 and W-ME107-27.

[0188] Furthermore, results from HDX-MS epitope mapping studies correlated well with analysis of mouse and human sequence alignments, including data from Example 12 and binding and non-binding data for scFvs from Examples 5 and 9. Clones W-ME107-117 mapped to one region of the receptor, which is asynchronous to the IL-13 binding site. Clones W-ME107-10, W-ME107-27, and W-ME107-75 all mapped to similar regions on hIL13Rα2, but W-ME107-10 and W-ME107-27 possessed a common peptide, which was absent in W-ME107-75. This peptide had a small region overlapping with the IL-13 ligand binding site. Differences in how these three scFvs bind to the mouse receptor and where conserved residues are located may also help in further identifying different epitopes.

[0189] Example 14 - Epitope mapping of scFv using a peptide array Epitope mapping of W-ME107-10, W-ME107-27, W-ME107-75, and W-ME107-117 scFv clones to human IL13Rα2 revealed two distinct regions of the receptor as epitopes for the four clones (Example 13). W-ME107-10, W-ME107-27, and W-ME117-75 appeared to bind to similar, overlapping regions on domain 3, while W-ME107-117 bound to a different epitope on domain 1. Initial results of HDX-MS mapping of clone W-ME107-117 yielded several different peptides spread across both sides of domain 1. To narrow down the epitope, a 15aa long peptide with a 1aa shift containing aa(amino acids) 39-102 was synthesized on IL13Rα2 and assayed using an ELISA-based method. Binding of W-ME107-117 scFv to any of the 60 peptides included in the array was not detected. Binding was detected only to proteins containing a complete extracellular domain. This reinforces the findings in Example 13, which indicated that W-ME107-117 scFv possesses a structural epitope rather than a linear peptide epitope in the IL13Rα2 receptor.

[0190] Materials and methods Sixty N-terminal biotinylated peptides, corresponding to 74 consecutive aa units (aa39-102) in IL13Rα2, were ordered and synthesized by JPT Peptide Technologies (Germany). Upon arrival, the peptides were dissolved in sterile DMSO (dimethyl sulfoxide) to a final concentration of 0.5 mg / ml. 384-well ELISA plates were coated overnight at 4°C with 1 μg / ml streptavidin and 1 μg / ml hIL13Rα2-Fc in PBS. After washing and blocking the plates in blocking buffer (PBS supplemented with 0.5% BSA and 0.05% Tween 20®), biotinylated peptides and biotinylated versions of IL13Rα2 ECD and hIL13Rα2-avi, diluted to 0.25 μg / ml and 1 μg / ml respectively in blocking buffer, were conjugated for 30 minutes. Purified W-ME107-117 and W-ME107-75, diluted to 1 μg / ml in blocking buffer, were conjugated for 1 hour. Binding detection was enabled by HRP-labeled anti-FLAG M2 antibody (Sigma-Aldrich #A8592). Binding signal generation was initiated by adding TMB ELISA substrate (Thermofisher Scientific #34029) and terminated by adding 1M sulfuric acid. The plates were analyzed at 450 nm. Each sample was assayed in duplicate, and the average absorbance value was obtained after subtracting the blankwell value.

[0191] result Binding of W-ME107-117 scFv to any of the peptides in the array was not detected. Binding was detected only to hIL13Rα2-avi and hIL13Rα2-Fc (Figure 9). As predicted, W-ME107-75 scFv, which has an epitope that does not appear in the peptide array, showed binding only to the ECD domain of IL13Rα2. The experiment was repeated with 10-fold higher concentrations of peptides and the same results were obtained.

[0192] conclusion Epitope mapping of W-ME107-117 scFv revealed an epitope located on the opposite side of IL13Rα2 from the epitopes found in W-ME107-10, W-ME107-27, and W-ME107-75 scFv clones. The structure shown in Figure 10 shows that W-ME107-117 binds to a portion of IL13Rα2 consisting of three beta chains linked by a loop region that folds into a beta-sheet domain. The beta-sheet and two loops contained in the epitope are absent from the portion of IL13Rα2 that binds ligand IL-13. Therefore, W-ME107-117 does not interfere with ligand binding. The absence of binding to the peptide array, along with the results obtained from Example 12, reinforces the results of Example 13, which revealed that W-ME107-117 has a conformational epitope.

[0193] Example 15 - Lentivirus construction and T cell modification In this example, chimeric antigen receptor (CAR) T cells were constructed based on selected scFv, and the CAR T cells were tested in a target cell killing assay.

[0194] Materials and methods Construction of a viral vector for T cell transduction Selected scFv was incorporated into a second-generation CAR construct containing a co-stimulatory domain derived from CD137(4-1BB) and a stimulatory domain derived from CD3ζ. The CAR cassette was inserted into a third-generation self-inactivating (SIN) lentiviral vector (SBI, System Biosciences, Mountain View, CA) under the control of the elongation factor-1α (EF1α) promoter (Figure 11). Green fluorescent protein (GFP) was incorporated after the CAR cassette and isolated by the self-cleaved T2A sequence. All recombinant sequences were purchased from GenScript (Piscataway, NJ). Viral particle production was performed by transient gene transfer of 293T cells using the constructed CAR lentiviral plasmid and the corresponding helper plasmid. The supernatant containing the viral particles was collected, ultracentrifuged, and then used in experiments.

[0195] T cell modification Human peripheral blood mononuclear cells (PBMCs) are divided into 2 x 10 6 T cells were activated over 3 days at a concentration of 10 cells / ml using OKT-3 (50 ng / ml, BioLegend San Diego, CA) and IL-2 (100 IU / ml, Proleukin, Novartis, Basel, Switzerland). After activation, T cells (2 x 10) were activated. 6 T cells were resuspended in 30 μl of enriched lentivirus with 10 mg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) and IL-2 (100 IU) and incubated at 37°C for 4 hours. The T cells were transduced the following day using a similar method and resuspended in culture medium (RPMI1640 supplemented with 10% FBS, 1% PeSt, and 1% sodium pyruvate) at a final concentration of 100 IU / ml of IL-2. After 7 days, CAR T cells were enriched by sorting based on GFP expression (BD FACSAria III, BD Bioscience, San Jose, CA). The sorted cells were grown using immunocult reagent (STEMCELL Technologies, Vancouver, CA) according to the manufacturer's protocol.

[0196] Death assay Human glioblastoma cell line U-87MG, which naturally expresses hIL13Rα2, and melanoma cell line Mel526, which does not express hIL13Rα2, were first modified using lentiviral plasmids to express firefly luciferase, with the luciferase signal used as a readout signal for cell viability, and then exposed to CAR T cells. These target cells were co-cultured with CAR-modified T cells for 24 hours at a ratio of 0, 0.2, 1, 5, or 25 CAR T cells:1 target cell effector (CAR T cell):target (tumor cell) cell. CAR T cells and negative controls (CD19-targeted CAR T cells) were used in this example. Firefly luciferase activity was used as a measure of target cell viability and measured using the ONE-Glo Luciferase assay system (Promega Biotech AB, Sweden).

[0197] result All tested CAR T cell constructs specifically killed hIL13Rα2-expressing glioblastoma cells (U-87MG) but did not kill antigen-negative melanoma cells (Mel526) (Figure 12). The negative control (CD19-targeted CAR T cells) did not kill any cancer cell lines, as predicted since none of the cancer cell lines express CD19. W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells, based on W-ME107-10 scFv, W-ME107-75 scFv, and W-ME107-117 scFv, respectively, already showed sufficient cytotoxicity at low effector:target cell ratios. On the other hand, W-ME107-27 CAR and W-ME107-55 CAR T cells, based on W-ME107-27 scFv and W-ME107-55 scFv, showed death only at higher effector:target ratios (Figure 12). Reference X-ME107-47(mAb47)CAR T cells, produced based on the previously reported mAb47 scFv (J Biol Chem 2012,287:30215-30227,Mol Ther 2016,24(2):354-363), showed only minimal target cell death.

[0198] Example 16 - Growth Assay This example investigated the proliferative capacity of various CAR T cells when co-cultured with target cells.

[0199] Materials and methods CAR T cells produced were labeled with CellTrace Violet Label (ThermoFisher) to track cell proliferation upon encounter with antigen-positive target cells. CellTrace Violet-labeled T cells were left unstimulated or co-cultured with target U-87MG cells (1:1 ratio). The cultures were left untreated or treated with 10 μM lovastatin (Sigma-Aldrich, as a control to prevent T cell proliferation) for 4 days, and then analyzed by flow cytometry (BD FACSCantoII, BD Bioscience). The dilution of the blue-violet pigment observed in the histogram (peak pattern) was considered to indicate cell proliferation.

[0200] result Approximately half of the W-ME107-55 and W-ME107-27 cells divided when co-cultured with tumor cells. Impressively, most W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells divided two or more times upon encountering target cells (Figure 13). Reference mAb47 (X-ME107-47) CAR T cells also proliferated when co-cultured with target tumor cells, but their proliferation was less than that of W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells, considering that only half of the mAb47 CAR T cells divided two or more times. As predicted, CD19-targeted CAR T cells did not proliferate when cultured with U-87MG cells. In summary, W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells showed high proliferative capacity upon target cell recognition. These results indicate that, unlike reference mAb47 CAR T cells, W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells can efficiently interact with target cells, thereby achieving high proliferative capacity.

[0201] Example 17 - Profiling and Characterization of CAR T Cells In this example, CAR T cells were investigated and characterized based on cytokine release profiles, surface marker expression, and CAR expression status.

[0202] Materials and methods Human CAR T cells were modified using a lentiviral construct as described in Example 15. The cells were cultured in cell medium supplemented with IL-2 (25 IU / mL) until analysis, or were grown using a rapid growth protocol with PBMCs from three donors as stimulants prior to analysis.

[0203] IFNγ secretion from unstimulated CAR T cells Mock (control), W-ME107-55 CAR, W-ME107-27 CAR, W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells were modified using lentiviral vectors and cultured in standard cell medium (Example 15). Seven days after lentiviral transduction, CAR T cells (2 x 10⁶ cells in 200 μL of medium) were cultured. 5 Cells (1 per well) were seeded into a 96-well plate, added to the plate, and cultured for 1 day. The supernatant was then collected. IFNγ secreted by CAR T cells into the cell culture supernatant was quantified by ELISA (Mabtech, Sweden).

[0204] IFNγ secretion from tumor-stimulated CAR T cells Mock (control) W-ME107-55 CAR, W-ME107-27 CAR, W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells were modified using lentiviral vectors (Example 15) and propagated using a rapid proliferation protocol. After proliferation, the CAR T cells were incubated for 3 days in cell medium supplemented with IL-2 (25 IU / mL). The CAR T cells were then seeded in 96-well plates with various ratios of U87UU or U343MG tumor cells and cultured for a further 2 days before the supernatant was collected. IFNγ secreted into the cell culture supernatant was quantified by ELISA (Mabtech, Sweden).

[0205] CAR expression levels on the cell surface As a mock (control), W-ME107-55 CAR, W-ME107-27 CAR, W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells were modified using lentiviral vectors and cultured in standard cell medium (Example 15). On days 3, 6, and 12 after lentiviral transduction, CAR T cells were stained for CD3 and CAR (using anti-human Ig(H+L) antibody) and analyzed by flow cytometry. The time-course CAR expression levels for each CAR T cell are shown as histograms.

[0206] Expression of CAR T cell surface activation markers before and after tumor stimulation Mock (control) W-ME107-27 CAR and W-ME107-117 CAR T cells were modified using lentiviral vectors (Example 15) and grown using a rapid proliferation protocol. Modified CAR T cells were incubated in cell medium supplemented with IL-2 (25 IU / mL) for 3 days prior to assay. The incubated CAR T cells were directly seeded in 96-well plates, cultured alone, and analyzed; or co-cultured with U87UU tumor cells for 1 day. CAR T cells were stained for PD1, Tim-3, LAG-3, CD69, CD25, and CD3 before analysis by flow cytometry. Data are presented as the percentage of cells positive for specific markers in CAR T cells (gated as CD3-positive and GFP-positive cells).

[0207] result Under steady state, immediately after T cell transduction with a lentiviral vector, W-ME107-55 CAR and W-ME107-27 CAR T cells secreted large amounts of IFNγ, while W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells secreted relatively small amounts of IFNγ compared to mock CAR T cell controls (Figure 15A). On the other hand, when modified CAR T cells were co-cultured with tumor cells, W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells expressed dose-dependently larger amounts of IFN-gamma (IFNγ), while W-ME107-55 CAR and W-ME107-27 CAR T cells secreted almost no IFNγ in response to tumor cell stimulation (Figure 15B). By analyzing surface CAR expression levels over time, we observed that all CAR T cells possessed CAR molecules expressed on the cell surface after lentiviral transduction. However, the expression of W-ME107-55 CAR and W-ME107-27 CAR decreased over time, while W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR maintained similar levels on the cell surface from the beginning (Figure 15C). By investigating surface activation markers on modified CAR T cells in the presence and absence of tumor cell stimulation, we found that W-ME107-27 CAR T cells already possessed higher levels of these activation markers than those expressed without tumor cell stimulation, and additional tumor stimulation did not affect marker expression levels. On the other hand, these markers remained at low levels in unstimulated W-ME107-117 CAR T cells, while their expression dramatically increased upon tumor cell stimulation (Figure 15D). In summary, these results indicate that W-ME107-55 CAR and W-ME107-27 CAR T cells possess target-independent basal-level activation, and that this basal-level activation leads to a lower responsiveness of CAR T cells to target tumor cells.

[0208] Example 18 - CAR T cells regulate glioblastoma tumor growth in vivo. This example investigated the efficacy of various CAR T cells in regulating glioblastoma tumor growth in an animal model. Since previous data suggested that W-ME107-55 CAR and W-ME107-27 CAR T cells possess nonspecific, target-independent basal-level activation, we analyzed the inhibitory efficacy of W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR T cells.

[0209] Materials and methods Human CAR T cells were modified using lentiviral constructs as described in Example 15. Previous data have shown that W-ME107-10 CAR, W-ME107-75 CAR, and W-ME107-117 CAR cells exhibited better proliferation and lower basal activation levels; therefore, we evaluated these CAR T cells in vivo. Human glioblastoma cells U343MG-Luc (1 x 10⁶ cells in 5 μL) 5 Cells were modified to express firefly luciferase and transplanted intracranially into nude mice. Injections were performed using a Hamilton syringe and stereotactic injection frame at a depth of 1 mm anterior, 1.5 mm right, and 2.7 mm from the bregma. Seven days after tumor transplantation, mice were treated with mock T cells (as a control) or various CAR T cells (2 million cells) administered intracranially at the same site. Mice were tracked by imaging of luciferase signaling using the IVIS system (NightOWL) and euthanized upon development of severe symptoms in strict accordance with locally approved animal ethics permits. A schematic diagram of the experimental procedure is shown in Figure 16A. Luciferase signaling (mean + SEM) was shown as an indicator of tumor growth, and mouse survival in each treatment group was shown as Kaplan-Meier curves, which were compared using the Gehan-Breslow-Wilcoxon test.

[0210] result All three CAR T cell lines tested (W-ME107-10 CAR T, W-ME107-75 CAR T, and W-ME107-117 CAR T) were able to modulate tumor growth compared to the mock T cell treatment group, as indicated by lower luciferase signaling in the treatment groups (Figure 16B). In addition, both W-ME107-75 CAR T cell therapy and W-ME107-117 CAR T cell therapy showed a significant improvement in mouse survival under current assay conditions (Figure 16C).

[0211] Example 19 - CDR differentially affects CAR expression and function. This example investigated the mechanism of differences in CAR expression levels between different constructs by substituting amino acids in the CDR region with alanine. Since previous data suggested that W-ME107-27 and W-ME107-117 have the greatest differences in CAR surface expression, we investigated the substituted amino acids in W-ME107-27 that are different from these two clones and W-ME107-117.

[0212] Materials and methods Virus construction and T cell modification DNA constructs containing amino acid substitutions (see Table 14 for details) were ordered from GenScript and subcloned into lentiviral vectors. The lentiviral constructs are described in Example 15. Human Jarcutt T cell lines were modified with the lentiviral constructs. The modified cells were cultured in cell medium (RPMI1640 supplemented with 10% FBS, 1% PeSt, and 1% sodium pyruvate) until flow cytometry analysis four days after viral transduction. [Table 14]

[0213] CAR expression analysis Modified cells were stained for CAR (using anti-human Ig(H+L) antibody) and analyzed by flow cytometry. The percentage of CAR-positive cells from GFP-positive cells was gated and presented.

[0214] result W-ME107-27-Ala2, W-ME107-27-Ala4, and W-ME107-27-Ala5 showed significantly higher CAR expression from GFP-positive transduced T cells. W-ME107-27-Ala1 and W-ME107-27-Ala3 showed similar levels of CAR expression to W-ME107-27 (Figures 17A, 17B). These results indicate that CDR2 in the heavy chain and CDR3 in the light chain were most involved in influencing CAR surface expression.

[0215] Example 20 - Basal level activation is associated with intracellular signaling domains. This example investigated the mechanism of basal-level activation of modified CAR T cells by removing the intracellular signaling domain of the CAR molecule (decoy CAR). Previous data suggested that W-ME107-27 and W-ME107-117 have the greatest differences in CAR surface expression, so we investigated these two clones.

[0216] Materials and methods Virus construction and T cell modification A DNA construct containing a decoy CAR was ordered from GenScript and subcloned into a lentiviral vector (Figure 18A), which produced W-ME107-27dCAR and W-ME107-117dCAR. The lentiviral construct is described in Example 15. Human T cells were modified with the lentiviral construct.

[0217] IFNγ secretion Modified cells were cultured in cell medium (RPMI1640 supplemented with 10% FBS, 1% PeSt, and 1% sodium pyruvate) supplied with 25 IU of IL-2 / mL for 7 days after viral transduction. Modified cells (2 x 10) 5 The cells (1 per well) were then seeded into 200 μL of cell medium in a 96-well plate without further cytokine supplementation. The cell culture supernatant was collected after 24 hours, and IFNγ in the supernatant was measured by ELISA (Mabtech, Sweden).

[0218] result Both W-ME107-27 and W-ME107-117 CAR T cells secreted IFNγ at steady state, while W-ME107-27 CAR T cells secreted significantly higher amounts of IFNγ (Figure 18C). When the intracellular signaling domain was removed, both W-ME107-27dCAR and W-ME107-117dCAR cells showed significantly less IFNγ secretion compared to their counterparts (Figure 18C). Mock T cells did not secrete IFNγ (Figure 18C). These results suggest that basal-level activation of modified CAR T cells is associated with their intracellular signaling domain.

[0219] Example A1 - Preparation of recombinant human IL13Rα2 (hIL13Rα2-avi) Biotinylated hIL13Rα2 was cloned and isolated. Materials and methods

[0220] material MultiBac Expression System Kit, Geneva Biotech, NaN SalI,G|TCGAC Thermo Fischer Scientific,FD0644 XhoI,C|TCGAG Thermo Fischer Scientific,FD0694 KpnI,GGTAC|C Thermo Fischer Scientific,FD0524 PstI,CTGCA|G,Thermo Fischer Scientific,FD0615 Rapid DNA Ligation Kit,Thermo Fischer Scientific,K1422 Cre recombinase, NEB, M0298 GeneJET Plasmid Miniprep Kit,Thermo Fischer Scientific,K0503 PureLink HiPure Plasmid DNA Mini kit,Thermo Fisher Scientific,K210002 LA, Sigma, L7025 LB Broth, Sigma, L03022 One Shot Mach1 T1 Phage-Resistant Chemically Competent E.coli,Thermo Fischer Scientific,C862003 One Shot PIR1 Chemically Competent E.coli,Thermo Fischer Scientific,C101010 Spectamycin, Sigma, S4014 ゲンタマイシン、Sigma,G1397 IPTG, ThermoFisher, 15529019 BluoGal, ThermoFisher, 15519028 テトラサイクリン、Bioline,87030 DH10EmBacyコンピテントcell (multibac kitからmodulation) Stellar Competent Cells,Clontech,636763. 17AEAMOP_ME107h_pMA-T,Gene Art 17AEAMPP_BirA_pMA-T,Gene Art pFastBac cis direction, GGA TTA TTC ATA CCG TCC CA pACEBac1 reverse direction (SV40 polyA), TGA AAT TTG TGA TGC TAT TGC pIDS in the direction of, CGA TAC TAG TAT ACG GAC C pIDS reverse direction, CCG TGC GTT TTA TTC TGT C ヘペス、VWR,441487M グリセロール、VWR,444485B ツイーン80 (registered trademark) (Surfact-Amps 80), Pierce, 0028328 イミダゾール、Merck,1.04716.0250 NaCl, VWR, 27800.360 HisTrap excel 1 ml, GE Healthcare, 17-3712-05 HiLoad Superdex 200 16 / 60,GE Healthcare,28-9893-35 Novex Sharp Pre-stained Protein Standard, Invitrogen, LC5800 NuPAGE antioxidant, Invitrogen, NP0005 NuPAGE LDS sample buffer, Invitrogen, NP0007 NuPAGE MES SDS Running Buffer 20X, Invitrogen, NP000202 NuPAGE Novex 4-12% Bis-Tris Protein Gels 1.0 mm 10-well,Invitrogen,NP0321BOX NuPAGE Novex 4-12% Bis-Tris Protein Gels 1.0 mm 15-well,Invitrogen,NP0323BOX Pierce Protein Concentrator 10K 5-20 ml,Thermo Scientific,88527 Anti-6X histidine-tagged antibody, Abcam, ab18184 Commassie Protein Assay Reagent,Thermo Fisher Scientific,1856209

[0221] Cloning of hIL13Rα2-avi The ECD domain of human IL13Rα2 (uniplot Q14627 aa29~331) was co-expressed with BirA in a baculovirus / Sf9 system to produce a biotinylated receptor. In the gene construct, the native signal peptides (aa1~28) were replaced with the gp67 baculovirus signal peptide for protein secretion into the culture medium. The Avi tag was incorporated into the C-terminus of the receptor to promote targeted biotinylation by BirA, followed by the His6 tag for purification. This gave the C-terminal amino acid sequence GLNDIFEAQKIEWHEHHHHHH (SEQ ID NO: 111) added after Trp331. The construct was codon-optimized for cutworm by flanking the cloning sites SalI and XhoI. Similarly, the E. coli BirA ligase gene was codon-optimized by flanking XhoI and KpnI. The genes were ordered from GeneArt and ThermoFisher (vectors 17AEAMOP_ME107h_pMA-T and 17AEAMPP_BirA_pMA-T). Multibac constructs of the receptor vector pACEBac1 in the donor vector pIDS and hIL13Rα2-avi in ​​BirA were prepared according to the manufacturer's protocol. The vector sequences were confirmed by GATC sequencing. The two vectors were fused by Cre-Lox recombination to form the hIL13Rα2-AVIhis / BirA construct. This construct was transformed into DH10EmbacY for gene translocation into bacmid. Positive clones were selected by blue / white screening of these cells. Finally, bacmid was isolated and analyzed for gene integration by PCR.

[0222] hIL13Rα2-avi expression and characterization Bacmid was introduced into Sf9 cells to produce baculovirus. Human IL13Rα2 was expressed in 2.3 L of transgenic Sf9 culture medium for 48 hours and collected from the medium by capturing it on a HisTrap Exel column. The column was equilibrated with buffer A (50 mM HEPES pH 7.0, 150 mM NaCl, 9 mM imidazole, 10% glycerol, and 10 μM Tween 80®). After washing the column with buffer A, the protein was eluted with buffer B (50 mM HEPES pH 7.0, 150 mM NaCl, 9 mM imidazole, 10% glycerol, 10 μM Tween 80®, and 300 mM imidazole).

[0223] After pooling and concentrating the protein, including the fractionated portion, the sample was finally purified on a Superdex 200 16 / 60 column using 50 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, and 10 μM Tween 80®. The purest protein fraction was selected, pooled, and concentrated by SDS-PAGE. The purified hIL13Rα2 receptor was electrophoresed on an SDS gel to determine its purity and size. Binding to the cloned mAb47 single-chain control was confirmed by ELISA and Western blotting. The receptor was also sent to Xiaofang Cao, Clinical Proteomics Mass Spectrometry, Science for Life Laboratory for MS analysis. The sample was experimented with three times, but only a portion of the sequence was covered. We were able to conclude that the protein ID was correct.

[0224] The embodiments described above should be understood as some exemplary examples of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, and changes can be made to these embodiments without departing from the scope or spirit of the invention. In particular, solutions of different parts in different embodiments can be combined in other configurations where technically possible. However, the scope of the invention is defined by the appended claims.

Claims

1. An antibody, or its antigen-binding fragment, that can bind to the interleukin-13 receptor subunit alpha-2 (IL13Rα2), Variable heavy chain (VH) domain complementarity determination region 1 (CDR1) consisting of the amino acid sequence GTFFSGSY (SEQ ID NO: 105), The VH domain CDR2 consists of the amino acid sequence IYGSGGYT (SEQ ID NO: 7). The VH domain CDR3 consists of the amino acid sequence ARYSPFYMDY (SEQ ID NO: 10), The variable light chain (VL) domain CDR1 consists of the amino acid sequence QSISSY (SEQ ID NO: 12). The VL domain CDR2 consists of the amino acid sequence AAS, and VL domain CDR3 consisting of amino acid sequence QQGYSFPPT (SEQ ID NO: 11) An antibody, or its antigen-binding fragment, containing such an antibody.

2. The antibody according to claim 1, or its antigen-binding fragment, wherein the extended VH domain CDR1 comprises the amino acid sequence GTFFSGSYMS (SEQ ID NO: 6).

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the extended VH domain CDR2 comprises the amino acid sequence SIYGSGGGYTY (SEQ ID NO: 8).

4. A variable heavy chain (VH) domain consisting of the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSGSYMSWVRQAPGKGLEWVSSIYGSGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSPFYMDYWGQGTLVTVSS (SEQ ID NO: 13); and Variable light chain (VL) domain consisting of the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAAPKLLLIYAASSLQSGVPPSRFSGSGSGTDFTLTISSSLQPEDFATYYCQQGYSFPPTFGQGTKLEIK (SEQ ID NO: 14) An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising:

5. An antibody capable of binding to interleukin-13 receptor subunit alpha-2 (IL13Rα2), or an antigen-binding fragment thereof, is: Amino acid sequence GFTFX １ X ２ X ３ X ４ A variable heavy chain (VH) domain complementarity determination region 1 (CDR1) consisting of each X ｎ n=1 to 4 are variable heavy chain (VH) domain complementarity determination region 1 (CDR1), independently selected from the group consisting of G, A, S, and Y; Amino acid sequence IB １ B ２ B ３ B ４ B ５ B ６ A VH domain CDR2 consisting of T, where each B ｍ , m = 1 - 6, is independently selected from the group consisting of G, S, and Y, the VH domain CDR2; Amino acid sequence AR-Z Ｈ -Z １ A VH domain CDR3 consisting of DY, Z １ Z is selected from the group consisting of F, M, I, and L. Ｈ The VH domain CDR3 is an amino acid sequence selected from the group consisting of VVRSTYGY (SEQ ID NO: 15), YGHYAYGSY (SEQ ID NO: 16), YSSSGWYYGF (SEQ ID NO: 17), TPYSAY (SEQ ID NO: 18), RYRSHRPGLS (SEQ ID NO: 19), FHPRYGY (SEQ ID NO: 20), GSYSHYGAHY (SEQ ID NO: 21), YYHYDYGYYY (SEQ ID NO: 22), YSPFY (SEQ ID NO: 3), RNYWEHGGGS (SEQ ID NO: 24), HHYGYYPPGSVYY (SEQ ID NO: 25), and VEYTYYYGSEGSPV (SEQ ID NO: 26); Variable light chain (VL) domain CDR1 consisting of amino acid sequence QSISSY (SEQ ID NO: 12); VL domain CDR2 consisting of amino acid sequence AAS; and Amino acid sequence QQ-Z Ｌ - A VL domain CDR3 consisting of T, Z Ｌ This is an amino acid sequence selected from the group consisting of TYYSPH (SEQ ID NO: 28), DYYLF (SEQ ID NO: 29), SYSTPY (SEQ ID NO: 30), FYSYPL (SEQ ID NO: 31), AFSPS (SEQ ID NO: 32), SYDTLL (SEQ ID NO: 33), ALSSLP (SEQ ID NO: 34), FSTRLS (SEQ ID NO: 35), GYSFPP (SEQ ID NO: 4), STYPF (SEQ ID NO: 37), YGSNPL (SEQ ID NO: 38), and RYNGLF (SEQ ID NO: 39), which is the VL domain CDR3. Includes, VH CDR1 consists of the amino acid sequence of SEQ ID NO: 100, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 53, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 64, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 75, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 101, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 53, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 65, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 76, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 101, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 54, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 66, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 77, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 102, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 55, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 67, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 78, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 101, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 53, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 68, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 79, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 103, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 56, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 69, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 80, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 104, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 54, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 70, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 81, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 101, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 53, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 71, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 82, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 105, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 7, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 10, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 11, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 104, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 57, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 72, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 83, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 101, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 53, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 73, and VL CDR3 consists of the amino acid sequence of SEQ ID NO: 84, or VH CDR1 consists of the amino acid sequence of SEQ ID NO: 106, VH CDR2 consists of the amino acid sequence of SEQ ID NO: 54, VH CDR3 consists of the amino acid sequence of SEQ ID NO: 74, and VL CDR3 consists of the amino acid sequence of SEQ ID NO:

85. An antibody, or its antigen-binding fragment.

6. VH domain CDR1 consisting of the amino acid sequence of SEQ ID NO: 101, VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 53, VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 65, VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, VL CDR2 consisting of the AAS amino acid sequence, and VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 76; or VH domain CDR1 consisting of the amino acid sequence of SEQ ID NO: 102, VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 55, VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 67, VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, VL CDR2 consisting of the AAS amino acid sequence, and VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 78; or VH domain CDR1 consisting of the amino acid sequence of SEQ ID NO: 104, VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 54, VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 70, VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, VL CDR2 consisting of the AAS amino acid sequence, and VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 81; or VH domain CDR1 consisting of the amino acid sequence of SEQ ID NO: 105, VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 7, VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 10, VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, VL CDR2 consisting of the AAS amino acid sequence, and VL CDR3 consisting of the amino acid sequence of SEQ ID NO:

11. The antibody according to claim 5, or its antigen-binding fragment.

7. VH domain consisting of the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGHYAYYGSYFDYWGQGTLVTVSS (SEQ ID NO: 86); and VL domain consisting of the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAAPKLLIYAASSLQSGVPPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYYLFTTFGQGTKLEIK (SEQ ID NO: 87); or VH domain consisting of the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFYGSYMGWVRQAPGKGLEWVSYISGYGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTPYSAYIDYWGQGTLVTVSS (SEQ ID NO: 88); and VL domain consisting of the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAAPKLLIYAASSLQSGVPPSRFSGSGSGTDFTLTISSSLQPEDFATYYCQQFYSYPLTFGQGTKLEIK (SEQ ID NO: 89); or VH domain consisting of the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFYSYSYGMSWVRQAPGKGLEWVSYISGGGGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYSHYGAHYLDYWGQGTLVTVSS (SEQ ID NO: 90); and VL domain consisting of the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALSSLPTTFGQGTKLEIK (SEQ ID NO: 91); or VH domain consisting of the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSGSYMSWVRQAPGKGLEWVSSIYGSGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSPFYMDYWGQGTLVTVSS (SEQ ID NO: 13); and The VL domain consists of the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSFPPTFGQGTKLEIK (SEQ ID NO: 14), The antibody according to claim 5 or 6, or its antigen-binding fragment, comprising:

8. The antibody according to any one of claims 1 to 7, wherein the antigen-binding fragment is a single-chain variable fragment (scFv).

9. An antigen recognition domain comprising an antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof; Transmembrane domain; and Intracellular signal transduction domains, A chimeric antigen receptor (CAR) that includes this receptor.

10. The CAR according to claim 9, wherein the transmembrane domain is selected from the group consisting of all or part of the transmembrane domain of surface antigen classification 28 (CD28), all or part of the transmembrane domain of CD8α, all or part of the transmembrane domain of CD27, all or part of the transmembrane domain of CD137, all or part of the transmembrane domain of CD134, all or part of the transmembrane domain of CD3ε, all or part of the transmembrane domain of CD3ζ, all or part of the transmembrane domain of CD3γ, all or part of the transmembrane domain of CD3δ, all or part of the transmembrane domain of TCRα, and all or part of the transmembrane domain of TCRβ.

11. The CAR according to claim 9 or 10, wherein the intracellular signaling domain is selected from the group consisting of the zeta chain of surface antigen classification 3 (CD3ζ), CD28, CD137, ICOS, CD27, CD40, CD134, and / or Myd88.

12. A T cell receptor (TCR) complex comprising an antibody according to any one of claims 1 to 8, or an antigen recognition domain comprising an antigen-binding fragment thereof.

13. The antibody according to any one of claims 1 to 8, or its antigen-binding fragment; and Effector molecule, selected from the group consisting of a detectable label, a cytotoxin, a metal, another antibody or its antigen-binding fragment, a nucleic acid sequence, and a lipid bilayer docking portion. A conjugate that includes this.

14. An antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof, or a nucleic acid molecule encoding a CAR according to any one of claims 9 to 11 and / or a TCR complex according to claim 12.

15. A vector comprising the nucleic acid molecule described in claim 14.

16. A cell comprising an antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof, a CAR according to any one of claims 9 to 11, a TCR complex according to claim 12, a nucleic acid molecule according to claim 14, and / or a vector according to claim 15.

17. The cell according to claim 16, wherein the cell is selected from the group consisting of T cells, natural killer (NK) cells, B cells, monocytes, and macrophages.

18. An antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof, a CAR according to any one of claims 9 to 11, a TCR complex according to claim 12, a conjugate according to claim 13 in which the effector molecule is a cytotoxin, a nucleic acid molecule according to claim 14, a vector according to claim 15, or a cell according to claim 16, for use as a drug.

19. An antibody according to any one of claims 1 to 8, or its antigen-binding fragment, a CAR according to any one of claims 9 to 11, a TCR complex according to claim 12, a conjugate according to claim 13 in which the effector molecule is a cytotoxin, a nucleic acid molecule according to claim 14, a vector according to claim 15, or a cell according to claim 16, for use in treating or delaying the onset of a disease selected from the group consisting of glioblastoma, medulloblastoma, breast cancer, head and neck cancer, pancreatic cancer, kidney cancer, ovarian cancer, colon cancer, liver cancer, lung cancer, urothelial carcinoma, and Kaposi's sarcoma.

20. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof, a CAR according to any one of claims 9 to 11, a TCR complex according to claim 12, a conjugate according to claim 13, a nucleic acid molecule according to claim 14, a vector according to claim 15 and / or a cell according to claim 16, and a pharmaceutically acceptable carrier.

21. Contacting a biological sample with an antibody or antigen-binding fragment according to any one of claims 1 to 8; and To measure the amount of the antibody or its antigen-binding fragment bound to at least one cell of the biological sample, thereby identifying the at least one cell as an IL13Rα2-positive cell, A method for identifying interleukin-13 receptor subunit alpha-2 (IL13Rα2) positive cells, including [specific cells].

Images