Interleukin-13 receptor subunit alpha-2 conjugate and its use

Abstract

This disclosure provides IL13Rα2 conjugates (antibodies including monospecific antibodies and multispecific antibodies such as bispecific antibodies, for example) and uses thereof. In some embodiments, such conjugates to the same epitope of IL13Rα2 (or IL13Rα2:IL13 complex) (e.g., human IL13Rα2) as an antibody containing a CDR as described herein (e.g., Tables 1-3). In some embodiments, such conjugates to the same epitope of IL13Rα2 (or IL13Rα2:IL13 complex) (e.g., human IL13Rα2) as an antibody containing heavy chain variable regions and light chain variable regions as described herein (e.g., Tables 1-3).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 514,790, filed July 20, 2023, the disclosures of which U.S. Provisional Patent Application are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes an electronic sequence listing (whose entire contents are incorporated herein by reference) submitted in XML file format. The sequence listing in XML file submitted with this application is named "14529-144-228_SEQ_LISTING.xml", was created on July 15, 2024, and has a size of 110,551 bytes.

[0003] 1. Technical field This disclosure generally relates to a binder, for example, an antibody (including a fragment thereof) that binds to interleukin-13 receptor α2 (IL13Rα2) (including human IL13Rα2), and a method of using the same. [Background technology]

[0004] 2.Background technology Interleukin-13 receptor subunit alpha-2 (IL-13Rα2 or IL13Rα2), also known as CD213A2 (differentiation antigen group 213A2), is a membrane-bound protein encoded in humans by the IL13Rα2 gene. IL-13Rα2 is a high-affinity membrane receptor for the anti-inflammatory cytokine interleukin-13 (IL-13). IL-13Rα2 is known to be overexpressed in a variety of cancers, including but not limited to malignant melanoma, malignant glioma, pancreatic cancer, ovarian cancer, breast cancer, liver cancer, head and neck cancer, and renal cancer. [Overview of the project]

[0005] 3. Outline of the Invention This disclosure provides IL13Rα2 conjugates (including human IL13Rα2 conjugates). Such conjugates include antibodies that bind to IL13Rα2 (or a complex comprising IL13Rα2 and IL13, referred herein to as the IL13Rα2:IL13 complex), such as monospecific or polyspecific antibodies (e.g., bispecific antibodies) that bind to IL13Rα2 (or the IL13Rα2:IL13 complex). In some embodiments, such conjugates to the same epitope of IL13Rα2 (or the IL13Rα2:IL13 complex) (e.g., human IL13Rα2) as an antibody containing a CDR as described herein (e.g., Tables 1-3). In some embodiments, such conjugates to the same epitope of IL13Rα2 (or the IL13Rα2:IL13 complex) (e.g., human IL13Rα2) as an antibody containing heavy chain variable regions and light chain variable regions as described herein (e.g., Tables 1-3).

[0006] This disclosure also provides nucleic acids encoding IL13Rα2 conjugates provided herein (e.g., antibodies or fragments thereof, e.g., antigen-binding fragments), vectors comprising one or more such nucleic acids, and cells comprising the nucleic acids, the vector, or both (e.g., cells expressing the conjugate).

[0007] This disclosure also provides compositions comprising an IL13Rα2 conjugate. In some embodiments, such compositions include an antibody that binds to IL13Rα2 (or IL13Rα2:IL13 complex), for example, a monospecific or polyspecific antibody (e.g., a bispecific antibody) that binds to IL13Rα2 (or IL13Rα2:IL13 complex). In some embodiments, such compositions include an antibody that binds to essentially the same epitope as IL13Rα2 (or IL13Rα2:IL13 complex) (e.g., human IL13Rα2) as an antibody comprising a CDR as described herein (e.g., Tables 1-3). In some embodiments, such compositions include an antibody that binds to essentially the same epitope as IL13Rα2 (or IL13Rα2:IL13 complex) (e.g., human IL13Rα2) as an antibody comprising a heavy chain variable region and a light chain variable region as described herein (e.g., Tables 1-3).

[0008] In some embodiments, the binder binds to IL13Rα2. In addition to or instead of this, the binder binds to a complex containing IL13Rα2 and IL13 (referred to herein as the IL13Rα2:IL13 complex). In addition to or instead of this, the binder does not bind to IL13Rα1. In some embodiments, the binder binds to a complex containing IL13Rα2 and IL13 but not IL13Rα1. In some embodiments, the binder does not inhibit the binding between IL13 and IL13Rα2 (e.g., human IL13Rα2 and / or cynoIL13Rα2).

[0009] This disclosure also provides nucleic acids encoding IL13Rα2 conjugates provided herein (e.g., antibodies or fragments thereof, e.g., antigen-binding fragments), vectors comprising one or more nucleic acids, or compositions comprising such nucleic acids, such vectors, or both (e.g., cells expressing the conjugate).

[0010] This disclosure further provides various uses of the conjugates and compositions, including, for example, methods for identifying cells expressing IL13Rα2, methods for reducing (including, but not limited to, eliminating) the viability or proliferation of IL13Rα2-expressing cells, and methods for killing IL13Rα2-expressing cells. In a further embodiment, the IL13Rα2-expressing cells are cancer cells. In a further embodiment, these cancer cells are melanoma cells, lung cancer (including non-small cell lung cancer) cells, or colorectal cancer cells. Other embodiments provided herein include methods for treating a disease or disorder in a subject with the IL13Rα2 conjugates or compositions provided herein. Such compositions include antibodies that bind to IL13Rα2 (or IL13Rα2:IL13 complex) (e.g., human IL13Rα2), for example, monospecific or polyspecific antibodies (e.g., bispecific antibodies) that bind to IL13Rα2 (or IL13Rα2:IL13 complex). In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is melanoma, lung cancer (including non-small cell lung cancer) cells, or colorectal cancer. In some embodiments, the cancer treated according to the method herein expresses (including overexpression) IL13Rα2. [Brief explanation of the drawing]

[0011] 4. Brief explanation of the drawing [Figure 1A] As further explained in Example 2, exemplary monovalent Kd results for A22 are provided. [Figure 1B] As further explained in Example 2, exemplary monovalent Kd results for A33 are provided. [Figure 1C] As further explained in Example 2, exemplary monovalent Kd results for A52 are provided. [Figure 1D] As further explained in Example 2, exemplary monovalent Kd results for benchmark antibody 1 are provided. [Figure 1E] As further explained in Example 2, exemplary monovalent Kd results for benchmark antibody 2 are provided.

[0012] [Figure 2-1] Figure 2A: Provides an exemplary SEC result for A22, as further described in Example 4. [Figure 2-2] Figure 2B: Provides an exemplary SEC result for A33, as further explained in Example 4. [Figure 2-3] Figure 2C: Provides an exemplary SEC result for A52, as further explained in Example 4. [Figure 2-4] Figure 2D: Provides exemplary SEC results for benchmark antibody 1, as further explained in Example 4. [Figure 2-5] Figure 2E: Provides exemplary SEC results for benchmark antibody 2, as further explained in Example 4.

[0013] [Figure 3-1] Figure 3A: Provides an exemplary SCX result for A22, as further described in Example 4. [Figure 3-2] Figure 3B: Provides an exemplary SCX result for A33, as further explained in Example 4. [Figure 3-3] Figure 3C: Provides exemplary SCX results for A52, as further explained in Example 4. [Figure 3-4] Figure 3D: Provides exemplary SCX results for benchmark antibody 1, as further explained in Example 4. [Figure 3-5] Figure 3E: Provides exemplary SCX results for benchmark antibody 2, as further explained in Example 4.

[0014] [Figure 4-1] Figure 4A: Provides exemplary SMAC results for A22, as further described in Example 4. [Figure 4-2] Figure 4B: Provides exemplary SMAC results for A33, as further described in Example 4. [Figure 4-3] Figure 4C: Provides exemplary SMAC results for A52, as further explained in Example 4. [Figure 4-4]As further explained in Figure 4D Example 4, exemplary SMAC results for benchmark antibody 1 are provided. [Figure 4-5] Figure 4E: Provides exemplary SMAC results for benchmark antibody 2, as further explained in Example 4.

[0015] [Figure 5-1] Figure 5A: Provides exemplary HIC results for A22, as further explained in Example 4. [Figure 5-2] Figure 5B: Provides exemplary HIC results for A33, as further explained in Example 4. [Figure 5-3] Figure 5C: Provides exemplary HIC results for A52, as further explained in Example 4. [Figure 5-4] Figure 5D: Provides exemplary HIC results for benchmark antibody 1, as further explained in Example 4. [Figure 5-5] Figure 5E: Provides exemplary HIC results for benchmark antibody 2, as further explained in Example 4.

[0016] [Figure 6-1] Figure 6A: Provides exemplary binding results of A22 to IL13Rα2 endogenously expressed in A375 cells, as further explained in Example 5. [Figure 6-2] Figure 6B: Provides exemplary binding results of A33 to IL13Rα2 endogenously expressed in A375 cells, as further explained in Example 5. [Figure 6-3] Figure 6C: Provides exemplary binding results of A52 to IL13Rα2 endogenously expressed in A375 cells, as further explained in Example 5. [Figure 6-4] Figure 6D: Provides exemplary binding results of benchmark antibody 1 against IL13Rα2 endogenously expressed in A375 cells, as further explained in Example 5. [Figure 6-5]Figure 6E: Provides exemplary binding results of benchmark antibody 2 against IL13Rα2 endogenously expressed in A375 cells, as further explained in Example 5.

[0017] [Figure 7-1] Figure 7A: Provides exemplary binding results of A22 to human IL13Rα2 expressed in HEK cells, as further explained in Example 5. [Figure 7-2] Figure 7B: Provides exemplary binding results of A33 to human IL13Rα2 expressed in HEK cells, as further explained in Example 5. [Figure 7-3] Figure 7C: Provides exemplary binding results of A52 to human IL13Rα2 expressed in HEK cells, as further explained in Example 5. [Figure 7-4] Figure 7D: Provides exemplary binding results of benchmark antibody 1 against human IL13Rα2 expressed in HEK cells, as further explained in Example 5. [Figure 7-5] Figure 7E: Provides exemplary binding results of benchmark antibody 2 against human IL13Rα2 expressed in HEK cells, as further explained in Example 5.

[0018] [Figure 8-1] Figure 8A: Provides exemplary binding results of A22 to cynoIL13Rα2 expressed in HEK cells, as further explained in Example 5. [Figure 8-2] Figure 8B: Provides exemplary binding results of A33 to cynoIL13Rα2 expressed in HEK cells, as further explained in Example 5. [Figure 8-3] Figure 8C: Provides exemplary binding results of A52 to cynoIL13Rα2 expressed in HEK cells, as further explained in Example 5. [Figure 8-4] Figure 8D: Provides exemplary binding results of benchmark antibody 1 against cynoIL13Rα2 expressed in HEK cells, as further explained in Example 5. [Figure 8-5]Figure 8E: Provides exemplary binding results of benchmark antibody 2 against cynoIL13Rα2 expressed in HEK cells, as further explained in Example 5.

[0019] [Figure 9-1] Figure 9A: Provides exemplary binding results of A22 to endogenously expressed IL13Rα2 in A375 cells pretreated with human IL13, as further described in Example 5. [Figure 9-2] Figure 9B: Provides exemplary binding results of A33 to endogenously expressed IL13Rα2 in A375 cells pretreated with human IL13, as further described in Example 5. [Figure 9-3] Figure 9C: Provides exemplary binding results of A52 to endogenously expressed IL13Rα2 in A375 cells pretreated with human IL13, as further explained in Example 5. [Figure 9-4] Figure 9D: Provides exemplary binding results of benchmark antibody 1 against endogenously expressed IL13Rα2 in A375 cells pretreated with human IL13, as further described in Example 5. [Figure 9-5] Figure 9E: Provides exemplary binding results of benchmark antibody 2 against endogenously expressed IL13Rα2 in A375 cells pretreated with human IL13, as further described in Example 5.

[0020] [Figure 10-1] Figure 10A: Provides exemplary binding results of A22 to human IL13Rα2 expressed in HEK cells pretreated with human IL13, as further described in Example 5. [Figure 10-2] Figure 10B: Provides exemplary binding results of A33 to human IL13Rα2 expressed in HEK cells pretreated with human IL13, as further explained in Example 5. [Figure 10-3] Figure 10C: Provides exemplary binding results of A52 to human IL13Rα2 expressed in HEK cells pretreated with human IL13, as further explained in Example 5. [Figure 10-4] Figure 10D: Provides exemplary binding results of benchmark antibody 1 against human IL13Rα2 expressed in HEK cells pretreated with human IL13, as further explained in Example 5. [Figure 10-5] Figure 10E: Provides exemplary binding results of benchmark antibody 2 against human IL13Rα2 expressed in HEK cells pretreated with human IL13, as further explained in Example 5.

[0021] [Figure 11-1] Figure 11A: Provides exemplary ADC piggyback assay results for A22 in A375 cells endogenously expressing IL13Rα2, as further described in Example 6. [Figure 11-2] Figure 11B: Provides exemplary ADC piggyback assay results for A33 in A375 cells endogenously expressing IL13Rα2, as further described in Example 6. [Figure 11-3] Figure 11C: Provides exemplary ADC piggyback assay results for A52 in A375 cells endogenously expressing IL13Rα2, as further explained in Example 6. [Figure 11-4] Figure 11D: Provides exemplary ADC piggyback assay results for benchmark antibody 1 in A375 cells endogenously expressing IL13Rα2, as further described in Example 6. [Figure 11-5] Figure 11E: Provides exemplary ADC piggyback assay results for benchmark antibody 2 in A375 cells endogenously expressing IL13Rα2, as further explained in Example 6.

[0022] [Figure 12-1] Figure 12A: Provides exemplary ADC piggyback assay results for A22 in A375 cells endogenously expressing IL13Rα2 and pretreated with human IL13, as further described in Example 6. [Figure 12-2]Figure 12B: Provides exemplary ADC piggyback assay results for A33 in A375 cells endogenously expressing IL13Rα2 and pretreated with human IL13, as further described in Example 6. [Figure 12-3] Figure 12C: Provides exemplary ADC piggyback assay results for A52 in A375 cells endogenously expressing IL13Rα2 and pretreated with human IL13, as further described in Example 6. [Figure 12-4] Figure 12D: Provides exemplary ADC piggyback assay results for benchmark antibody 1 in A375 cells endogenously expressing IL13Rα2 and pretreated with human IL13, as further described in Example 6. [Figure 12-5] Figure 12E: Provides exemplary ADC piggyback assay results for benchmark antibody 2 in A375 cells endogenously expressing IL13Rα2 and pretreated with human IL13, as further described in Example 6.

[0023] [Figure 13-1] Figure 13A: Provides exemplary ADC piggyback assay results for A22 in HEK cells expressing cynoIL13Rα2, as further described in Example 6. [Figure 13-2] Figure 13B: Provides exemplary ADC piggyback assay results for A33 in HEK cells expressing cynoIL13Rα2, as further described in Example 6. [Figure 13-3] Figure 13C: Provides exemplary ADC piggyback assay results for A52 in HEK cells expressing cynoIL13Rα2, as further described in Example 6. [Figure 13-4] Figure 13D: Provides exemplary ADC piggyback assay results for benchmark antibody 1 in HEK cells expressing cynoIL13Rα2, as further described in Example 6. [Figure 13-5]Figure 13E: Provides exemplary ADC piggyback assay results for benchmark antibody 2 in HEK cells expressing cynoIL13Rα2, as further explained in Example 6.

[0024] [Figure 14-1] Figure 14A: Provides exemplary binding results of A22 to human IL13Rα1 expressed in HEK cells, as further explained in Example 5. [Figure 14-2] Figure 14B: Provides exemplary binding results of A33 to human IL13Rα1 expressed in HEK cells, as further explained in Example 5. [Figure 14-3] Figure 14C: Provides exemplary binding results of A52 to human IL13Rα1 expressed in HEK cells, as further explained in Example 5. [Figure 14-4] Figure 14D: Provides exemplary binding results of benchmark antibody 1 against human IL13Rα1 expressed in HEK cells, as further explained in Example 5. [Figure 14-5] Figure 14E: Provides exemplary binding results of benchmark antibody 2 against human IL13Rα1 expressed in HEK cells, as further explained in Example 5.

[0025] [Figure 15-1] Figure 15A: Provides exemplary ADC piggyback assay results for A22 in HEK cells expressing human IL13Rα1, as further described in Example 6. [Figure 15-2] Figure 15B: Provides exemplary ADC piggyback assay results for A33 in HEK cells expressing human IL13Rα1, as further described in Example 6. [Figure 15-3] Figure 15C: Provides exemplary ADC piggyback assay results for A52 in HEK cells expressing human IL13Rα1, as further explained in Example 6. [Figure 15-4]Figure 15D: Provides exemplary ADC piggyback assay results for benchmark antibody 1 in HEK cells expressing human IL13Rα1, as further explained in Example 6. [Figure 15-5] Figure 15E: Provides exemplary ADC piggyback assay results for benchmark antibody 2 in HEK cells expressing human IL13Rα1, as further described in Example 6.

[0026] [Figure 16-1] Figure 16A: Provides exemplary SDS-PAGE results for A22, as further explained in Example 4. [Figure 16-2] Figure 16B: Provides an exemplary SDS-PAGE result for A33, as further explained in Example 4. [Figure 16-3] Figure 16C: Provides exemplary SDS-PAGE results for A52, as further explained in Example 4. [Figure 16-4] Figure 16D: Provides exemplary SDS-PAGE results for benchmark antibody 1, as further explained in Example 4. [Figure 16-5] Figure 16E: Provides exemplary SDS-PAGE results for benchmark antibody 2, as further explained in Example 4.

[0027] [Figure 17-1] Figure 17A: This figure shows the results of an analysis comparing free IL13Rα2 from A375 cells with IL13Rα2:1L-13 from A375 cells in terms of EC50. All EC50 values ​​are plotted on the y-axis. [Figure 17-2] Figure 17B: This figure shows the results of an analysis comparing the EC50 of free IL13Rα2 from A375 cells and IL13Rα2:1L-13 from A375 cells. The EC50 of IL13Rα2 is plotted along the y-axis, and the EC50 of IL13Rα2:1L-13 is plotted along the x-axis.

[0028] [Figure 18]The results of an analysis comparing free human IL13Rα2 from A375 cells with free cynoIL13Rα2 from cynoIL13Rα2-overexpressing cells in terms of EC50 are shown.

[0029] [Figure 19-1] Figure 19A: Comparison of Bmax in the adaptation group. The analysis results comparing the Bmax of free IL13Rα2 of A375 and IL-13-bound IL13Rα2 of A375 are shown. [Figure 19-2] Figure 19B: Comparison of Bmax in the adaptation group. This shows the results of an analysis comparing Bmax between free human IL13Rα2 from A375 and free cynoIL13Rα2 from cynoIL13Rα2 overexpressing cells.

[0030] [Figure 20-1] Figure 20A: This figure shows the results of a comparative analysis of the cytotoxic IC50 of free IL13Rα2 from A375 cells and IL13Rα2:1L-13 from A375 cells. All IC50 values ​​are plotted on the y-axis. [Figure 20-2] Figure 20B: This figure shows the results of an analysis comparing the cytotoxic IC50 of free IL13Rα2 from A375 cells and IL13Rα2:1L-13 from A375 cells. The IC50 of IL13Rα2 is plotted along the y-axis, and the IC50 of IL13Rα2:1L-13 is plotted along the x-axis.

[0031] [Figure 21-1] Figure 21A: Comparison of cell binding ability and cytotoxicity. The results of a comparative analysis of cell binding EC50 and cytotoxic IC50 for free IL13Rα2 in A375 cells are shown. [Figure 21-2] Figure 21B: Comparison of cell binding ability and cytotoxicity. The results of a comparative analysis of cell binding EC50 and cytotoxic IC50 for IL-13-bound IL13Rα2 in A375 cells are shown.

[0032] [Figure 22-1]Figure 22A: This shows the results of an analysis comparing the IC50 of free human IL13Rα2 from A375 and free cynoIL13Rα2 from cynoIL13Rα2-overexpressing cells. [Figure 22-2] Figure 22B: This shows the results of a comparative analysis of cell-bound EC50 and cytotoxic IC50 for free cynoIL13Rα2 in cynoIL13Rα2-overexpressing cells.

[0033] [Figure 23-1] Figure 23A: This figure shows the results of a comparative analysis of A375 cell-bound EC50 to free human IL13Rα2 and its binding Kd affinity. Both EC50 and Kd are plotted on the y-axis. [Figure 23-2] Figure 23B: This figure shows the results of a comparative analysis of A375 cell-bound EC50 to free human IL13Rα2 and its Kd affinity. Kd is plotted along the y-axis, and EC50 is plotted along the x-axis.

[0034] [Figure 24-1] Figure 24A: This figure shows the results of a comparative analysis of EC50 binding to IL13-treated A375 cells and Kd affinity binding to the human IL13Rα2:IL13 complex. Both EC50 and Kd are plotted on the y-axis. [Figure 24-2] Figure 24B shows the results of a comparative analysis of EC50 binding to IL13-treated A375 cells and Kd affinity binding to the human IL13Rα2:IL13 complex. Kd is plotted along the y-axis, and EC50 is plotted along the x-axis.

[0035] [Figure 25-1] Figure 25A: This figure shows the results of a comparative analysis of EC50 binding to overexpressed cynoIL13Rα2 in HEK cells and Kd affinity binding to free cynoIL13Rα2. Both EC50 and Kd are plotted on the y-axis. [Figure 25-2] Figure 25B: This figure shows the results of a comparative analysis of EC50 binding to overexpressed cynoIL13Rα2 in HEK cells and Kd affinity binding to free cynoIL13Rα2. Kd is plotted along the y-axis, and EC50 is plotted along the x-axis. [Modes for carrying out the invention]

[0036] 5. Detailed explanation This disclosure is based, at least in part, on novel IL13Rα2 conjugates and their properties. Such agents include antibodies that bind to IL13Rα2 (or IL13Rα2:IL13 complex) (e.g., monospecific or polyspecific antibodies (including bispecific antibodies)), including antibodies that bind to human IL13Rα2 (or IL13Rα2:IL13 complex). In certain embodiments, such conjugates are useful for compositions and methods to identify cells expressing IL13Rα2, to reduce (including but not limited to eliminating) the viability or proliferation of IL13Rα2-expressing cells, or to kill IL13Rα2-expressing cells (e.g., cancer cells). In addition, the IL13Rα2 conjugates described herein, such as IL13Rα2-binding antibodies (e.g., monospecific or polyspecific antibodies (including bispecific antibodies)), are useful for killing and / or removing tumor cells and inhibiting tumor invasion and metastasis. In further embodiments, tumor cells express (including overexpression) IL13Rα2. The IL13Rα2 conjugates described herein, such as IL13Rα2-binding antibodies (e.g., monospecific or polyspecific antibodies (including bispecific antibodies)), are useful in compositions and methods for treating diseases or disorders, such as cancer.

[0037] To be understood, when used herein, chapter or section headings are for organizational purposes only and should not be construed as limiting and / or separating the subject matter described herein.

[0038] 5.1.Definition The techniques and procedures described or referenced herein include those generally well understood and / or commonly used by those skilled in the art, such as Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have meanings generally understood by those skilled in the art. For the purpose of interpreting this specification, the following definitions of terms shall apply, and where appropriate, the singular form of a term shall also include the plural form, and the plural form of a term shall also include the singular form. In the event of any conflict between the definitions of terms provided and any reference incorporated herein by reference, the definitions provided below shall prevail.

[0039] Unless otherwise indicated, the term "IL13Rα2" refers to a polypeptide ("polypeptide" and "protein" are used synonymously herein) or any native form of IL13Rα2 derived from any vertebrate source, including mammals, such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats).

[0040] An example amino acid sequence of human IL13Rα2 is shown below: MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQDFEIVDPGYLGYLYLQWQPPLSLDHFKECTVEYELKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEVQSSWAETTYWISPQGIPETKVQDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDHALQCVDYIKADGQNIGCRFPYLEASDYKDFYICVNGSSENKPIRSSYFTFQLQNIVKPLPPVYLTFTRESSCEIKLKWSIPLGPIPARCFDYEIEIREDDTTLVTATVENETYTLKTTNETRQLCFVVRSKVNIYCSDDGIWSEWSDKQCWEGEDLSKKTLLRFWLPFGFILILVIFVTGLLLRKPNTYPKMIPEFFCDT (Sequence ID 83, UniProt:Q14627). In some embodiments, the exemplary amino acid sequence of human IL13Rα2 is amino acids (aa)27-aa380 of SEQ ID NO: 83. In some embodiments, the extracellular domain (ECD) of human IL13Rα2 is aa1-aa343 of SEQ ID NO: 83. In some embodiments, the ECD of human IL13Rα2 is aa27-aa343 of SEQ ID NO: 83.

[0041] The following is an example amino acid sequence of cynomolgus (cyno) IL13Rα2 (identical to rhesus macaque IL13Rα2): MDFVYLAIRCLCTFLISTTFGYTSSSDTEIKVNPPQDFEIVDPGYLGYLYLQWQPPLSLDNFKECTVEYELKYRNIGSETWTTIITKNLHYKDGFDLNKGIEA KIHTLLPWQCTNGSEVQSSWAEATYWISPQGIPETKVQDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDRALQCVDYIKVDGQNIGCRFPYLESSD YKDFYICVNGSSETKPIRSSYFTFQLQNIVKPLPPVCLTCTQESLYEIKLKWSIPLGPIPARCFVYEIEIREDDTTLVTTTVENETYTLKITNETRQLCFVVRSKVNIYCSDDGIWSEWSDKQCWEVEELLKKTLLLFLLPFGFILILVIFVTGLLLCKRDSYPKMNFSVIDEDFPYQETWY (Sequence ID 84, UniProt:F6Z890). In some embodiments, the exemplary amino acid sequence of cynomolgus monkey / rhesus monkey IL13Rα2 is aa26-aa388 in Sequence ID 84. In some embodiments, the extracellular domain (ECD) of cynomolgus monkey / rhesus monkey IL13Rα2 is aa1-aa340 in Sequence ID 84. In some embodiments, the ECD of cynomolgus macaque / rhesus macaque IL13Rα2 is aa26-aa340 of sequence number 84.

[0042] The following is an example amino acid sequence of mouse IL13Rα2: MAFVHIRCLCFILLCTITGYSLEIKVNPPQDFEILDPGLLGYLYLQWKPPVVIEKFKGCTLEYELKYRNVDSDSWKTIITRNLIYKDGFDLNKGIEGKIRTHLSEHCTNGSEVQSPWIEASYGISDEGSLETKIQDMKCIYYNWQYLVCSWKPGKTVYSDTNYTMFFWYEGLDHALQCADYLQHDEKNVGCKLSNLDSSDYKDFFICVNGSSKLEPIRSSYTVFQLQNIVKPLPPEFLHISVENSIDIRMKWSTPGGPIPPRCYTYEIVIREDDISWESATDKNDMKLKRRANESEDLCFFVRCKVNIYCADDGIWSEWSEEECWEGYTGPDSKIIFIVPVCLFFIFLLLLLCLIVEKEEPEPTLSLHVDLNKEVCAYEDTLC (Sequence ID 85, UniProt:O88786). In some embodiments, the exemplary amino acid sequence of mouse IL13Rα2 is aa21-aa383 in SEQ ID NO: 85. In some embodiments, the exemplary amino acid sequence of mouse IL13Rα2 is aa22-aa383 in SEQ ID NO: 85. In some embodiments, the ECD of mouse IL13Rα2 is aa1-aa334 in SEQ ID NO: 85. In some embodiments, the ECD of mouse IL13Rα2 is aa21-aa334 in SEQ ID NO: 85. In some embodiments, the ECD of mouse IL13Rα2 is aa22-aa334 in SEQ ID NO: 85. In some embodiments, the ECD of mouse IL13Rα2 is aa1-aa344 in SEQ ID NO: 85. In some embodiments, the ECD of mouse IL13Rα2 is aa21-aa344 in SEQ ID NO: 85. In some embodiments, the ECD of mouse IL13Rα2 is aa22-aa344 of sequence number 85.

[0043] The following is an example amino acid sequence of rat IL13Rα2: MALMAVNTRCLCLFLLCTITGHSLEIKVNPPQDFEILDPGLLGYLYLQWKPPVVMDNFKECKLEYELKYRNVDSDSWKTIITRNLIYKDGFDLNKGIEGKIRTHLSEHCTNGSEVQSPWTEASYGIADEGSLGTKIQDMKCIYYNWQYLVCSWKPGKTVHSDTNYTMFFWYEGLDHALQCADYLQDNEKNVGCKLSNLDSSDYKDFFIRVNGSSKLEPIRSSYMVFQLQNIVKPLPPEFLHISVENSIDIRMKWSTPGGPIPPSCYTYEIVVREDDISWESATDKNDMKLKRRANESEDLCFFVRCKINIYCADDGIWSEWSEEECWEGYTGPDSKIVFIVPVCLFFIFLLLLLCLIVEKEDPEPTLSLHVDLNKEMYAYEETLC (Sequence ID 86, UniProt:Q8VHK6). In some embodiments, the exemplary amino acid sequence of rat IL13Rα2 is aa24-aa385 in SEQ ID NO: 86. In some embodiments, the ECD of rat IL13Rα2 is aa1-aa336 in SEQ ID NO: 86. In some embodiments, the ECD of rat IL13Rα2 is aa24-aa336 in SEQ ID NO: 86.

[0044] IL13Rα2 is a single-pass type I transmembrane protein containing three fibronectin type III (FNIII)-like domains (101, 97, and 94aa), a single transmembrane (TM) domain (20aa), and a short cytoplasmic domain (17aa). There is one native mutant, W111R. Four N-linked glycosylation sites are predicted, including N115, N215, N290, and N299. IL13Rα2 is primarily tumor-limited, with protein expression in normal spermatocytes. IL13Rα2 is also upregulated in malignant melanoma, malignant glioma, pancreatic cancer, ovarian cancer, breast cancer, liver cancer, head and neck cancer, and renal cancer. Its soluble form is detectable in human and mouse serum / plasma (ng / mL), although conflicting reports have been found regarding its detection in humans. Its soluble form is produced in mice by alternative splicing and MMP-8 cleavage, but in humans by MMP-8 cleavage alone. IL13Rα2 is considered a high-affinity (fM) Th2 cytokine receptor and decoy receptor for IL-13, inhibiting IL-13 signaling, regulating serum and tissue levels of IL-13, and mediating biological effects such as tumor growth, cell survival, cell adhesion, and metastasis. The classical pathway of IL-13 activation is JAK / STAT via the binding of heterodimers (type II complexes) of IL-13Rα1 and IL-4Rα, rather than IL-13Rα2. In other words, IL-13Rα2 does not bind to IL-4, but has extremely high affinity (<10). -15 It binds to IL-13 at M). IL-13Rα2 may function as a negative regulator of IL-4, but it does not function in IL-13-induced signaling via type II IL-4R, although the mechanism is unknown.

[0045] In some embodiments, the term IL13Rα2 as used herein refers to the IL13Rα2 epitope. In further embodiments, the term IL13Rα2 as used herein refers to the epitope of the ECD of IL13Rα2. In some embodiments, the term IL13Rα2 as used herein refers to a complex comprising IL13Rα2 and IL13. In further embodiments, the term IL13Rα2 as used herein refers to a complex comprising the ECD of IL13Rα2 and IL13. In yet another embodiment, the term IL13Rα2 as used herein refers to the ECD of a complex comprising the ECD of IL13Rα2 and IL13.

[0046] IL-13Rα1 belongs to the same family as IL-13Rα2, but shares a low degree of sequence identity (20%). An exemplary amino acid sequence of human IL-13Rα1 is shown below: MEWPARLCGLWALLLCAGGGGGGGGAAPTETQPPVTNLSVSVENLCTVIWTWNPPEGASSNCSLWYFSHFGDKQDKKIAPETRRSIEVPLNERICLQVGSQCSTNESEKPSI LVEKCISPPEGDPESAVTELQCIWHNLSYMKCSWLPGRNTSPDTNYTLYYWHRSLEKIHQCENIFREGQYFGCSFDLTKVKDSSFEQHSVQIMVKDNAGKIKPSFNIVPLTSR VKPDPPHIKNLSFHNDDLYVQWENPQNFISRCLFYEVEVNNSQTETHNVFYVQEAKCENPEFERNVENTSCFMVPGVLPDTLNTVRIRVKTNKLCYEDDKLWSNWSQEMSIGKKRNSTLYITMLLIVPVIVAGAIIVLLLYLKRLKIIIFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIYEKQTKEETDSVVLIENLKKASQ (Sequence ID 87, UniProt:P78552). In some embodiments, the exemplary amino acid sequence of human IL13Rα1 is aa22-aa427 in Sequence ID 87. In some embodiments, the exemplary amino acid sequence of the ECD of human IL13Rα1 is aa1-aa343 in Sequence ID 87. In some embodiments, the exemplary amino acid sequence of the ECD of human IL13Rα1 is aa22-aa343 in Sequence ID 87. In some embodiments, the term IL13Rα1 as used herein refers to the IL13Rα1 epitope. In even further embodiments, the term IL13Rα1 as used herein refers to the ECD of IL13Rα1. In even further embodiments, the term IL13Rα1 as used herein refers to the epitope of the ECD of IL13Rα1.

[0047] An example amino acid sequence of human IL-13 is shown below: MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN (Sequence ID 88, UniProt: P35225). In some embodiments, the exemplary amino acid sequence of human IL-13 is aa25-aa146 of Sequence ID 88. In some embodiments, the exemplary amino acid sequence of human IL-13 is as follows: MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN (SEQ ID NO: 89, GenBank: AAK53823.1).

[0048] An example amino acid sequence of cynoIL13 is shown below: MALLLTMVIALTCLGGFASPSPVPPSTALKELIEELVNITQNQKAPLCNGSMVWSINLTAGVYCAALESLINVSGCSAIEKTQRMLNGFCPHKVSAGQFSSLRVRDTKIEVAQFVKDLLVHLKKLFREGQFN (Sequence ID 90, GenBank: BG75889.1).

[0049] As used herein, the term “conjugate” or its grammatical equivalent refers to a molecule (e.g., an antibody) having one or more antigen-binding sites that bind to an antigen. In some embodiments, IL13Rα2 conjugates as described herein include antibodies (including antibody fragments, e.g., antigen-binding fragments or epitope-binding fragments), or other peptide-based molecules, as well as conjugates of antibodies, antibody fragments, or peptide-based molecules (e.g., antibody-drug conjugates) that bind to IL13Rα2, e.g., human IL13Rα2.

[0050] The terms “antibody,” “immunoglobulin,” and “Ig” are used synonymously and in their broadest sense herein, specifically encompassing, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having polyepitope specificity or monoepitope specificity, recombinant antibodies, single-domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full-length heavy and / or light chains. As used herein, VHH refers to a domain antibody derived from the variable region of an antibody consisting only of a heavy chain. Exemplary single-domain antibodies include, but are not limited to, antibodies that naturally lack a light chain, e.g., those derived from camelids (e.g., llamas), single-domain antibodies derived from conventional four-chain antibodies, manipulated antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies may originate from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. VHHs may also originate from other non-camelid species that can produce heavy-chain antibodies naturally lacking the light chain. Antibodies also include antibody fragments (and / or polypeptides containing antibody fragments) that retain IL13Rα2 binding properties. Non-limiting examples of antibody fragments include antigen-binding regions and / or effector regions of antibodies, e.g., Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain post-antibody molecules, bivariate domain antibodies, single variable domains, linear antibodies, V regions, polyspecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabodies, didiabodies, disulfide-bonded Fvs(dsFv), single-domain antibodies (e.g., nanobodies) or other fragments (e.g., fragments consisting of non-covalently bonded heavy and light chain variable regions). Generally speaking, the variable (V) region domain may be the heavy chain (VH) variable domain and / or light chain (VL) variable domain of an immunoglobulin in any appropriate configuration. For example, antibodies also include tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers.Therefore, for example, the V region domain may be a dimer and may include VHH-VHH, VH-VH, VH-VL, or VL-VL dimers that bind to IL13Rα2. If desired, VH and VL may be covalently linked either directly or via a linker to form a single-stranded Fv (scFv). For ease of reference, scFv proteins are referred to herein in the category of “antibody fragments.” Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also called “minimum recognition units” or “hypervariable regions”) can be obtained by constructing polynucleotides encoding the one or more CDRs in question. Such polynucleotides can be prepared, for example, by using a polymerase chain reaction that synthesizes the variable region using mRNA from antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991), Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995), and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)).The antibody fragment may be incorporated into, for example, a single-domain antibody, a maxibody, a minibody, an intrabody, a diabody, a triabody, a tetrabody, a variable domain (v-NAR) of a novel antigen receptor, and a bis-single-chain Fv region (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, the antibody comprising VH and / or VL further comprises a light chain constant region and / or a heavy chain constant region, for example, one or more constant regions (including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions). In some embodiments, the antibody may contain any of the above epitope-binding fragments. The antibodies described herein may be antibodies against immunoglobulin molecules of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0051] When the term "monospecific" is used in reference to a binder (e.g., an antibody), it refers to a binder having one or more binding sites that each bind to the same epitope of the same antigen.

[0052] When the term "multispecific" is used in reference to a conjugate (e.g., an antibody), it means that the conjugate can specifically bind to at least two distinct epitopes, for example, two binding sites formed by pairs of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), or by VHH domains, each binding to different antigens or different epitopes on the same antigen. Such a bispecific conjugate (e.g., an antibody) may have a 1+1 configuration (containing one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific conjugates (e.g., antibodies) may be in the form of 2+1 or 1+2 (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope), or 2+2 (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific conjugate (e.g., antibody) contains two antigen-binding sites, each may bind to a different epitope. Such a bispecific conjugate (e.g., antibody) may bind to two different epitopes on the same antigen (e.g., epitopes on IL13Rα2).

[0053] The term “identical” or “identity” percentage, in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are identical when compared and aligned to the greatest extent possible (with gaps introduced where necessary), and when no conserved amino acid substitutions are considered as part of sequence identity, or two or more sequences or subsequences that have a predetermined percentage of identical nucleotides or amino acid residues. The identity percentage can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain sequence alignment of amino acids or nucleotides are well known in the art. Examples of algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their variants. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning that when compared and aligned to the maximum extent possible, the nucleotide or amino acid residue identity is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99%. In some embodiments, the identity extends over an amino acid sequence region that is at least about 10 residues long, at least about 20 residues long, at least about 40–60 residues long, at least about 60–80 residues remaining, or any integer value in between. In some embodiments, the identity extends over a region longer than 60–80 residues, for example, at least about 80–100 residues, and in some embodiments, the sequence is substantially identical over the full length of the sequence being compared, for example, the coding region of the target protein or antibody. In some embodiments, identity exists across nucleotide sequence regions that are at least about 10 nucleotides long, at least about 20 nucleotides long, at least about 40–60 nucleotides long, at least about 60–80 nucleotides long, or any integer value in between.In some embodiments, the identity exists over a region longer than 60–80 bases, for example, at least about 80–1000 bases or more, and in some embodiments, the sequence is substantially identical over the full length of the sequence being compared, for example, the nucleotide sequence encoding the protein in question.

[0054] A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a side chain with similar chemical properties. Families of amino acid residues with similar side chains are generally defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine with phenylalanine is a conservative substitution. In general, conservative substitutions in the sequences of polypeptides, soluble proteins, and / or antibodies of this disclosure do not prevent the polypeptide, soluble protein, or antibody containing the amino acid sequence from binding to a target binding site. Methods for identifying non-binding conservative amino acid substitutions are well known in the art.

[0055] The term “polypeptide” refers to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, or may contain non-amino acids (e.g., may be interrupted by non-amino acids). The term also includes amino acid polymers that are modified in nature or by intervention, e.g., by the formation of disulfide bonds, glycosylation, lipidization, acetylation, phosphorylation, or any other operation or modification, e.g., by binding to or conjugation (directly or indirectly) of a moiety, e.g., a labeling component or a drug (e.g., a toxin). Within the definition, for example, polypeptides containing one or more analogues of amino acids (e.g., non-natural amino acids) and polypeptides containing other modifications known in the art. Since the polypeptides of this disclosure may be based on an immunoglobulin superfamily of an antibody or other member, it will be found that in some embodiments, the polypeptides may exist as single-stranded or single-stranded dimers.

[0056] As used herein, “antigen” is a portion or molecule containing an epitope to which a binder (e.g., an antibody) can bind. Thus, an antigen to which an antibody can bind. In some embodiments, the antigen to which the binder (e.g., an antibody) described herein binds is IL13Rα2 (e.g., human IL13Rα2) or a fragment thereof, the fragment containing one or more domains of IL13Rα2.

[0057] As used herein, “epitope” is a term used in the art and refers to a local region of an antigen to which an antibody can bind. An epitope can be a linear epitope, a structural epitope, a nonlinear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, the epitope can be a sequence of amino acids in the polypeptide (a “linear” epitope), or it can contain amino acids derived from two or more discontinuous regions of the polypeptide (a “structural” epitope, a nonlinear” epitope, or a “discontinuous” epitope), such as human IL13Rα2. Generally, it will be apparent to those skilled in the art that linear epitopes may or may not depend on the secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether the group is folded into a native three-dimensional protein structure. In other embodiments, the antibody requires amino acid residues that constitute the epitope to adopt a specific three-dimensional structure (e.g., bend, twist, turn, or fold) in order to recognize the epitope and bind to it.

[0058] When two antibodies recognize the same, overlapping, or adjacent epitopes in three-dimensional space, the antibodies bind to the "epitope," or to an "essentially identical epitope" or "same epitope" as the reference antibody. The most widely used rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is the competition assay, which can be constructed in a considerable number of different forms, for example, using either a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized in a 96-well plate or expressed on the cell surface, and the ability of an unlabeled antibody to block the binding of a labeled antibody is measured using radiolabeling, fluorescent labeling, or enzymatic labeling.

[0059] Epitope binning is a process of grouping antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for identifying the epitope recognition characteristics of various antibodies by using a competitive assay that clusters antibodies based on their epitope recognition characteristics and combines this with a computational process that identifies antibodies with distinct binding specificities.

[0060] As used herein, the terms “specifically bind,” “specifically recognize,” “immunospecifically bind,” “selectively bind,” “immunospecifically recognize,” and “immunospecific” are synonyms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope) in the same way that those skilled in the art would understand binding. In some embodiments, “specifically bind” means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, rapidly, for longer periods, with higher affinity, or some combination of the above, than alternative substances (including related and unrelated proteins). For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined by immunoassays, Biacore®, KinExA3000 (Sapidyne Instruments, Boise, ID), OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds to or specifically binds to an antigen when it binds to the antigen with higher affinity than any cross-reactive antigen, as determined by experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, specific or selective reactions result in reactions with at least 2x background signal or background noise, and sometimes more than 10x background. For example, see Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for considerations regarding binding specificity. In some embodiments, the extent to which an antibody or antigen-binding domain binds to a “non-target” protein is less than about 10% of the extent to which that antibody or antigen-binding domain binds to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. In some embodiments, a molecule that specifically binds to an antigen binds to that antigen with a Ka of at least 2log, 2.5log, 3log, or 4log greater than the Ka of the molecule when it binds to another antigen.In some embodiments, molecules that specifically bind to an antigen do not cross-react with other proteins. In another specific embodiment, molecules that specifically bind to an antigen do not cross-react with other proteins other than IL13Rα2. In some embodiments, "specifically binds" means, for example, that a polypeptide or molecule binds to a protein or target at a K level of about 0.1 mM or less (more typically less than about 1 μM). D This means binding by K. In some embodiments, "specifically binding" means that the polypeptide or molecule binds to the target at a concentration of at least about 0.1 μM, at least about 0.01 μM, or at least about 1 nM. D This means binding. Due to sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize more than one protein or target. Similarly, due to homology within specific regions of polypeptide sequences of different proteins, specific binding can include polypeptides or molecules that recognize more than one protein or target. In some embodiments, a polypeptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not require an exclusive binding, e.g., binding to one target (this binding may be included). Thus, in some embodiments, a polypeptide or molecule can specifically bind to more than one target. In some embodiments, the same antigen-binding site on its polypeptide or molecule can bind to multiple targets. For example, an antibody may, in certain cases, contain two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, an antibody may be bispecific, containing at least two antigen-binding sites with different specificities. Generally, though not always, the term "binding" refers to "specific binding."

[0061] "Binding affinity" generally refers to the total strength of the non-covalent interactions between a single binding site of a molecule (e.g., a binder such as an antibody) and its binding partner (e.g., an antigen such as IL13Rα2). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art (including the methods described herein). Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present disclosure. In one embodiment, the "K D " or "K D value" may be measured by biolayer interferometry (BLI), for example, using an OctetQK384 system (ForteBio, Menlo Park, CA). Alternatively, K D may be measured by a radioisotope-labeled antigen binding assay (RIA), for example, an RIA performed with the Fab version of the corresponding antibody and its antigen (Chen, et al., (1999) J. Mol Biol 293:865-881), or by surface plasmon resonance (SPR) assay using Biacore™, for example, Biacore™-2000 or Biacore™-3000 (Biacore™, Inc., Piscataway, NJ). "Association rate" or "k on " and "dissociation rate" or "k off " can also be determined by the same SPR or BLI techniques described above, for example, using an OctetQK384 system (ForteBio, Menlo Park, CA) (in the case of SPR), or Biacore™-2000 (in the case of SPR) or Biacore™-3000 (Biacore™, Inc., Piscataway, NJ) (in the case of BLI).

[0062] When used in the context of IL13Rα2 conjugates (e.g., antibodies), the term "compete" or any grammatical variation thereof means conjugates that compete for the same epitope or binding site on the target, and this competition includes competition among such conjugates as determined by assays in which the conjugate under consideration prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand or reference antigen-binding protein, e.g., a reference antibody) to a common antigen (e.g., IL13Rα2). Numerous types of competitive binding assays can be used to determine whether a test conjugate competes with a reference molecule for binding to IL13Rα2 (e.g., human IL13Rα2). Examples of assays that can be used include solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (e.g., see Stahli et al., (1983) Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (e.g., see Kirkland et al., (1986) J.Immunol. 137:3614-3619 or Cheung et al., (1990) Virology 176:546-552); solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (e.g., see Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (e.g., see Morel et al.) al., (1988) Molec. Immunol. 25:7-15); and directly labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen (e.g., IL13Rα2, e.g., human IL13Rα2) bound to a solid surface or cell having either an unlabeled test antigen-binding protein (e.g., test IL13Rα2 antibody) or a labeled reference antigen-binding protein (e.g., reference IL13Rα2 antibody).Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and / or antibodies that bind to an adjacent epitope (e.g., a similar or overlapping epitope) that is close enough to the epitope to which the reference antibody binds that the antibody would be sterically hindrance. Typically, when competitive antibodies are present in excess, they inhibit the specific binding of the reference antibody to the common antigen by at least 20%, e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more.

[0063] As used herein, the terms “constant region” or “constant domain” are well-known antibody technology terms referring to an antibody portion that does not directly participate in the binding of the antibody to an antigen but can exhibit various effector functions, such as interaction with Fc receptors, e.g., the carboxyl-terminal portions of the light and / or heavy chains. This term includes portions of an immunoglobulin molecule that have an amino acid sequence that is generally more conserved than the immunoglobulin variable domain.

[0064] Antibody "effector function" refers to the biological activity resulting from the antibody's Fc region (e.g., the Fc region of the native sequence or the Fc region of an amino acid sequence variant), and varies depending on the antibody isotype. Examples of antibody effector functions include binding to C1q and complement-dependent cytotoxicity, binding to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0065] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, the native Fc region, recombinant Fc region, and variant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 (according to the EU numbering system) or Pro230 (according to the EU numbering system) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue at position 447 according to the EU numbering system) may be removed, for example, during the production or purification of the antibody, or by recombinant operation of the nucleic acid encoding the heavy chain of the antibody. Exemplary Fc region sequences are shown below (CH2 domain = bold text, CH3 domain = underlined text). [ka]

[0066] A "functional Fc region" possesses the "effector function" of the Fc region in the native sequence. Examples of such "effector functions" include binding to C1q, complement-dependent cytotoxicity (CDC), binding to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis (antibody-dependent cell phagocytosis, i.e., ADCP), and downregulation of cell surface receptors (e.g., B cell receptors (BCRs)). Such effector functions generally require an Fc region combined with a binding region or binding domain (e.g., an antibody-variable region or antibody-variable domain) and can be evaluated using various assays as disclosed.

[0067] "Natural-type Fc regions" contain the same amino acid sequence as the naturally occurring Fc region and have not been manipulated, modified, and / or altered by humans (e.g., not isolated, purified, or selected (and not containing other sequences, such as variable region sequences, or combined with other sequences)). Examples of natural-type human Fc regions include natural-type human IgG1 Fc regions (allotypes other than A and A), natural-type human IgG2 Fc regions, natural-type human IgG3 Fc regions, and natural-type human IgG4 Fc regions, as well as their natural variants.

[0068] A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the Fc region of the native sequence based on at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, and preferably about 1 to about 5 amino acid substitutions, compared to the Fc region of the native sequence or the Fc region of the parent polypeptide. The variant Fc regions described herein may have at least about 80% homology to the Fc region of the native sequence and / or the Fc region of the parent polypeptide, or at least about 90%, e.g., at least about 95% homology. The variant Fc regions described herein may lack effector function (e.g., silent Fc). An example variant Fc region ("silent Fc") sequence is shown below (CH2 domain = bold text (amino acid changes are underlined), CH3 domain = underlined text). [ka]

[0069] As used herein, the term “heavy chain,” when used in reference to antibodies, refers to a polypeptide chain of approximately 50–70 kDa, wherein the amino-terminal portion contains a variable region of approximately 120–130 or more amino acids, and the carboxy-terminal portion contains one or more constant regions. A “heavy chain” can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of its constant domain, where alpha gives rise to the IgA class of antibody, delta to the IgD class of antibody, epsilon to the IgE class of antibody, gamma to the IgG class of antibody, and mu to the IgM class of antibody, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0070] As used herein, the term “light chain” can refer to a polypeptide chain of approximately 25 kDa when used in relation to antibodies, wherein the amino-terminal portion contains a variable region of approximately 100 to 110 or more amino acids, and the carboxy-terminal portion contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of its constant domain, there are two distinct types, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art.

[0071] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to the portion of an antibody that contains amino acid residues that interact with an antigen, giving the binding fragment, binding domain, or binding region specificity and affinity for that antigen (e.g., CDR). As used herein, “antigen-binding fragment” includes, for example, an antibody portion containing one or more CDRs, such as an “antibody fragment” containing the antigen-binding region or variable region of that antibody.

[0072] Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, polyspecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fv(scFv) (e.g., monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies, and any of the above epitope-linked fragments.

[0073] In some embodiments, the antibodies described herein include an immunoglobulin molecule and an immunoactive portion of the immunoglobulin molecule, including a molecule having one or more antigen-binding sites that bind to the IL13Rα2 antigen.

[0074] The antibody may be an antibody of any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In some embodiments, the antibody described herein is an IgG antibody (e.g., human IgG), or a class thereof (e.g., human IgG1, human IgG2, human IgG3, or IgG4) or a subclass thereof.

[0075] In some embodiments, the antibody is a four-chain antibody unit containing two heavy (H) chain / light (L) chain pairs. In further embodiments, the amino acid sequences of the H chains are identical, and the amino acid sequences of the L chains are identical. In other embodiments, the amino acid sequences of the H chains are different from each other. In addition to or instead of this, the amino acid sequences of the L chains are different from each other. For example, the antibody contains a first H chain / L chain pair and a second H chain / L chain pair, the first H chain / L chain pair binding to the IL13Rα2 antigen, and the second H chain / L chain pair binding to another IL13Rα2 antigen or an antigen other than IL13Rα2. In some embodiments, the antibody is a two-chain antibody unit containing a VHH-VHH pair. In further embodiments, the amino acid sequences of the VHH are identical. In other embodiments, the amino acid sequences of the VHH are different from each other. For example, the antibody comprises a first VHH and a second VHH, the first VHH binding to the IL13Rα2 antigen and the second VHH binding to another IL13Rα2 antigen or an antigen other than IL13Rα2. In some embodiments, the H chain and / or L chain include a constant region, e.g., a human constant region. In some embodiments, the L chain constant region of such an antibody is a kappa light chain constant region or a lambda light chain constant region, e.g., a human kappa light chain constant region or a human lambda light chain constant region. In some embodiments, the H chain constant region of such an antibody includes a gamma heavy chain constant region, e.g., a human gamma heavy chain constant region. In some embodiments, such an antibody includes an IgG constant region, e.g., a human IgG constant region (e.g., constant regions of IgG1, IgG2, IgG3, and / or IgG4).

[0076] An antibody or fragment may preferentially bind to IL13Rα2 (or IL13Rα2:IL13 complex), e.g., human IL13Rα2, meaning that the antibody or fragment binds to IL13Rα2 with a higher affinity than it binds to a control protein (e.g., an irrelevant control protein, e.g., chicken egg white lysozyme), and / or to human IL13Rα2 with a higher affinity than it binds to an irrelevant control protein. For example, the antibody or fragment may specifically recognize and bind to IL13Rα2 or a portion thereof. "Specific binding" means that the antibody or fragment binds to IL13Rα2 with an affinity at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times higher than its affinity to an irrelevant control protein (e.g., chicken egg white lysozyme). In some embodiments, the antibody or a fragment thereof may bind substantially exclusively to IL13Rα2 (for example, IL13Rα2 can be distinguished from other known polypeptides based on a measurable difference in binding affinity). In some embodiments, the IL13Rα2 conjugate (e.g., antibody) may react with IL13Rα2 sequences other than human IL13Rα2 sequences (e.g., cynomolgus monkey IL13Rα2 sequences). In other embodiments, the IL13Rα2 conjugate (e.g., antibody) does not react with non-human (e.g., cynomolgus monkey) sequences.

[0077] The term "variable region" or "variable domain" refers to a portion of the antibody's light or heavy chain, generally located at the amino terminus, approximately 120–130 amino acids in the heavy chain and approximately 100–110 amino acids in the light chain. This region is used for the binding and specificity of each particular antibody to its specific antigen. The variable region of the heavy chain may be referred to as "VH," and the variable region of the light chain may be referred to as "VL." The term "variable" refers to the significant differences in the sequence of a particular segment of the variable region between antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody to its specific antigen. However, this variability is not evenly distributed across the 110-amino acid range of the variable region. Instead, the V region consists of a less variable (e.g., relatively immutable) region called a framework region (FR) of approximately 15-30 amino acids, separated by a shorter, more variable (e.g., highly variable) region called a "hypervariable region" or, instead, a "complementarity-determining region (CDR)." The variable regions of the heavy and light chains each contain four frameworks (FR1, FR2, FR3, and FR4) that primarily consist of β-sheet structures, each linked by three hypervariable regions. These hypervariable regions form connecting loops and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are closely linked by frameworks and, together with the hypervariable regions of other chains, contribute to the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). The constant region does not directly participate in antibody binding to the antigen, but it indicates the antibody's involvement in various effector functions, such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region shows significant sequence differences between different antibodies. Sequence variability is concentrated in the CDR, but the less variable portion within the variable region is called the framework region (FR).The CDRs of the light and heavy chains are primarily involved in the interaction between the antibody and its antigen. In specific embodiments, the variable region is the human variable region.

[0078] The terms “hypervariable region,” “HVR,” “HV,” “complementarity-determining region,” or “CDR,” as used herein, refer to regions within the antibody variable region that are sequence-hypervariable and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions: three in the VH region (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3) and three in the VL region (L1 or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). A considerable number of descriptions of hypervariable regions are used and are included herein. Kabat CDRs are sequence-variable and are the most commonly used (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Instead, Chothia refers to the location of the structural loop (see, for example, Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). The end of a Chothia CDR-H1 loop, when numbered using the Kabat numbering rules, varies between H32 and H34 depending on the length of the loop (this variation is because the Kabat numbering scheme inserts at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; and if both 35A and 35B exist, the loop ends at 34). The hypervariable region of AbM represents a compromise between Kabat's CDR and Chothia's structural loop and is used in Oxford Molecula's AbM antibody modeling software (see, for example, Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The hypervariable region of "contact" is based on an analysis of available complex crystal structures. The residues derived from each of these hypervariable regions or CDRs are shown below.

[0079] A universal numbering system has been developed and is widely adopted (ImMunoGeneTics (IMGT® Information System (Lefranc et al., Dev.Comp.Immunol.27(1):55-77(2003))). IMGT is an integrated information system specifically for human and other vertebrate immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complexes (MHC). In this specification, CDRs are referred to in terms of both amino acid sequence and position within the light or heavy chain. The "position" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides in structures called loops; therefore, CDRs and framework residues can be easily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into acceptor frameworks typically derived from human antibodies. An additional numbering system (AHon) has been developed by Honegger and Developed in Pluckthun, J. Mol. Biol. 309:657-670 (2001). Correspondence between numbering systems (e.g., including Kabat numbering and IMGT-specific numbering systems) is well known to those skilled in the art (see, for example, the above-mentioned Kabat, Chothia and Lesk, Martin, and Lefranc et al.), and is illustrated below. Various systems known in the art or described herein represent various ways of describing CDRs and are often considered equivalent when used to define the same antibody. The exemplary systems shown herein combine Kabat and Chothia. Residues derived from each of these hypervariable regions or CDRs are illustrated in the table below. [Table 10]

[0080] The hypervariable region may include the "ultra-ultravariable region" of VL at positions 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and VH at positions 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). As used herein, the terms "hypervariable region," "HVR," "HV," "complementarity-determining region," or "CDR" are used synonymously.

[0081] Where used synonymously herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or modified bases and / or analogs thereof, or any substance that can be incorporated into the polymer by DNA polymerase or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides, such as methylated nucleotides, and their analogs. Cells producing the binding molecules of this disclosure may include parental hybridoma cells into which the nucleic acid encoding the antibody has been introduced, as well as bacterial host cells and eukaryotic host cells. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left direction of a double-stranded polynucleotide sequence is the 5' direction. The direction in which a newly generated RNA transcript is added from 5' to 3' is called the transcription direction. Of the sequence regions on the DNA strand that have the same sequence as the RNA transcript, the region located 5' to the 5' end of the RNA transcript is called the "upstream sequence," and of the sequence regions on the DNA strand that have the same sequence as the RNA transcript, the region located 3' to the 3' end of the RNA transcript is called the "downstream sequence."

[0082] The term "vector" refers to a substance used to carry or contain nucleic acid sequences (for example, for the purpose of introducing nucleic acid sequences into host cells). Examples of vectors applicable to use include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selectable sequences or selectable markers that can function to be stably incorporated into the chromosomes of host cells. In addition, the vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included may, for example, confer resistance to antibiotics or toxins, compensate for nutritional deficiencies, or supply essential nutrients that are not present in the culture medium. Expression control sequences may include constitutive promoters and / or inductive promoters, transcriptional enhancers, transcriptional terminators, etc., which are well known in the art. When two or more nucleic acid molecules are co-expressed (for example, both the heavy and light chains of an antibody or both the VH and VL of an antibody), both nucleic acid molecules may be inserted into, for example, one expression vector or separate expression vectors. In single-vector expression, the coding nucleic acid can be functionally ligated to one common expression control sequence, or to different expression control sequences, such as one inductive promoter and one constitutive promoter. The introduction of the nucleic acid molecule into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting for gene product expression, amplification or immunoblotting of mRNA by polymerase chain reaction (PCR), or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will find that the nucleic acid molecule is expressed in sufficient quantities to produce the desired product (e.g., an IL13Rα2 binder as described herein), and will further find that the expression level can be optimized using methods well known in the art to obtain sufficient expression.

[0083] The term "pharmaceutically acceptable," as used herein, means that it is approved by a federal or state regulatory authority or is listed for use in animals, and more specifically in humans, in the United States Pharmacopeia, the European Pharmacopeia, or any other generally accepted pharmacopoeia.

[0084] "Excipient" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulant. Examples of excipients include encapsulants or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coatings, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizers, sweeteners, solubilizers, humectants, and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (full or incomplete adjuvant)), or vehicle. In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers, e.g., phosphates, citrates, and other organic acids; antioxidants (including ascorbic acid); low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other sugar chains (including glucose, mannose, or dextrin); chelating agents, e.g., EDTA; sugar alcohols, e.g., mannitol or sorbitol; salt-forming counterions, e.g., sodium; and / or nonionic surfactants, e.g., TWEEN®, polyethylene glycol (PEG), and PLURONICS®. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990). In one embodiment, each component is "pharmaceutically acceptable" in the sense that it is compatible with other components of the pharmaceutical formulation, and is suitable for use in contact with human and animal tissues or organs in a manner that balances a reasonable benefit-risk ratio without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications.For example, see Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009, Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007, Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed to the excipient at the dose and concentration used. In some embodiments, the pharmaceutically acceptable excipient is a pH-buffered aqueous solution. In some embodiments, the excipients are sterile liquids, such as water and oil, and the oils include petroleum oils (of animal, plant, or synthetic origin), such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is an exemplary excipient when the composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solution, dextrose aqueous solution, and glycerol aqueous solution can also be used as liquid excipients, particularly for injection solutions. Other possible excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The compositions of this disclosure may also contain small amounts of wetting agents, emulsifiers, or pH buffers, if desired. The compositions may take the form of solvents, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.Oral compositions (including formulations) may contain standard excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Compositions (including pharmaceutical compounds) may contain a prophylactic or therapeutically effective amount of IL13Rα2 conjugate (e.g., antibody), for example in isolated or purified form, along with appropriate amounts of excipients, to provide a form suitable for administration to a target (e.g., a patient). The formulation must be suitable for the administration method.

[0085] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, prevent or delay the onset of symptoms and / or their underlying causes, and / or improve or repair damage caused by or associated with a disease, disorder, or condition. In some embodiments, the effective dose is a therapeutic effective dose or a preventive effective dose.

[0086] The term “therapeutic dose,” as used herein, means an amount of a drug (e.g., the antibody described herein or any other drug described herein) that is sufficient to reduce and / or improve the severity and / or duration of a given disease, disorder, or condition and / or symptoms associated therewith. A therapeutic dose of a drug (including therapeutic agents) may be the amount necessary to (i) reduce, delay, or improve the exacerbation or progression of a given disease, disorder, or condition; (ii) reduce, delay, or improve the recurrence, progression, or onset of a given disease, disorder, or condition; and / or (iii) improve or enhance the preventive or therapeutic effect of another therapy (e.g., therapy other than administering the drug described herein). The “therapeutic dose” of a substance / molecule / drug of this disclosure (e.g., IL13Rα2 antibody) may vary depending on factors such as the individual’s condition, age, sex, and weight, and the ability of the substance / molecule / drug to induce a desired response in the individual. A therapeutic dose includes an amount in which the therapeutically beneficial effects of the substance / molecule / drug outweigh any toxic or adverse effects. In certain embodiments, the term “therapeutic dose” refers to the amount of drug that is effective in “treating” a disease, disorder, or condition in a subject or mammal.

[0087] The term “to treat,” or any grammatical variation thereof, means reducing and / or improving the severity and / or duration of a given disease, disorder or condition and / or symptoms associated therewith, for example, (i) reducing, delaying or improving the advancement or progression of a given disease, disorder or condition; (ii) reducing, delaying or improving the recurrence, progression or onset of a given disease, disorder or condition; and / or (iii) improving or enhancing the preventive or therapeutic effect of another therapy (e.g., therapy other than administering the drugs described herein).

[0088] The "preventive effective dose" is the amount of a pharmaceutical composition that, when administered to a target, produces the intended preventive effect, such as preventing or delaying the onset (or recurrence) of a disease, disorder, or condition, or reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or related symptoms(s).

[0089] Complete therapeutic or preventive effects may not necessarily occur with a single dose, and may only occur after a series of doses. Therefore, the therapeutic or preventive dose may be administered in more than one dose.

[0090] The terms "approximately" and "about" refer to variations of 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% or less of a given value or range.

[0091] As used herein, comparative terms, such as reduction, decrease, increase, or any of these grammatical variations, can refer to a particular variation from a reference value. In some embodiments, such variation can refer to a value that is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than the reference value, or about 1x, 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, or 100x higher. In some embodiments, such variation can refer to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the reference value.

[0092] As used in this disclosure and claims, the singular forms with “a,” “an,” and “the” include the plural form unless the context clearly indicates otherwise.

[0093] In some embodiments, the terms “first,” “second,” “third,” “fourth,” and synonyms in the names of components are used to distinguish and identify one or more components that share a particular identity in those names. For example, “first antibody” and “second antibody” are used to distinguish two antibodies.

[0094] Wherever embodiments are described herein using the phrase "including," it is understood that other similar embodiments are also provided, described using the phrases "consisting of" and / or "essentially consisting of." Wherever embodiments are described herein using the phrase "essentially consisting of," it is understood that other similar embodiments are also provided, described using the phrase "consisting of."

[0095] The term "between" or "between" refers to a range that includes both A and B, as used in phrases such as "between A and B" or "between A and B."

[0096] When the term "and / or" is used in this specification in phrases such as "A and / or B," it is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, when the term "and / or" is used in phrases such as "A, B and / or C," it is intended to include each of the embodiments of A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0097] The terms "optional" or "at will" mean that the situation described below may or may not occur, so this description includes both cases where the situation occurs and cases where it does not.

[0098] 5.2. IL13Rα2 binder In some embodiments, the present disclosure provides IL13Rα2 conjugates that can be used herein as therapeutic agents for the treatment of cancer. Examples of cancers, but not limited to, include melanoma, lung cancer (including non-small cell lung cancer, NSCLC), colorectal cancer, malignant glioma, pancreatic cancer, ovarian cancer, breast cancer, liver cancer, head and neck cancer, and renal cancer. Such agents include antibodies that bind to IL13Rα2 (or IL13Rα2:IL13 complex) (e.g., monospecific antibodies or polyspecific antibodies (including bispecific antibodies)). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, bispecific antibodies, and heteroconjugate antibodies, as well as variants thereof with improved or reduced affinity or other properties.

[0099] In some embodiments, IL13Rα2 conjugates (e.g., antibodies) that bind to IL13Rα2 are described herein, comprising an IL13Rα2 polypeptide, an IL13Rα2 polypeptide fragment, an IL13Rα2 peptide, an IL13Rα2:IL13 complex, or an IL13Rα2 epitope. In some embodiments, the IL13Rα2 conjugate is a human antibody or humanized antibody (e.g., comprising a human constant region) that binds to IL13Rα2, comprising an IL13Rα2 polypeptide, an IL13Rα2 polypeptide fragment, an IL13Rα2 peptide, or an IL13Rα2 epitope. In some embodiments, the IL13Rα2 conjugate (e.g., antibody), e.g., a human IL13Rα2 conjugate, can bind to IL13Rα2 expressed on the surface of mammalian (e.g., human) cells (including cancer cells expressing IL13Rα2). In some embodiments, an IL13Rα2 conjugate (e.g., an antibody), such as a human IL13Rα2 conjugate, can bind to IL13Rα2 expressed on the surface of mammalian (e.g., human) cells (including cancer cells that overexpress IL13Rα2). In some embodiments, the IL13Rα2 conjugate (e.g., an antibody) binds to an extracellular epitope of IL13Rα2 exposed on a cell, such as a cancer cell. In some embodiments, the IL13Rα2 conjugate (e.g., an antibody) binds to a complex comprising IL13Rα2 and IL13. In some embodiments, the IL13Rα2 conjugate (e.g., an antibody) binds to a complex comprising the extracellular domains (ECDs) of IL13Rα2 and IL13. In even further embodiments, the IL13Rα2 conjugate (e.g., an antibody) binds to an epitope of a complex comprising the ECDs of IL13Rα2 and IL13. In some embodiments, IL13Rα2 conjugates (e.g., antibodies) that bind to IL13Rα2, such as human IL13Rα2 or a portion thereof, are described herein. In some embodiments, IL13Rα2 is human IL13Rα2. In some embodiments, the IL13Rα2 conjugate is a human IL13Rα2 conjugate (e.g., an antibody that binds to human IL13Rα2). In some embodiments, the IL13Rα2 conjugate (e.g., an antibody) binds to both human IL13Rα2 and cynoIL13Rα2.In other embodiments, the IL13Rα2 conjugate (e.g., an antibody) binds to human IL13Rα2 but not to cynoIL13Rα2.

[0100] In some embodiments, the IL13Rα2 binding agent (e.g., antibody) provided herein has a dissociation constant (K) for IL13Rα2 (e.g., human IL13Rα2 and / or cynoIL13Rα2). D ) 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It binds at M). Various methods for measuring binding affinity are known in the art and any of them can be used for the purposes of this disclosure, such as RIA using the Fab version of the antibody in question and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81), by Octet® using, for example, the Octet® Red96 system, or by Biacore® biolayer interferometry (BLI) or surface plasmon resonance (SPR) assay using, for example, Biacore® TM-2000 or Biacore® TM-3000. The "association rate" or "kon" may be determined using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) method as described above, for example, with the Octet® Red96, Biacore® TM-2000, Biacore® TM-3000 system, Biacore® TM-8K, or Biacore® TM-8K+ system.

[0101] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided herein do not bind to IL13Rα1 (e.g., human IL13Rα1 and / or cynoIL13Rα1). In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided herein do not bind to human IL13Rα1. In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided herein do not bind to human IL13Rα1 or cynoIL13Rα1. In other embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided herein bind to IL13Rα2 (e.g., human IL13Rα2) with higher affinity than to IL13Rα1 (e.g., human IL13Rα1). In some embodiments, the binding affinity of the IL13Rα2 conjugate (e.g., antibody) provided herein to IL13Rα2 (e.g., human IL13Rα2) is at least twice as high as the binding affinity to IL13Rα1 (e.g., human IL13Rα1). In some embodiments, the binding affinity of the IL13Rα2 conjugate (e.g., antibody) provided herein to IL13Rα2 (e.g., human IL13Rα2) is at least five times as high as the binding affinity to IL13Rα1 (e.g., human IL13Rα1). In some embodiments, the binding affinity of the IL13Rα2 conjugate (e.g., antibody) provided herein to IL13Rα2 (e.g., human IL13Rα2) is at least ten times as high as the binding affinity to IL13Rα1 (e.g., human IL13Rα1). In some embodiments, the binding affinity of the IL13Rα2 conjugate (e.g., antibody) provided herein to IL13Rα2 (e.g., human IL13Rα2) is at least 100 times greater than the binding affinity to IL13Rα1 (e.g., human IL13Rα1). In some embodiments, the binding affinity of the IL13Rα2 conjugate (e.g., antibody) provided herein to IL13Rα2 (e.g., human IL13Rα2) is at least 1000 times greater than the binding affinity to IL13Rα1 (e.g., human IL13Rα1).

[0102] Furthermore, or alternatively, the IL13Rα2 conjugates provided herein (e.g., antibodies) do not inhibit the binding between IL13 and IL13Rα2 (e.g., human IL13Rα2 and / or cynoIL13Rα2).

[0103] In some embodiments, the IL13Rα2 conjugate (e.g., antibody) described herein includes the amino acid sequence of any one of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of any one of the antibodies described herein, for example, the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3, which is shown in Tables 1-3. Thus, in some embodiments, the IL13Rα2 conjugate (e.g., antibody) described herein includes any one, any two and / or all three heavy chain CDRs derived from any one of the antibodies referred to as (a) A22, (b) A33 and (c) A52, as shown in Tables 1-3, and / or any one, any two and / or all three light chain CDRs. In some embodiments, the IL13Rα2 binders (e.g., antibodies) described herein include, as shown in Tables 1-3, one, two, and / or all three heavy chain CDRs derived from (a) the antibody designated A22, (b) the antibody designated A33, and (c) the antibody designated A52, as well as one, two, and / or all three light chain CDRs.

[0104] In some embodiments, the IL13Rα2 conjugate (e.g., antibody) comprises one of the VH regions (including VH CDR1, VH CDR2 and / or VH CDR3) and / or VL regions (including VL CDR1, VL CDR2 and / or VL CDR3) from the conjugates described herein (see, for example, any one of Tables 1-3). Thus, in some embodiments, the IL13Rα2 conjugate (e.g., antibody) described herein comprises one, any two and / or all three of the heavy chain CDRs in Table 1, and / or one, any two and / or all three of the light chain CDRs. In some embodiments, the IL13Rα2 conjugate (e.g., antibody) described herein comprises one, any two and / or all three of the heavy chain CDRs in Table 2, and / or one, any two and / or all three of the light chain CDRs. In some embodiments, the IL13Rα2 binders (e.g., antibodies) described herein include one, two, and / or all three of the heavy chain CDRs in Table 3, and / or one, two, and / or all three of the light chain CDRs.

[0105] In some embodiments, the IL13Rα2 binders (e.g., antibodies) provided in this disclosure include (i) VH CDR1, VH CDR2 and VH CDR3 as shown in VH, which includes the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 48 or SEQ ID NO: 73, and / or (ii) VL CDR1, VL CDR2 and VL CDR3 as shown in VL, which includes the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 49 or SEQ ID NO: 74.

[0106] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure include VH CDR1, VH CDR2 and / or VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 25, and / or VL CDR1, VL CDR2 and / or VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 26. The CDR sequences can be determined by a well-known numbering system or a combination thereof. In some embodiments, the CDRs are numbered using IMGT numbering. In some embodiments, the CDRs are numbered using Kabat numbering. In some embodiments, the CDRs are numbered using AbM numbering. In other embodiments, the CDR is numbered by Chothia numbering. In other embodiments, the CDR is numbered by Contact numbering. In some embodiments, the CDR sequence is determined by any combination of two or more of the numbering systems described above, for example, a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and illustrated in Chapter 5.1 above.

[0107] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including (a) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 5, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, and 10, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14, and / or (b) a VL region including a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 24.

[0108] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 6, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 22.

[0109] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 2, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 7, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 12, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 22.

[0110] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 3, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 6, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 22.

[0111] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 13, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 17, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 23.

[0112] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 5, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 9, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 14, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 18, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 21, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 24.

[0113] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 10, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 22.

[0114] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure include VH CDR1, VH CDR2 and / or VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 48, and / or VL CDR1, VL CDR2 and / or VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 49. The CDR sequences can be determined by a well-known numbering system or a combination thereof. In some embodiments, the CDRs are numbered by IMGT numbering. In some embodiments, the CDRs are numbered by Kabat numbering. In some embodiments, the CDRs are numbered by AbM numbering. In other embodiments, the CDR is determined by Chothia numbering. In other embodiments, the CDR is determined by Contact numbering. In some embodiments, the CDR sequence is determined by any combination of two or more of the numbering systems described above, for example, a combination of Kabat and Chothia.

[0115] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including (a) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, 30, and 31, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, and 36, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40, and / or (b) a VL region including a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, and 44, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 46, and 47.

[0116] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 45.

[0117] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 28, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 33, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 38, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 42, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 45.

[0118] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 29, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 45.

[0119] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 30, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 39, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 43, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 46.

[0120] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 35, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 44, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 21, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 47.

[0121] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 45.

[0122] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure include VH CDR1, VH CDR2 and / or VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO: 73, and / or VL CDR1, VL CDR2 and / or VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO: 74. The CDR sequences can be determined by a well-known numbering system or a combination thereof. In some embodiments, the CDRs are numbered by IMGT numbering. In some embodiments, the CDRs are numbered by Kabat numbering. In some embodiments, the CDRs are numbered by AbM numbering. In other embodiments, the CDR is determined by Chothia numbering. In other embodiments, the CDR is determined by Contact numbering. In some embodiments, the CDR sequence is determined by any combination of two or more of the numbering systems described above, for example, a combination of Kabat and Chothia.

[0123] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including (a) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, and 54, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 56, 57, 58, and 59, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 61, 62, and 63, and / or (b) a VL region including a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66, and 67, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 20, and 69, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 71, and 72.

[0124] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 50, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 55, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 70.

[0125] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 51, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 56, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 61; and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 70.

[0126] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 52, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 55, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 70.

[0127] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 53, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 57, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 62, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 66, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 71.

[0128] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 54, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 58, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 63, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 69, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 72.

[0129] In some embodiments, the IL13Rα2 binder (e.g., an antibody) provided in this disclosure comprises a VH region including a VH CDR1 containing the amino acid sequence of SEQ ID NO: 50, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 59, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region including a VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 70.

[0130] In some embodiments, the antibody further comprises one or more framework regions of SEQ ID NOs: 25, 26, 48, 49, 73 and / or 74. In some embodiments, the antibody or a fragment thereof further comprises the sequences of framework 1 (FR1), framework 2 (FR2), framework 3 (FR3) and / or framework 4 (FR4), as shown in any one of SEQ ID NOs: 25, 26, 48, 49, 73 and 74. In some embodiments, the antibody provided in this disclosure is a humanized antibody. The framework regions described herein are determined based on the boundaries of a CDR numbering system. In other words, when a CDR is determined by, for example, Kabat, IMGT or Chothia, the framework regions are the amino acid residues around the CDR in the variable region in the form FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. For example, FR1 is defined as the N-terminal amino acid residue relative to the amino acid residue of CDR1, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residue between the amino acid residues of CDR1 and CDR2, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residue between the amino acid residues of CDR2 and CDR3, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the C-terminal amino acid residue relative to the amino acid residue of CDR3, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0131] In some embodiments, the IL13Rα2 conjugate (e.g., an antibody such as a monospecific or bispecific antibody) (including the human IL13Rα2 conjugates described herein) includes a VH region or VH domain. In addition to or instead of this, in some embodiments, the IL13Rα2 conjugate (e.g., an antibody such as a monospecific or bispecific antibody) (including the human IL13Rα2 conjugates described herein) includes a VL region or VL domain. In some embodiments, the IL13Rα2 conjugate (e.g., an antibody such as a monospecific or bispecific antibody) (including the human IL13Rα2 conjugates described herein) has a combination of (i) a VH domain or VH region and (ii) a VL domain or VL region.

[0132] In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises VH containing the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises VL containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises VH containing the amino acid sequence of SEQ ID NO: 25 and VL containing the amino acid sequence of SEQ ID NO: 26.

[0133] In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises VH containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises VL containing the amino acid sequence of SEQ ID NO: 49. In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises VH containing the amino acid sequence of SEQ ID NO: 48 and VL containing the amino acid sequence of SEQ ID NO: 49.

[0134] In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises a VH containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises a VL containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, the IL13Rα2 conjugate (e.g., antibody) provided in this disclosure comprises a VH containing the amino acid sequence of SEQ ID NO: 73 and a VL containing the amino acid sequence of SEQ ID NO: 74.

[0135] In certain embodiments, the IL13Rα2 conjugates provided in this disclosure (e.g., antibodies or fragments thereof) include an amino acid sequence that is identical in a specific percentage (e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more) to any of the antibodies or fragments provided in this disclosure, e.g., the CDR, VH, or VL of Tables 1-3, or any full-length antibody chain as disclosed herein. In further embodiments, the IL13Rα2 conjugates provided in this disclosure include an amino acid sequence that is identical to any antibody or fragment thereof provided in this disclosure, for example, VH or VL in Tables 1-3, or any full-length antibody chain as disclosed herein, in a specific percentage (e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more).

[0136] Determining the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using mathematical algorithms. A non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), which has been modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). By performing a BLAST nucleotide search with the NBLAST nucleotide program parameter set, e.g., score=100, word length=12, nucleotide sequences homologous to the nucleic acid molecules described herein can be obtained. Protein searches using BLAST can be performed with the XBLAST program parameter set, for example, score 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). In some embodiments, the percentage of identity between two sequences is calculated by dividing the number of residues (possibly multiple) that are different between the two sequences in the alignment (excluding or including conserved amino acid substitutions (possibly multiple) or degenerate nucleotide substitutions (possibly multiple)) by the number of residues in any one of the following: (i) the full length of the shorter sequence, (ii) the full length of the longer sequence, (iii) the average length of the two sequences, (iv) the total length of the gapless portion of the alignment, (v) the length of the alignment (excluding overhangs), or (vi) the length of the alignment (including overhangs).As used herein, an overhang refers to a sequence alignment where, with respect to the alignment, one or both ends of the alignment contain residues that do not align with the residues of the other sequence (e.g., a gap). Alternatively, iterative searches can be performed using PSI BLAST to detect segregated relationships between molecules (see the same reference). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4 can be used. The percentage of identity between two sequences can be determined by whether or not gaps are allowed, using techniques similar to those described above. When calculating the percentage of identity, typically only exact matches are counted.

[0137] In some embodiments, the binders provided in this disclosure (e.g., antibodies) include substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the binders include sequences that retain the ability to bind to IL13Rα2. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions are made in regions outside the CDR (e.g., the FR and / or constant regions).

[0138] In some embodiments, as long as binding to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%), the positions of one or more CDRs (e.g., CDR1, CDR2, or CDR3) along the VH region and / or one or more CDRs (e.g., CDR1, CDR2, or CDR3) along the VL region of the IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) described herein may vary by only 1, 2, 3, 4, 5, or 6 amino acids. For example, in some embodiments, as long as binding to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%), the position defining any of the CDRs in Tables 1, 2, or 3 may be varied by shifting the N-terminal and / or C-terminal boundaries of the CDR by 1, 2, 3, 4, 5, or 6 amino acids relative to the current CDR position. In addition to or instead of this, in some embodiments, as long as binding to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%), the lengths of one or more CDRs along the VH region (e.g., CDR1, CDR2, or CDR3) and / or one or more CDRs along the VL region (e.g., CDR1, CDR2, or CDR3) of the IL13Rα2 conjugates (e.g., antibodies) described herein (including human IL13Rα2 conjugates) can vary by 1, 2, 3, 4, 5 or more amino acids (e.g., they can be shorter or longer).For example, in some embodiments, as long as binding to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%), the VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described by SEQ ID NOs: 1-24, 27-47, or 50-72. In other embodiments, as long as binding to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%), the VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described by SEQ ID NOs: 1-24, 27-47, or 50-72. In some embodiments, as long as binding to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%), the amino-terminuses of CDR1, CDR2, and / or CDR3 of VH and / or VL described herein may be elongated or shortened by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-24, 27-47, or 50-72.In addition to or instead of this, in some embodiments, the carboxyl termini of CDR1, CDR2, and / or CDR3 of VH and / or VL described herein may be elongated or shortened by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-24, 27-47, or 50-72, as long as binding to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Whether binding to IL13Rα2 (e.g., human IL13Rα2) is maintained can be confirmed using any method known in the art, for example, the binding assays and binding conditions described in the "Examples" chapter described herein.

[0139] In other embodiments, the IL13Rα2 binders (e.g., antibodies) provided in this disclosure (including human IL13Rα2 binders), which bind to IL13Rα2, further include conservative sequence modifications. With respect to polypeptides that are IL13Rα2 binders (e.g., antibodies), such as human IL13Rα2 binders, the conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. Thus, in some embodiments, a putative non-essential amino acid residue in IL13Rα2 is replaced by another amino acid residue derived from the same side chain family. Methods for identifying conserved amino acid substitutions that do not exclude antigen binding and the nucleotides encoding those amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al. Protein Eng. 12(10):879-884 (1999), and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the conserved sequence modifications described herein modify the amino acid sequence of its IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, amino acid sequence modification refers to up to one, two, three, four, five, or six amino acid substitutions in a CDR, for example, a CDR listed in any one of Tables 1 to 3. Thus, for example, each such CDR may contain up to five conservative amino acid substitutions, for example, up to four (or less) conservative amino acid substitutions, for example, up to three (or less) conservative amino acid substitutions, for example, up to two (or less) conservative amino acid substitutions, or one or fewer conservative amino acid substitutions.In some embodiments, the IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) comprises one or more CDRs (including six) whose identity to A22, A33, or A52 CDRs (see, for example, Tables 1, 2, or 3) is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0140] In some embodiments, the IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) comprises VH and VL containing the same CDR as CDR A22, A33, or A52 (see, for example, Tables 1, 2, or 3). In some embodiments, the amino acid sequence modification does not include any modification within the SDR. In some embodiments, the amino acid sequence modification does not include any modification within the CDR (e.g., any of CDR1, CDR2, CDR3, or any combination thereof). In addition to or instead of this, the amino acid sequence modification is located in the framework, constant region, and / or crystallizable fragment region (Fc).

[0141] In some embodiments, the antibody or fragment provided in this disclosure comprises a VH domain having at least 75%, at least 80%, at least 85%, 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 the amino acid sequence of SEQ ID NO: 25, and / or a VL domain having at least 75%, at least 80%, at least 85%, 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 the amino acid sequence of SEQ ID NO: 26, and the binding of the antibody or fragment to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0142] In some embodiments, the antibody or fragment provided in this disclosure comprises a VH domain having at least 75%, at least 80%, at least 85%, 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 the amino acid sequence of SEQ ID NO: 48, and / or a VL domain having at least 75%, at least 80%, at least 85%, 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 the amino acid sequence of SEQ ID NO: 49, and the binding of the antibody or fragment to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0143] In some embodiments, the antibody or fragment provided in this disclosure comprises a VH domain having at least 75%, at least 80%, at least 85%, 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 the amino acid sequence of SEQ ID NO: 73, and / or a VL domain having at least 75%, at least 80%, at least 85%, 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 the amino acid sequence of SEQ ID NO: 74, and the binding of the antibody or fragment to IL13Rα2 (e.g., human IL13Rα2) is maintained (e.g., substantially maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0144] In some embodiments, functional epitopes can be mapped, for example, by combinatorial alanine scanning to identify amino acids in the IL13Rα2 protein necessary for interaction with the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure. In some embodiments, the epitope can be identified using the three-dimensional and crystalline structures of the IL13Rα2 conjugates (such as antibodies) that bind to IL13Rα2. In some embodiments, this disclosure provides antibodies that specifically bind to the same epitopes as any of the IL13Rα2 conjugates (e.g., antibodies or fragments thereof) provided in this disclosure.

[0145] For example, in some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure bind to the same epitopes as an anti-IL13Rα2 antibody, which includes VH CDR1, VH CDR2, and VH CDR3, as shown in VH containing the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3, as shown in VL containing the amino acid sequence of SEQ ID NO: 26.

[0146] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure bind to the same epitopes as anti-IL13Rα2 antibodies, including VH CDR1, VH CDR2, and VH CDR3, as shown in VH containing the amino acid sequence of SEQ ID NO: 48, and VL CDR1, VL CDR2, and VL CDR3, as shown in VL containing the amino acid sequence of SEQ ID NO: 49.

[0147] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure bind to the same epitopes as anti-IL13Rα2 antibodies, including VH CDR1, VH CDR2, and VH CDR3, as shown in VH containing the amino acid sequence of SEQ ID NO: 73, and VL CDR1, VL CDR2, and VL CDR3, as shown in VL containing the amino acid sequence of SEQ ID NO: 74.

[0148] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided herein specifically bind to IL13Rα2 in a competitive manner with any one of the anti-IL13Rα2 antibodies or fragments thereof described herein.

[0149] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure specifically bind to IL13Rα2 in a competitive manner with anti-IL13Rα2 antibodies comprising VH CDR1, VH CDR2, and VH CDR3, as shown in VH comprising the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3, as shown in VL comprising the amino acid sequence of SEQ ID NO: 26.

[0150] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure specifically bind to IL13Rα2 in a competitive manner with anti-IL13Rα2 antibodies comprising VH CDR1, VH CDR2, and VH CDR3, as shown in VH containing the amino acid sequence of SEQ ID NO: 48, and VL CDR1, VL CDR2, and VL CDR3, as shown in VL containing the amino acid sequence of SEQ ID NO: 49.

[0151] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) provided in this disclosure specifically bind to IL13Rα2 in a competitive manner with anti-IL13Rα2 antibodies comprising VH CDR1, VH CDR2, and VH CDR3, as shown in VH comprising the amino acid sequence of SEQ ID NO: 73, and VL CDR1, VL CDR2, and VL CDR3, as shown in VL comprising the amino acid sequence of SEQ ID NO: 74.

[0152] In some embodiments, the IL13Rα2 conjugate contains six CDRs of the antibody A22. In even further embodiments, the IL13Rα2 conjugate contains six CDRs listed in one column of Table 1. In some embodiments, the IL13Rα2 conjugate contains three heavy chain variable region CDRs as shown in SEQ ID NO: 25 and three light chain variable region CDRs as shown in SEQ ID NO: 26. In some embodiments, the IL13Rα2 conjugate contains a heavy chain variable region as shown in SEQ ID NO: 25 and a light chain variable region as shown in SEQ ID NO: 26.

[0153] In some embodiments, the IL13Rα2 conjugate contains six CDRs of the antibody A33. In even further embodiments, the IL13Rα2 conjugate contains six CDRs listed in one column of Table 2. In some embodiments, the IL13Rα2 conjugate contains three CDRs of the heavy chain variable region as shown in SEQ ID NO: 48 and three CDRs of the light chain variable region as shown in SEQ ID NO: 49. In some embodiments, the IL13Rα2 conjugate contains a heavy chain variable region as shown in SEQ ID NO: 48 and a light chain variable region as shown in SEQ ID NO: 49.

[0154] In some embodiments, the IL13Rα2 conjugate contains six CDRs of the antibody A52. In even further embodiments, the IL13Rα2 conjugate contains six CDRs listed in one column of Table 3. In some embodiments, the IL13Rα2 conjugate contains three heavy chain variable region CDRs as shown in SEQ ID NO: 73 and three light chain variable region CDRs as shown in SEQ ID NO: 74. In some embodiments, the IL13Rα2 conjugate contains a heavy chain variable region as shown in SEQ ID NO: 73 and a light chain variable region as shown in SEQ ID NO: 74.

[0155] In some embodiments, the conjugated antibody has good development potential based on assays known in the art, such as various chromatographic methods (including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and stand-up monolayer adsorption chromatography (SMAC)). In some embodiments, the conjugate has good development potential based on measurements of monomer percentage, solubility, and / or antibody aggregation or precipitation.

[0156] In some embodiments, the IL13Rα2 conjugates described herein (e.g., antibodies such as monospecific or bispecific antibodies) include human IL13Rα2 conjugates and comprise a heavy chain having a combination of (i) a VH described herein, e.g., in any one of Tables 1-3, and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3). An exemplary IgG heavy chain comprises any VH sequence described herein and the following CH1, hinge, CH2, and CH3 amino acid sequences: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 77)

[0157] In further embodiments, the carboxyl terminus (C-terminus) of VH is directly or indirectly conjugated to the amino terminus (N-terminus) of one or more heavy chain constant domains.

[0158] In some embodiments, the IL13Rα2 conjugates described herein (e.g., antibodies such as monospecific or bispecific antibodies) include human IL13Rα2 conjugates and comprise a light chain having a combination of (i) a VL described herein, e.g., any one of Tables 1-3, and (ii) a light chain constant domain (CL). An exemplary light chain (e.g., a light chain paired with an IgG heavy chain) comprises any VL sequence described herein and the following CL amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 78)

[0159] In further embodiments, the C-terminus of the VL is directly or indirectly conjugated to the N-terminus of the CL.

[0160] In some embodiments, the IL13Rα2 conjugates described herein (e.g., antibodies such as monospecific or bispecific antibodies) include human IL13Rα2 conjugates and comprise (a) a heavy chain having a combination of (i) a VH as described herein, e.g., in any one of Tables 1 to 3, and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3); and (b) a light chain having a combination of (i) a VL as described herein, e.g., in any one of Tables 1 to 3, and (ii) a light chain constant domain (CL or CL1) in IgG form. An exemplary IL13Rα2 conjugate (e.g., antibody) comprises an IgG heavy chain having any VH sequence as described herein and the amino acid sequence of SEQ ID NO: 77, and a light chain having any VL sequence as described herein and the amino acid sequence of SEQ ID NO: 78.

[0161] In some embodiments, an IL13Rα2 binding agent (e.g., an antibody such as a monospecific antibody or a bispecific antibody) comprising a human IL13Rα2 binding agent described herein comprises (a) a heavy chain having the amino acid sequence of SEQ ID NO: 75, and (b) a light chain having the amino acid sequence of SEQ ID NO: 76. In some embodiments, an IL13Rα2 binding agent (e.g., an antibody such as a monospecific antibody or a bispecific antibody) comprising a human IL13Rα2 binding agent described herein comprises (a) a heavy chain having the amino acid sequence of SEQ ID NO: 79, and (b) a light chain having the amino acid sequence of SEQ ID NO: 80. In some embodiments, an IL13Rα2 binding agent (e.g., an antibody such as a monospecific antibody or a bispecific antibody) comprising a human IL13Rα2 binding agent described herein comprises (a) a heavy chain having the amino acid sequence of SEQ ID NO: 81, and (b) a light chain having the amino acid sequence of SEQ ID NO: 82.

[0162] In some embodiments, provided by the present disclosure is an IL13Rα2 binding protein comprising any one of the anti-IL13Rα2 antibodies described herein. In some embodiments, the IL13Rα2 binding protein is an antibody comprising two heavy chains and two light chains. In some embodiments, the IL13Rα2 binding protein is an antibody comprising two heavy chains comprising the same VH region and two light chains comprising the same VL region.

[0163] In some embodiments, the IL13Rα2 binding protein is a monoclonal antibody comprising a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody. In some embodiments, the anti-IL13Rα2 antibody is an antibody fragment, e.g., scFv. In some embodiments, the IL13Rα2 binding protein is a fusion protein comprising an anti-IL13Rα2 antibody provided by the present disclosure. In other embodiments, the IL13Rα2 binding protein is a multispecific antibody comprising an anti-IL13Rα2 antibody provided by the present disclosure or a fragment thereof. Other exemplary IL13Rα2 binding molecules are described in more detail in the following chapters. In some embodiments, the anti-IL13Rα2 antibodies or antigen-binding proteins according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in Sections 5.2.1 to 5.2.4 below. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2]

[0164] 5.2.1. Antibody Fragments Even though the term "antibody" may be used herein in the phrase "antibody or fragment thereof", as used herein, the term "antibody" should be understood to include various antibody fragments, such as antigen-binding fragments or epitope-binding fragments. Thus, when the term "antibody" is used alone without the continuation of the term "fragment thereof" or a similar term, the term "antibody" should be understood to include antibody fragments, such as antigen-binding fragments or epitope-binding fragments. Antibodies provided by the present disclosure include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.

[0165] Antibody variants and derivatives include functional antibody fragments that retain the ability to bind to an antigen. Examples of antibody fragments include, but are not limited to, those described in Chapter 5.1 above. Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen-binding domain and in which parts of the light and heavy chains are crosslinked by disulfide bonds), Fab' (e.g., antibody fragments containing a single antigen-binding domain including Fab and an additional portion of the heavy chain up to the hinge region), F(ab')2 (e.g., two Fab' molecules linked by interchain disulfide bonds at the hinge region of the heavy chain, which may be for the same or different epitopes), and bispecific Fa b (e.g., a Fab molecule having two antigen-binding domains, each potentially for a different epitope), a single-chain containing a variable region (also known as scFv) (e.g., a single light chain and a single heavy chain of an antibody with variable antigen-binding determination regions linked together by a chain of, for example, 10-25 amino acids), a disulfide-bonded Fv, i.e., dsFv (e.g., a single light chain and a single heavy chain of an antibody with variable antigen-binding determination regions linked together by a disulfide bond), and a bispecific scFv. (For example, an scFv or dsFv molecule having two antigen-binding domains (each of which may be for different epitopes), a diabody (for example, a dimerized scFV formed when the VH domain of a first scFv is assembled with the VL domain of a second scFv, and the VL domain of the first scFv is assembled with the VH domain of a second scFv, where the two antigen-binding regions of the diabody may be for the same or different epitopes), a triabody This includes (for example, a diabody formed in a similar manner to a diabody, but whose three antigen-binding domains are trimerized scFvs made from a single complex, and whose three antigen-binding domains may be directed to the same or different epitopes), as well as a tetrabody (for example, a diabody formed in a similar manner to a diabody, but whose four antigen-binding domains are tetramerized scFvs made from a single complex, and whose four antigen-binding domains may be directed to the same or different epitopes).

[0166] Various techniques have been developed for the generation of antibody fragments. Traditionally, these fragments were obtained via the proteolysis of intact antibodies (see, for example, Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17 and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be generated directly by recombinant host cells. For example, since Fab, Fv, and scFv antibody fragments can all be expressed and secreted from E. coli, yeast, or insect cells, large quantities of these fragments can be easily generated. Antibody fragments can be isolated from the antibody phage library mentioned above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). Alternatively, the F(ab')2 fragment can be isolated directly from recombinant host cell culture medium according to another approach. Fab and F(ab')2 fragments with extended in vivo half-lives, including salvage receptor-binding epitope residues, are described, for example, in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-stranded Fv fragment (scFv) (see, for example, WO93 / 16185, U.S. Patents No. 5,571,894 and 5,587,458). Fv and scFv are combined intact sites with a deletion of the constant region, which may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to fuse effector proteins at either the amino or carboxyl terminus of scFv (see, for example, the above literature edited by Borrebaeck). The antibody fragment may be, for example, a "linear antibody," as described in the reference cited above. Such a linear antibody may be monospecific or polyspecific, for example, bispecific.

[0167] 5.2.2. Humanized Antibodies This disclosure provides humanized antibodies that bind to IL13Rα2 (including human IL13Rα2). The humanized antibodies of this disclosure may contain one or more CDRs derived from VH and / or VL disclosed herein, for example, CDRs shown in Tables 1-3. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced into the antibody from a non-human source. These non-human amino acid residues are often called “import” residues, and these residues are typically obtained from “import” variable domains. Humanized antibodies that bind to IL13Rα2 can be produced using techniques known to those skilled in the art (Zhang et al., Molecular Immunology, 42(12):1445-1451, 2005; Hwang et al., Methods, 36(1):35-42, 2005; Dall'Acqua et al., Methods, 36(1):43-60, 2005; Clark, Immunology Today, 21(8):397-402, 2000; and U.S. Patents No. 6,180,370, No. 6,054,927, No. 5,869,619, No. 5,861,155, No. 5,712,120 and No. 4,816,567).

[0168] In some cases, the humanized antibody is constructed by CDR transplantation, in which the six amino acid sequences of the VH and VL CDRs of the parent's non-human antibody (e.g., rodents) are transplanted into the human antibody framework. For example, Padlan et al. (FASEB J.9:133-139, 1995) determined that only about one-third of the residues in the CDR actually come into contact with the antigen, and referred to these residues as "specificity-determining residues" or "SDRs." In the SDR transplantation method, only the residues of the SDR are transplanted into the human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34, 2005).

[0169] The selection of human variable domains (both light and heavy chains) used to construct humanized antibodies can be crucial for reducing antigenicity. For example, the sequences of variable domains of non-human (e.g., rodent) antibodies can be screened against an entire library of known human variable domain sequences using a so-called "best-fit" method. The human sequence most closely resembling that rodent sequence can be selected as the human framework for the humanized antibody (Sims et al. (1993) J.Immunol.151:2296, Chothia et al. (1987) J.Mol.Biol.196:901). Alternatively, a specific framework derived from the consensus sequences of all human antibodies of a particular subgroup of light or heavy chains can be used. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, Presta et al. (1993) J. Immunol., 151:2623). In some cases, the framework is the most abundant human subclass, V. L 6 Subgroup I(V L 6I) and V H Subgroup III (V H It originates from the consensus sequence of (III). Alternatively, human germline genes are used as a source for the framework region.

[0170] In an alternative paradigm based on CDR comparison (called Superhumanization), framework homology is not important. The method involves comparing non-human sequences with a functional human germline gene repertoire. Next, genes encoding canonical structures identical to or closely related to mouse sequences are selected from among these genes. Then, among the genes that share this canonical structure with non-human antibodies, the gene with the highest homology in the CDR is selected as the framework donor. Finally, non-human CDRs are transplanted into these frameworks (see, for example, Tan et al., J.Immunol. 169:1119-1125, 2002).

[0171] Furthermore, it is generally desirable to humanize antibodies while maintaining their affinity for the antigen and other desirable biological properties. To achieve this goal, humanized antibodies are prepared by a process that analyzes the parent sequence and various conceptual humanization products using three-dimensional models of the parent and humanized sequences according to one method. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs that illustrate and display the putative three-dimensional structure of selected immunoglobulin sequence candidates are available. Examples of such programs include WAM (Whitelegg and Rees, Protein Eng. 13:819-824, 2000), Modeller (Sali and Blundell, J.Mol.Biol. 234:779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-2713, 1997). Verification of these indications allows for the analysis of the potential role of residues in the function of candidate immunoglobulin sequences, for example, the analysis of residues that influence the candidate immunoglobulin's ability to bind to its antigen. Thus, framework residues can be selected or combined from recipient and import sequences to achieve desired antibody properties, such as improved affinity for target antigens. Generally, hypervariable region residues are directly and substantially involved in the influence of antigen binding.

[0172] Another method for humanizing antibodies is based on a criterion called Human String Content (HSC). This method compares mouse sequences to a repertoire of human germline genes and scores the differences as HSC. Then, instead of using a comprehensive identity scale, the target sequence is humanized by maximizing its HSC, generating multiple diverse humanized variants. See, for example, Lazar et al., Mol.Immunol. 44:1986-1998, 2007.

[0173] In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include those based on the creation of large libraries of humanized variants and the selection of the best clones using enrichment techniques or high-throughput screening techniques. Antibody variants may be isolated from phage display libraries, ribosome display libraries, and yeast display libraries, as well as by screening bacterial colonies (see, for example, Hoogenboom, Nat. Biotechnol. 23:1105-1116, 2005; Dufner et al., Trends Biotechnol. 24:523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21:163-70, 2003; and Schlapschy et al., Protein Eng. Des. Sel. 17:847-60, 2004).

[0174] In the framework library approach, a set of residue variants is introduced into a designated position within the framework, and then the library is selected to choose the framework that best corresponds to the transplanted CDR. Substitution residues may include some or all of the "Vernier" residues identified as potentially contributing to the CDR structure (see, for example, Foote and Winter, J.Mol.Biol.224:487-499, 1992), or those derived from a more limited set of target residues identified by Baca et al. (J.Biol.Chem.272:10678-10684, 1997).

[0175] In framework shuffling, instead of creating a combinatorial library of selected residue variants, the entire framework is combined with non-human CDRs (see, for example, Dall’Acqua et al., Methods 36:43-60, 2005). The library may be screened for binding in a two-step selection process, first humanizing the VL and then the VH. Alternatively, a one-step framework shuffling process may be used. Such a process has been shown to be more efficient than two-step screening. This is because improved biochemical and physicochemical properties (including enhanced expression, increased affinity, and increased thermal stability) are seen in the resulting antibodies (see, for example, Damschroder et al., Mol. Immunol. 44:3049-60, 2007).

[0176] The “humanization” method is based on experimentally identifying the minimal essential determinants (MEDs) and sequentially replacing non-human fragments into a library of human frameworks and evaluating binding. Starting from the CDR3 regions of the non-human VH and VL chains, other regions of the non-human antibody are gradually replaced with human frameworks (including CDR1 and CDR2 of both VH and VL). Typically, from this approach, epitopes of antibodies from multiple subclasses with distinct human V segment CDRs are maintained and identified. Humanization enables the isolation of antibodies that are 91-96% homologous to human germline antibodies. See, for example, Alfenito, Cambridge Healthtech Institute’s Third Annual PEGS, The Protein Engineering Summit, 2007.

[0177] The "human engineering" method involves modifying a non-human antibody or non-human antibody fragment, such as a mouse antibody, chimeric antibody, mouse antibody fragment, or chimeric antibody fragment, by making predetermined changes to its amino acid sequence in order to produce a modified antibody that, despite its reduced immunogenicity in humans, retains the desired binding properties of the original non-human antibody. Generally, this technique involves classifying amino acid residues of non-human (e.g., mouse) antibodies as "low-risk," "medium-risk," or "high-risk" residues. This classification is performed using comprehensive risk / reward calculations that assess the benefits of making a particular substitution (e.g., for immunogenicity in humans) against the risk of the substitution affecting the folding of the resulting antibody and / or the risk of substituting with human residues. Specific human amino acid residues to be substituted at predetermined positions (e.g., low-risk or moderate-risk positions) in non-human (e.g., mouse) antibody sequences can be selected by aligning an amino acid sequence derived from the variable region of the non-human antibody with the corresponding region of a given human antibody sequence or a consensus human antibody sequence. According to this alignment, amino acid residues at low-risk or moderate-risk positions in the non-human sequence can be substituted with corresponding residues in the human antibody sequence. Techniques for producing human-modified proteins are described in further detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Patents 5,766,886, 5,770,196, 5,821,123 and 5,869,619, and WO93 / 11794.

[0178] 5.2.3. Antibody Variants The modification of antibodies that bind to IL13Rα2 as described herein is intended. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, such as specificity, thermal stability, expression level, effector function, glycosylation, decreased immunogenicity, or solubility. That is, variants of antibodies that bind to IL13Rα2 as described herein can be prepared, and these variants are intended to be included in this disclosure. In some embodiments, the antibody variant is an antibody having a variation in the amino acid sequence compared to the original antibody, for example, one or more amino acid substitutions, deletions, or insertions as described above. For example, the variation may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide, which alters the amino acid sequence compared to the original antibody or polypeptide (e.g., a conservative substitution). Relevant sites for substitutional mutagenesis include CDRs, FRs, and / or constant regions. For example, the antibody variant can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or by the synthesis of the desired antibody or polypeptide. Those skilled in the art will know that amino acid changes can alter the post-translational processes of antibodies.

[0179] chemical modification Other exemplary modifications include, for example, chemical modifications by covalently bonding any type of molecule to the antibody. Antibody derivatives may include antibodies chemically modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, linking to cell ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., Fc or part of Fc). Any of a number of chemical modifications may be carried out by known techniques, including, but not limited to, predetermined chemical cleavage, acetylation, compounding, and metabolic synthesis of tunicamycin. In addition, the antibody may contain one or more non-classical amino acids.

[0180] In some embodiments, the antibodies provided in this disclosure are modified to increase or decrease the degree to which they are glycosylated. Adding or deleting glycosylation sites from an antibody may conveniently be achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0181] When fusing the antibodies provided in this disclosure to the Fc region, the glycans bound to the antibody may be modified. Native antibodies produced by mammalian cells typically contain branched double-chain glycans bound by an N-bond to Asn297 in the CH2 domain of their Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). These glycans may include a variety of glycans, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the double-chain glycan structure. In some embodiments, modification of the glycans in the binding molecules provided in this disclosure may be performed to create variants with improved specific properties.

[0182] In other embodiments, when the antibody provided in this disclosure is fused to an Fc region, the antibody variant provided in this disclosure may have a glycan structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 relative to the total of all glycan structures bound to Asn297 (e.g., complex structures, hybrid structures, and high-mannose structures) when measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 refers to the asparagine residue located approximately 297th in the Fc region (Fc region in residue EU numbering). However, due to minor sequence variations in the antibody, Asn297 may be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publications 2003 / 0157108 and 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Publication Nos. 2003 / 0157108, WO2000 / 61739, WO2001 / 29246, U.S. Patent Publication Nos. 2003 / 0115614, U.S. Patent Publication Nos. 2002 / 0164328, U.S. Patent Publication Nos. 2004 / 0093621, and U.S. Patent Publication Nos. 20 Examples include 04 / 0132140, U.S. Patent Publication 2004 / 0110704, U.S. Patent Publication 2004 / 0110282, U.S. Patent Publication 2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application Publication No. 2003 / 0157108, and WO2004 / 056312), and knockout cell lines such as those containing the alpha-1,6-fucosyltransferase gene, FUT8, or knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107).

[0183] The antibody-containing binding molecules provided in this disclosure further comprise bisected glycans, for example, in which GlcNAc is bound to the branching of a double-chain glycan bound to the Fc region. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the glycan bound to the Fc region are also provided. Such variants may have improved CDC function. Such variants are described, for example, in WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0184] In the antibodies and molecules containing an Fc region of this disclosure, an Fc region variant may be generated by introducing one or more amino acid modifications into the Fc region. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes one or more amino acid modifications (e.g., substitutions) at the amino acid positions.

[0185] In some embodiments, this application envisions variants having some, but not all, effector functions, where the in vivo half-life of the binding molecule is important, but which are desirable candidate variants for applications where certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / loss of CDC activity and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the binding molecule lacks FcγR binding (i.e., is likely to lack ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays for evaluating the ADCC activity of the relevant molecules are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition to the above, the ADCC activity of the molecule in question may be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may be performed to confirm the lack of CDC activity, since the antibody cannot bind to C1q.For example, see C1q-binding ELISA and C3c-binding ELISA in WO2006 / 029879 and WO2005 / 100402. CDC assays may be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), Cragg, MS et al., Blood 101:1045-1052 (2003), and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0186] Examples of binding molecules with reduced effector function include molecules having one or more substitutions among the Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Examples of such Fc variants include Fc variants having two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, and so-called "DANA" Fc variants having alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581).

[0187] Specific variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0188] In some embodiments, the variant includes an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 (residues in EU numbering) of the Fc region. In some embodiments, the modification is made in an Fc region that alters (e.g., improves or decreases) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642 and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0189] Binding molecules exhibiting extended half-life and improved binding to the fetal Fc receptor (FcRn) (which is responsible for the transfer of maternal IgG to the fetus) (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). These molecules contain an Fc region having one or more substitutions in that region that improve binding to FcRn. Examples of such Fc variants include substitutions in one or more Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, a variant with a substitution in Fc region residue 434 (US Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), US Patent Nos. 5,648,260, 5,624,821, and WO94 / 29351.

[0190] In some embodiments, it may be desirable to produce a cysteine-modified antibody in which one or more residues of the antibody are substituted with cysteine ​​residues. In some embodiments, the substituted residue is located in a contactable site on the antibody. By substituting these residues with cysteine, a reactive thiol group is positioned in a contactable site on the antibody, and this thiol group may be used to conjugate the antibody to other parts, such as a drug part or a linker-drug part, as further described herein, to produce an immunoconjugate.

[0191] Other known covalent modifications of antibodies are included within the scope of this disclosure. Covalent modifications include reacting a target amino acid residue of an antibody with an organic derivatizer that can react with a given side chain or N-terminal or C-terminal residue of the antibody. Other modifications include deamidation of glutaminyl residues to their corresponding glutamyl residues and deamidation of asparaginyl residues to their corresponding aspartyl residues, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0192] The antibodies described herein that bind to IL13Rα2 may be modified to form a chimeric molecule containing an antibody that binds to IL13Rα2, which is fused or conjugated to another heterogeneous polypeptide, amino acid sequence, or small molecule compound, such as an immunoactivator (e.g., cytokine), an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

[0193] Provided in this disclosure is also a fusion protein comprising an antibody that binds to IL13Rα2 and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is fused by genetic engineering or chemically conjugated is useful for directing the antibody to cells expressing IL13Rα2 on the cell surface. Antibodies fused by genetic engineering or chemically conjugated are described in more detail in the following chapters.

[0194] In vitro affinity maturation In some embodiments, antibody variants with improved properties, such as affinity, stability, or expression levels, compared to the parent antibody can be prepared by in vitro affinity maturation. Like natural prototypes, in vitro affinity maturation is based on the principles of mutation and selection. The antibody library is presented on the surface of an organism (e.g., a phage, bacterium, yeast, or mammalian cell) or presented in association with their coding mRNA or coding DNA (e.g., by covalent or non-covalent bonding). Affinity selection of the presented antibodies allows for the isolation of the organism or complex containing the genetic information encoding that antibody. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with affinity in a narrow nanomolar range. Affinity-matured antibodies can have nanomolar or even picomolar affinity for a target antigen.

[0195] Phage display is a widely used method for antibody presentation and selection. The antibody is presented on the surface of an Fd bacteriophage or M13 bacteriophage as a fusion to a bacteriophage coat protein. Selection involves exposure to an antigen to bind the antibody presented on the phage to its target; this process is called "panning." The antigen-bound phage is removed, and this phage is used to infect bacteria to generate phages for further selection. For an overview, see, for example, Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002), and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0196] In yeast display systems (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997) and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can fuse to the adhesion subunit of the yeast agglutinin protein Aga2p, which then binds to the yeast cell wall via a disulfide bond to Aga1p. Aga2p-mediated protein presentation minimizes the possibility of interaction with other molecules in the yeast cell wall by causing the protein to protrude away from its cell surface. The library is screened using magnetic separation and flow cytometry to select antibodies exhibiting improved affinity or stability. Binding to the relevant soluble antigen is determined by labeling the yeast with the biotinylated antigen and a secondary reagent conjugated to a fluorophore, such as streptavidin. The differences in antibody surface expression can be measured through immunofluorescence labeling of either hemagglutinin or c-Myc epitope tags adjacent to single-chain antibodies (e.g., scFv). Since expression has been shown to correlate with the stability of the presented protein, antibodies can be selected for improved stability and affinity (see, e.g., Shusta et al., J.Mol.Biol.292:949-56(1999)). An additional advantage of yeast display is that the presented protein folds in the endoplasmic reticulum of eukaryotic yeast cells, utilizing endoplasmic reticulum chaperones and quality control mechanisms. Once maturation is complete, antibody affinity can be conveniently "fine-tuned" while presented on the yeast surface, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is that the size of the functional library may be smaller than that of other display methods; however, recent approaches use the yeast cell conjugation system to achieve a size of 10 14 This creates a presumed combinatorial diversity (see, for example, U.S. Patent Application Publication No. 2003 / 0186374 and Blaise et al., Gene 342:211-18 (2004)).

[0197] In ribosome display, an antibody-ribosome-mRNA (ARM) complex is generated during selection in a cell-free system. A DNA library encoding an antibody from a specific library is genetically engineered and fused to a spacer sequence lacking a stop codon. This spacer sequence remains bound to the peptidyl-tRNA during translation, occupying the ribosome tunnel and allowing the corresponding protein to protrude from the ribosome and fold. The resulting complex, consisting of mRNA, ribosome, and protein, can bind to a surface-bound ligand, allowing for simultaneous isolation of the antibody and its encoding mRNA through affinity capture by the ligand. Subsequently, the ribosome-bound mRNA is reverse transcribed back to cDNA, and this cDNA can be mutagenesized for use in the next round of selection (see, for example, Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, puromycin is used as an adapter molecule to establish a covalent bond between the antibody and mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0198] These methods, being performed entirely in vitro, offer two main advantages over other selection techniques. First, library diversity is limited only by the number of ribosomes and different mRNA molecules present in the test tube, and not by the transformation efficiency of bacterial cells. Second, random mutations can be easily introduced after each selection round, for example, by non-proofreading polymerases, since no library should be transformed after any diversification step. In some embodiments, mammalian display systems may be used.

[0199] Diversity may be introduced into the CDRs of an antibody library either in a targeted manner or through random introduction. The former approach includes sequentially targeting the entire CDR of the antibody via high or low levels of mutagenesis, or targeting isolated hotspots of somatic hypermutations suspected to affect affinity for experimental or structural reasons (see, e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)) or residues. Diversity may also be introduced by substitution of naturally diverse regions via DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003), U.S. Patents 5,565,332 and 6,989,250). Alternative techniques include targeting hypervariable loops extending to framework region residues, utilizing loop deletions and insertions in CDRs (see, e.g., Bond et al., J.Mol.Biol.348:699-709 (2005)), or using hybridization-based diversification (see, e.g., U.S. Patent Application Publication 2004 / 0005709). Additional methods for creating diversification in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Further methods that can be used to bring about antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Patent Nos. 8,685,897 and 8,603,930, and U.S. Patent Application Publications 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855 and 2009 / 0075378 (each incorporated herein by reference).

[0200] Library screening can be achieved by various techniques known in the art. For example, antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed on host cells attached to adsorption plates, used for cell sorting, conjugated to biotin for capture by streptavidin-coated beads, or used in any other way for panning display libraries.

[0201] For an overview of in vitro affinity maturation methods, see, for example, Hoogenboom, Nature Biotechnology 23:1105-16 (2005), Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010), and the references in this specification.

[0202] Antibody internalization assays may be used to determine receptor-mediated endocytosis when bound to an antibody. In some embodiments, the efficacy of a particular antibody-based therapeutic agent depends on the antibody internalization process. In some embodiments, antibody internalization assays examine the rate and extent of antibody internalization to assess the antibody's ability to deliver the therapeutic agent to the relevant site or cell. Non-limiting exemplary assays are described below. The target cells are seeded at an appropriate seeding density (e.g., in a 96-well U-bottom plate), and the test antibody is labeled with a signal-reporting reagent, such as a fluorescent compound, horseradish peroxidase (HRP) reagent, a radiolabeled compound, or biotin. The test antibody and target cells are then incubated in an appropriate molar ratio. After incubation, unbound antibody is removed by washing. The cells can be left on ice for a period of time or incubated at 37°C to facilitate internalization. Subsequently, the cells may be incubated for a period of time in the presence of a stopping reagent to inhibit internalization. The cells are then washed and incubated with a signal-emitting reagent. The final signal can be examined using a plate reader or imaging device and analytical software. For example, the mean fluorescence intensity (MFI) of the cells can be measured using a flow cytometer, and a decrease in MFI may indicate antibody internalization, antibody dissociation, or a combination of both. Cell imaging can be scanned and acquired to analyze signal intensity, size, and shape. Alternatively, the cells are lysed to release the internalized antibody. This antibody is then captured in a microtiter well plate coated with a specific antigen that induces antibody production. The bound antibody in the well is detected using a secondary antibody conjugated with alkaline phosphatase or HRP and a chromogenic substrate. Alternative detectable labels for the antibody and their disclosures will be apparent to those skilled in the art. Any method known in the art can be used in this disclosure to determine antibody internalization.

[0203] 5.2.4. Other binders containing the antibodies of this disclosure In some embodiments, the antibody or fragment provided herein is part of a larger binder. Non-limiting exemplary binders, including the antibody or fragment provided herein, are described below.

[0204] This disclosure provides an IL13Rα2 conjugate (e.g., an antibody) having a masking portion and / or cleavable portion in which one or more of the IL13Rα2 binding domains of the IL13Rα2 conjugate (e.g., an antibody) are masked (e.g., via a masking portion) and / or activatable (e.g., via a cleavable portion). Techniques for masking IL13Rα2 conjugates (e.g., antibodies) are well known in the art, and such techniques include the masking technique SAFEbody (see, for example, U.S. Patent Application Publication 2019 / 0241886) and the masking technique Probody (see, for example, U.S. Patent Application Publication 2015 / 0079088). Using such techniques, IL13Rα2 conjugates (e.g., antibodies) that are masked and / or activatable can be produced. Such masked and / or activatable IL13Rα2 conjugates (e.g., antibodies) are also useful for preparing conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable ADCs (AADCs), which include conjugates directly or indirectly linked to another agent, such as a drug and / or immunoactivator (e.g., cytokine). For example, the IL13Rα2 conjugates (e.g., antibodies) of this disclosure, such as a human IL13Rα2 conjugate, may be covalently linked to one or more agents, such as a drug and / or immunoactivator (e.g., cytokine), by a synthetic linker.

[0205] If desired, an IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) may be linked or conjugated (directly or indirectly) to a moiety having effector function, such as cytotoxic activity (e.g., a chemotherapeutic moiety or radioisotope), immune mobilization activity, or immunomodulatory activity. Examples of linked or conjugated moieties include cytotoxic drugs (e.g., toxins such as aurilistatin), non-cytotoxic drugs (e.g., signaling regulators such as kinases), masking moieties that mask one or more binding domains of the IL13Rα2 conjugate (e.g., an antibody), or cleavable moieties that enable activation of the IL13Rα2 conjugate by cleaving a cleavable moiety to expose one or more binding domains of the IL13Rα2 conjugate (e.g., an antibody) in the tumor microenvironment of a masked conjugate. The immune mobilization-promoting moiety may include other antigen conjugates, such as viral proteins that selectively bind to cells of the innate and / or adaptive immune systems. Alternatively, or in addition to this, IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) are optionally linked or conjugated (directly or indirectly) to a portion (e.g., a tag) that facilitates isolation from the mixture or a portion having reporter activity (e.g., a detection label or reporter protein). It will be apparent that the features of IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) described herein also extend to polypeptides containing IL13Rα2 conjugate fragments.

[0206] In some embodiments, the IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) described herein, wherein the conjugate that binds to IL13Rα2 may be linked or conjugated (directly or indirectly) to a polypeptide, thereby enabling the generation of an activatable antibody. In some embodiments, the IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) may be linked or conjugated (directly or indirectly) to an additional agent. In some embodiments, the additional agent is a drug, resulting in an ADC, or an AADC when the antibody of the ADC includes a masking moiety and a cleavable moiety.

[0207] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) described herein (including human IL13Rα2 conjugates) are conjugated (directly or indirectly) or recombinantly linked to therapeutic agents (e.g., cytotoxic agents or cytokines), or diagnostic or detectable agents. The conjugated or recombinantly linked antibodies, including masked or activatable conjugates, may be useful, for example, for treating or preventing diseases, disorders, or conditions, such as IL13Rα2-mediated diseases, disorders, or conditions. The conjugated or recombinantly linked IL13Rα2 conjugates (e.g., antibodies), including masked or activatable conjugates, may be useful, for example, for monitoring or prognosticating the onset, progression, progression, and / or severity of IL13Rα2-mediated diseases, disorders, or conditions.

[0208] Such diagnosis and detection can be achieved, for example, by coupling an IL13Rα2 binder (e.g., an antibody) to a detectable substance, such as enzymes (horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, but not limited thereto), prosthetic groups (streptavidin / biotin or avidin / biotin, but not limited thereto), fluorescent substances (umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dancylcloride, or phycoerythrin, but not limited thereto), luminescent substances (luminol, but not limited thereto), bioluminescent substances (luciferase, luciferin, or aequorin, but not limited thereto), chemiluminescent substances (acridinium-based compounds or HALOTAG, but not limited thereto), radioactive substances (iodine, 131 I, 125 I, 123 I and 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 115 In, 113 In, 112 In and 111 In), Technonetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga and 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 PM, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re,142 Pr, 105 Rh, 97 Ru, 68 Enjoy, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, or 117 Examples include sn (but not limited to sn), positron-emitting metals obtained using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions.

[0209] Described herein are IL13Rα2 conjugates (e.g., antibodies) that are recombinantly linked or conjugated (covalent or non-covalent conjugation (direct or indirect)) to heterologous proteins or polypeptides, or fragments thereof, for example, polypeptides (e.g., consisting of about 10 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, or about 100 amino acids), and their use. In particular, described herein are fusion proteins comprising antigen-binding fragments (e.g., fragments containing VH and / or VL CDR1, CDR2 and / or CDR3) of the IL13Rα2 conjugates (e.g., antibodies) described herein (including human IL13Rα2 conjugates) and heterologous proteins, polypeptides, or peptides. In some embodiments, heterologous proteins, polypeptides, or peptides to which an IL13Rα2 conjugate (e.g., an antibody) is linked are useful for directing the IL13Rα2 conjugate to specific cells (e.g., IL13Rα2-expressing cells, including cancer cells). Other non-limiting heterologous proteins, polypeptides, or peptides to which an IL13Rα2 conjugate (e.g., an antibody) is linked may be useful as an internalization signal or for engaging tumor cells with immune cells.

[0210] Furthermore, the IL13Rα2 conjugates described herein (e.g., antibodies) (including human IL13Rα2 conjugates) can be linked (directly or indirectly) to marker sequences or "tag" sequences, such as peptides, to facilitate purification. In some embodiments, the amino acid sequence of the marker or tag is a hexahistidine peptide, such as a tag provided for pQE vectors (see, e.g., QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistidine facilitates the purification of fusion proteins. Other peptide tags useful for purification include, but are not limited to, hemagglutinin ("HA") tags (corresponding to an epitope derived from influenza hemagglutinin protein) (Wilson et al., 1984, Cell 37:767-78) and "FLAG" tags.

[0211] Methods for linking or conjugating a portion (including polypeptides) to an antibody (directly or indirectly) are well known in the art, and any one of these can be used to produce the antibody-drug conjugates or fusion proteins described herein.

[0212] In some embodiments, the IL13Rα2 conjugate (e.g., antibody) described herein is a fusion protein. The term "fusion protein," as used herein, refers to a polypeptide comprising the amino acid sequence of a conjugate (e.g., antibody) and the amino acid sequence of a heterogeneous polypeptide or protein (e.g., a polypeptide or protein that is not typically part of that antibody). In certain embodiments, the fusion protein retains the biological activity of the IL13Rα2 conjugate. In certain embodiments, the fusion protein comprises the VH region, VL region, VH CDR (one, two, or three VH CDRs), and / or VL CDR (one, two, or three VL CDRs) of an IL13Rα2 antibody, and the fusion protein binds to an IL13Rα2 epitope, an IL13Rα2 fragment, and / or an IL13Rα2 polypeptide. In some embodiments, the fusion protein comprises the VH or heavy chain of an IL13Rα2 antibody, the VL or light chain of an IL13Rα2 antibody, separated by a linker, such as a cleavable linker.

[0213] Fusion proteins may be generated, for example, through techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be used to modify the activity of IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) as described herein (e.g., including IL13Rα2 conjugates with higher affinity and lower dissociation rate) (e.g., U.S. Patents No. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458,91, 5,830,791, 5,834,252 and 5,837,458, Patents No. 5,605,793, 5,811,238, 5,830,791, 5,830,791, 5,830,791, See 24(2):308-13). In some embodiments, the IL13Rα2 conjugate (including the human IL13Rα2 conjugate) may be modified before recombination by random mutagenesis by error-prone PCR, random nucleotide insertion or other methods. The polynucleotides encoding the IL13Rα2 conjugates described herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0214] The IL13Rα2 conjugates (e.g., antibodies) described herein (including human IL13Rα2 conjugates) may be bound to a solid support, which may be useful for immunoassays or purification of target antigens. Examples of such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0215] The IL13Rα2 conjugates described herein (e.g., antibodies) (including human IL13Rα2 conjugates) can also be linked or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0216] The linker may be a “cleavable portion” that facilitates the release of a drug linked or conjugated in a cell, but non-cleavable linkers are also contemplated in this disclosure. Linkers for use in the conjugates of this disclosure (e.g., ADC or AADC) include, but are not limited to, acid-unstable linkers, disulfide-containing linkers, peptidase-sensitive linkers, light-unstable linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to avoid transporter-mediated multidrug resistance.

[0217] Antibody and drug conjugates (including those in which the drug is used for the preparation of ADCs or AADCs) can be prepared using a variety of bifunctional protein coupling agents.

[0218] This disclosure further envisions antibody-drug conjugates (including drugs for the preparation of ADCs or AADCs), which may be prepared using any suitable method disclosed in the art (see, for example, Bioconjugate Technique (Hermanson ed., 2d ed. 2008)).

[0219] Conventional conjugation strategies for antibodies and drugs (including when the drug is a drug for the preparation of ADCs or AADCs) are based on random conjugation chemical reactions involving ε-amino groups on Lys residues or thiol groups on Cys residues, resulting in heterogeneous conjugates. Recently developed techniques enable site-specific conjugation of antibodies, resulting in uniform loading and avoiding conjugate subpopulations with altered antigen binding or pharmacokinetics. These techniques include "Thiomab" manipulations involving cysteine ​​substitutions at heavy and light chain positions, which yield reactive thiol groups without inhibiting immunoglobulin folding and assembly or altering antigen binding (see, e.g., Junutula et al., 2008, J.Immunol.Meth.332:41-52 and Junutula et al., 2008, Nature Biotechnol.26:925-32). Alternatively, by recoding the stop codon UGA from a stop to a selenocysteine ​​insertion, selenocysteine ​​can be inserted into the antibody sequence simultaneously with translation, enabling site-specific covalent conjugation at the nucleophilic selenool group of selenocysteine ​​in the presence of other native amino acids (see, for example, Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56 and Hofer et al., 2009, Biochemistry 48(50):12047-57).

[0220] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) described herein are conjugated to drugs, such as immunoactivators or cytotoxic agents. In some embodiments, to produce ADCs or AADCs, the IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) disclosed herein may optionally be conjugated with one or more cytotoxic agents disclosed herein or known in the art. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. In some embodiments, the cytotoxic agent is an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin. In some embodiments, the cytotoxic agent is a radioactive conjugate or radioisotope for producing a radioconjugated drug. Conjugates of polypeptides or molecules with one or more small molecule toxins. Conjugates of polypeptides or molecules with cytotoxic agents are made using various bifunctional protein coupling agents.

[0221] In other embodiments, the IL13Rα2 conjugates described herein (e.g., antibodies) (including human IL13Rα2 conjugates) are conjugated to drugs, such as signaling regulators, pro-apoptotic agents, mitotic inhibitors, antitumor antibiotics, immunomodulators, nucleic acids for gene therapy, alkylating agents, angiogenesis inhibitors, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, corticosteroids, phototherapeutic agents, oligonucleotides, radionuclides, radiosensitizers, topoisomerase inhibitors, such as camptothecin or its analogues, and tyrosine kinase inhibitors.

[0222] The IL13Rα2 conjugates (e.g., antibodies) described herein (including human IL13Rα2 conjugates) may be monospecific, bispecific, tripspecific, or more than polyspecific. The agents may include monospecific or polyspecific antibodies. A polyspecific antibody, for example, a bispecific antibody, is a monoclonal antibody having binding specificity to at least two different targets (e.g., antigens), or a monoclonal antibody having two different epitopes on the same target (e.g., a bispecific antibody against IL13Rα2 having a first binding domain to a first epitope of IL13Rα2 and a second binding domain to a second epitope of IL13Rα2). In some embodiments, monospecific and polyspecific (e.g., bispecific) antibodies can be constructed based on the antibody sequences described herein, e.g., the CDR sequences listed in Tables 1-3. In some embodiments, the polyspecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibodies are mouse antibodies, chimeric antibodies, human antibodies, or humanized antibodies.

[0223] In some embodiments, one binding specificity of the multispecific antibody is for IL13Rα2, and the other is for any other target (e.g., an antigen). In some embodiments, a multispecific (e.g., bispecific) antibody may contain more than one target (e.g., antigen) binding domain, each of which is specific to a different target (e.g., a first binding domain that binds to IL13Rα2 and a second binding domain that binds to another target (e.g., an antigen)). In some embodiments, the second target is an immune checkpoint regulator (e.g., a negative checkpoint regulator). In some embodiments, the second target is expressed on immune cells. In some embodiments, the second target is expressed on tumor cells or cancer cells.

[0224] In some embodiments, a polyspecific (e.g., bispecific) antibody molecule can bind to more than one (e.g., two or more) epitopes on the same target (e.g., antigen).

[0225] Methods for producing polyspecific antibodies are known in the art, such as the co-expression of two immunoglobulin heavy-light chain pairs (the two heavy chains having different specificities) (see, for example, Milstein and Cuello, 1983, Nature 305:537-40). For further details on generating polyspecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0226] Exemplary structures of polyspecific antibodies are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10:1-18, Brinkman et al., 2017, MABS, 9:2, 182-212, Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276, and Spiess et al., 2015, Mol.Immunol.67 95-106.

[0227] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates. As a non-limiting example, the BsIgG form may include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assemblies, charge pairs, Fab-arm exchanges, SEED bodies, triomabs, LUZ-Y, Fcab, κλ-bodies, and / or orthogonal Fabs.

[0228] In some embodiments, BslgG includes a heavy chain that has been manipulated for heterodimerization. For example, the heavy chain can be manipulated for heterodimerization using the "knobs-into-holes" strategy, the SEED platform, a common heavy chain (e.g., κλ-body), and the use of the Fc region of the heterodimer. In BsIgG, strategies for avoiding homodimeric heavy chain pairing are known in the art, including knobs-into-holes, duobody, azymetric, chargepair, HA-TF, SEEDbody, and protein A affinity difference.

[0229] Another form of bispecific antibody is IgG with an additional antigen-binding moiety. For example, monospecific IgG can be made bispecific by manipulating it to add an additional antigen-binding unit, for example, at the N-terminus or C-terminus of either the heavy or light chain. Examples of additional antigen-binding units include single-domain antibodies (e.g., variable heavy or light chains), manipulated protein scaffolds, and paired antibody variable domains (e.g., single-stranded variable fragments or variable fragments). Non-limiting examples of added IgG forms include bivariable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIHIgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol.Immunol. 67(2015):95-106. In some embodiments, exemplary antibody forms are B-Body forms for monospecific or polyspecific (e.g., bispecific antibodies), as described, for example, in WO2018 / 075692 and U.S. Patent Application Publication No. 2018 / 0118811.

[0230] A bispecific (Bs) antibody (BsAb) fragment is a form of bispecific antibody molecule that lacks some or all of the antibody's constant domains. For example, some BsAbs lack the Fc region. In embodiments, the bispecific antibody fragment comprises a heavy-chain region and a light-chain region linked by a peptide linker, enabling efficient expression of its BsAb in a single host cell. Non-exclusive examples of bispecific antibody fragments include, but are not limited to, nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody.

[0231] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or function. In some embodiments, a dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher titers. For example, a fusion of a bispecific antibody to an albumin-binding protein or human serum albumin can be constructed to extend the serum half-life of the antibody fragment. In some embodiments, a BsAb molecule can be constructed using chemical conjugation, e.g., chemical conjugation of an antibody and / or antibody fragment. An exemplary bispecific antibody conjugate is the CovX-body form, in which a low molecular weight drug is site-specifically conjugated to a single reactive lysine in each Fab arm, or in the antibody or its fragment. In some embodiments, the conjugation improves the serum half-life.

[0232] Methods for producing polyspecific antibodies (including bispecific antibodies) are known in the art. For example, polyspecific antibodies (including bispecific antibodies) can be produced by separately expressing constituent antibodies in various host cells and then purifying / assembling them, or by expressing constituent antibodies in a single host cell. Purification of polyspecific (e.g., bispecific) antibody molecules can be carried out by various methods known in the art (including affinity chromatography).

[0233] In some embodiments, the IL13Rα2 conjugates (e.g., antibodies) disclosed herein (including human IL13Rα2 conjugates) can be provided in any antibody form disclosed herein or known in the art. As a non-limiting example, in some embodiments, the IL13Rα2 conjugate (e.g., antibody) (including human IL13Rα2 conjugate) is Fabs-in-tandem-1g (FIT-1g), DVD-1g, hybrid hybridoma (quadroma or tetradoma), antikalin platform (Pieris), diabody, single-stranded diabody, tandem single-stranded Fv fragment, TandAb, triple-specific Ab (Affimed), Dart (dual-affinity retargeting) (Macrogenics), bispecific Xmab (Xencor), bispecific T-cell engager (Bite, Amgen, 55kDa), triplebody, tribody (multifunctional recombinant antibody derivative that is a Fab-scFv fusion protein (CreativeBiolabs)), duobody platform (Genmab), dock and You can choose from the lock platform, knobs-into-holes (KIH) platform, humanized bispecific IgG antibody (REGN1979) (Regeneron), Mab2 bispecific antibody (F-Star), DVD-Ig bivariable domain immunoglobulin (Abbott), kappa-lambda body, TBTI (tetravalent bispecific tandem Ig), and CrossMab (Roche).

[0234] In some embodiments, the polyspecific (e.g., bispecific) antibodies disclosed herein include an IL13Rα2 binding domain and one or more additional binding domains that bind to one or more targets other than IL13Rα2. In some embodiments, the polyspecific (e.g., bispecific) antibodies disclosed herein include an IL13Rα2 binding domain comprising an amino acid sequence of VH and / or VL, such as those disclosed herein, e.g., the amino acid sequences in Table 1, Table 2, or Table 3.

[0235] In some embodiments, what is described herein is a polyspecific (e.g., bispecific) antibody containing a binding domain that binds to IL13Rα2, and includes VH CDRs and VL CDRs as described herein, for example, antibodies containing VH CDRs and VL CDRs as shown in Table 1, Table 2, or Table 3.

[0236] In some embodiments, the IL13Rα2 conjugate is a bispecific antibody comprising a first binding domain and a second binding domain. In further embodiments, the first binding domain comprises six CDRs of the antibody A22. In some embodiments, the first binding domain comprises six CDRs listed in one column of Table 1. In some embodiments, the first binding domain comprises three CDRs of the heavy chain variable region, as shown in SEQ ID NO: 25, and three CDRs of the light chain variable region, as shown in SEQ ID NO: 26. In some embodiments, the first binding domain comprises a heavy chain variable region, as shown in SEQ ID NO: 25, and a light chain variable region, as shown in SEQ ID NO: 26. In some embodiments, the second binding domain binds to a different epitope of the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to the first or second complex.

[0237] In some embodiments, the IL13Rα2 conjugate is a bispecific antibody comprising a first binding domain and a second binding domain. In further embodiments, the first binding domain comprises six CDRs of an antibody named A33. In some embodiments, the first binding domain comprises six CDRs listed in one column of Table 2. In some embodiments, the first binding domain comprises three CDRs of the heavy chain variable region, as shown in SEQ ID NO: 48, and three CDRs of the light chain variable region, as shown in SEQ ID NO: 49. In some embodiments, the first binding domain comprises a heavy chain variable region, as shown in SEQ ID NO: 48, and a light chain variable region, as shown in SEQ ID NO: 49. In some embodiments, the second binding domain binds to a different epitope of the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to the first or second complex.

[0238] In some embodiments, the IL13Rα2 conjugate is a bispecific antibody comprising a first binding domain and a second binding domain. In further embodiments, the first binding domain comprises six CDRs of the antibody A52. In some embodiments, the first binding domain comprises six CDRs listed in one column of Table 3. In some embodiments, the first binding domain comprises three CDRs of the heavy chain variable region, as shown in SEQ ID NO: 73, and three CDRs of the light chain variable region, as shown in SEQ ID NO: 74. In some embodiments, the first binding domain comprises a heavy chain variable region, as shown in SEQ ID NO: 73, and a light chain variable region, as shown in SEQ ID NO: 74. In some embodiments, the second binding domain binds to a different epitope of the first complex and does not bind to the second complex. In other preferred embodiments, the second binding domain does not bind to the first or second complex.

[0239] In another embodiment, the antibody or its antigen-binding fragment provided in this disclosure may be part of an engineered cell surface receptor, such as a chimeric antigen receptor (CAR). Typically, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0240] In some embodiments, the Disclosure provides a CAR comprising an extracellular domain containing one or more antibodies or fragments thereof provided in the Disclosure. In some embodiments, the extracellular domain of the CAR provided in the Disclosure includes VH CDRs and VL CDRs disclosed herein, for example, CDRs shown in Table 1, Table 2, or Table 3.

[0241] The CARs of this disclosure include a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain may be derived from either a natural or synthetic source. As used herein, “transmembrane domain” refers to any protein structure that is thermodynamically stable on the cell membrane, preferably the eukaryotic cell membrane. A transmembrane domain suitable for use in the CARs described herein may be obtained from a natural protein. Alternatively, the domain may be a synthetic, non-natural protein segment that is thermodynamically stable on the cell membrane, such as a hydrophobic protein segment. Transmembrane domains are classified based on their three-dimensional structure. For example, a transmembrane domain may form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure that can penetrate the cellular phospholipid bilayer.

[0242] The CARs of this disclosure include an intracellular signaling domain. This intracellular signaling domain is responsible for activating at least one of the normal effector functions of an immunoeffector cell expressing the CAR. The term "effector function" refers to a specialized function of the cell. For example, the effector function of a T cell may be cytolytic activity or helper activity (including cytokine secretion). That is, the term "cytoplasmic signaling domain" refers to the portion of the protein that transmits the effector function signal, instructing the cell to perform its specialized function. Usually, the entire cytoplasmic signaling domain is available, but in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such a truncated portion may be used in place of the intact chain, as long as it transmits the effector function signal. That is, the term cytoplasmic signaling domain is intended to include any portion of the truncated portion of the cytoplasmic signaling domain that is sufficient to transmit the effector function signal.

[0243] In some embodiments, the intracellular signaling domain includes the primary intracellular signaling domain of the immune effector cell. In some embodiments, the CAR includes an intracellular signaling domain that is essentially derived from the primary intracellular signaling domain of the immune effector cell. The “primary intracellular signaling domain” refers to a cytoplasmic signaling sequence that acts in the form of a stimulus to induce immune effector function.

[0244] Many immune effector cells require co-stimulation in addition to antigen-specific signaling to promote cell proliferation, differentiation, and survival, and to activate their effector function. In some embodiments, the CAR includes at least one co-stimulatory signaling domain. As used herein, the term “co-stimulatory signaling domain” refers to at least a portion of proteins that mediate signaling within a cell to induce an immune response, such as effector function.

[0245] The CARs of this disclosure may include a hinge domain located between an extracellular antigen-binding domain and a transmembrane domain. A hinge domain is generally an amino acid segment located between two domains of a protein, which can enable the flexibility of the protein and the movement of one or both of those domains relative to each other. Any amino acid sequence that results in such flexibility and movement of the extracellular antigen-binding domain can be used for the transmembrane domain of an effector molecule.

[0246] The CARs of this disclosure may include a signal peptide (also known as a signal sequence) at the N-terminus of their polypeptide. Generally, a signal peptide is a peptide sequence that directs a polypeptide to a desired site within a cell.

[0247] Other manipulated transmembrane receptors, including those comprising antibodies or fragments provided in this disclosure, are also included in this disclosure.

[0248] 5.3. Nucleic acids, vectors, and cells In addition, the provided materials include nucleic acids encoding an IL13Rα2 conjugate (e.g., an antibody or antibody fragment) or a fusion polypeptide as disclosed herein, nucleic acids complementary to the nucleic acid, vectors containing nucleic acids as disclosed herein, and cells comprising one or more of the IL13Rα2 conjugate, nucleic acids as disclosed herein, or vectors as disclosed herein. In some embodiments, the cells express the IL13Rα2 conjugate. In some embodiments, the cells replicate the nucleic acid or vector. In some embodiments, the provided materials are for generating an IL13Rα2 conjugate, e.g., a human IL13Rα2 conjugate, and fragments thereof. For example, isolated cells may produce an IL13Rα2 conjugate (e.g., an antibody or antibody fragment). In this regard, cells (e.g., isolated cells) may produce antibodies or fragments thereof containing VH and VL as disclosed herein. In some embodiments, the polynucleotides described herein may comprise one or more nucleic acid sequences encoding an IL13Rα2 binder (e.g., an antibody or antibody fragment). In some embodiments, the polynucleotide is an isolated polynucleotide and / or a recombinant polynucleotide. In various embodiments, the isolated polynucleotide comprises nucleotide sequences encoding VH and / or VL, wherein the VH and VL include a CDR identical to the complementarity-determining region (CDR) disclosed herein.

[0249] As used herein, the term “complementary” refers to specific binding between polynucleotides based on the sequence of the polynucleotides. As used herein, a first polynucleotide and a second polynucleotide are complementary when they bind to each other in a hybridization assay under stringent conditions, for example, when they produce a signal of a predetermined or detectable level in the hybridization assay. The polynucleotide moieties are complementary if they follow conventional base pairing rules, e.g., A pairs with T (or U) and G pairs with C (provided that small regions (e.g., less than about 3 bases) of mismatched sequences, insertions, or deletions are present). The term “stringent assay conditions” refers to conditions that are suitable for generating nucleic acid binding pairs that are sufficiently complementary to produce a desired level of specificity in the assay, e.g., a probe-target mRNA binding pair, but are generally unsuitable for the formation of binding pairs between binding members that are not sufficiently complementary to produce the desired specificity. The term "stringent assay conditions" generally refers to a combination of hybridization and washing conditions.

[0250] In some embodiments, one or more vectors (e.g., expression vectors) may contain one or more polynucleotides to express one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, for amplifying the polynucleotides in the host cell to produce a useful amount of the polynucleotides, and for expressing a binder, such as an antibody or antibody fragment, using recombinant methods.

[0251] In some embodiments, one or more vectors are expression vectors in which one or more polynucleotides are functionally ligated to one or more polynucleotides containing expression control sequences. Specifically intended are autonomously replicating recombinant expression constructs, such as plasmids and viral DNA vectors, that incorporate one or more polynucleotides encoding antibody sequences that bind to IL13Rα2. Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression system in which the expression construct is used. Promoter and enhancer sequences are generally selected for their ability to increase gene expression, and operator sequences are generally selected for their ability to regulate gene expression. The expression construct may also include sequences encoding one or more selectable markers that make it possible to identify host cells having the construct. The expression construct may also include sequences that facilitate, preferably promote, homologous recombination in host cells. In some embodiments, the expression construct may also include sequences necessary for replication in host cells.

[0252] Exemplary regulatory sequences include promoter / enhancer sequences, such as the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41:521-530, 1985); Roussarcoma virus promoter (Davis et al., Hum. Gene Ther., 4:151, 1993); Tie promoter (Korhonen et al., Blood, 86(5):1828-1835, 1995); Simian virus 40 promoter; DRA (downregulated in adenomas, Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293:G923-G934, 2007); MCT1 (monocarboxylic acid transporter 1; Cuff et al.) This includes al., Am.J.Physiol.Gastrointet. Liver Physiol., G977-G979.2005); and Math1 (mouse atnal homolog 1; Shroyer et al., Gastroenterology, 132:2477-2478, 2007), and in the case of expression in mammalian cells, the promoter is operably ligated upstream (e.g., 5') of the polypeptide coding sequence. In another variant, the promoter is an epithelial-specific promoter or an endothelial-specific promoter. The polynucleotide may also optionally include a suitable polyadenylated sequence (e.g., SV40 or the human growth hormone gene polyadenylated sequence) functionally ligated downstream (e.g., 3') of the polypeptide coding sequence.

[0253] If desired, one or more polynucleotides may optionally include a nucleotide sequence encoding a secretion signal peptide fused in-frame with its polypeptide sequence. This secretion signal peptide directs the secretion of an antibody polypeptide by cells expressing the one or more polynucleotides, which is then cleaved by the cells from the secreted polypeptide. The one or more polynucleotides may further optionally include sequences that facilitate the large-scale production of the vector, with only the intended function being addressed. For gene therapy, polynucleotides can be manufactured and administered using procedures described in the literature on various transgenes. See, for example, Isner et al., Circulation, 91:2687-2692, 1995 and Isner et al., Human Gene Therapy, 7:989-1011, 1996.

[0254] In some embodiments, the polynucleotide may further include additional sequences that facilitate uptake by host cells and expression of the antibody or its fragment (and / or any other peptide). In some embodiments, a “naked” transgene encoding the antibody or its fragment described herein (e.g., a transgene without a viral vector, liposome vector, or other vector to facilitate transfection) is utilized.

[0255] The polynucleotides of this disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA and synthetic DNA, and DNA may be double-stranded or single-stranded (where one strand may be a coding strand or a non-coding (antisense) strand). In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0256] This disclosure further relates to variants of the polynucleotides described herein, which, for example, encode fragments, analogs, and / or derivatives of the binding molecules of this disclosure. In certain embodiments, this disclosure provides polynucleotides having a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to the polynucleotide encoding the binding molecule of this disclosure, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical. As used herein, the phrase "polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to its reference sequence. However, the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of its reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations in the reference sequence can be made at the 5' or 3' end of the reference nucleotide sequence, or at any position between these ends, and can be scattered either individually among the nucleotides in the reference sequence or in one or more consecutive groups within the reference sequence.

[0257] The polynucleotide variant may include modifications in the coding region, non-coding region, or both. In some embodiments, the polynucleotide variant includes modifications that result in silent substitutions, additions, or deletions but do not alter the properties or activity of the encoded polypeptide. In some embodiments, the polynucleotide variant includes silent substitutions that do not alter the amino acid sequence of the polypeptide (due to degeneracy of the genetic code). For various reasons, for example, polynucleotide variants can be generated to optimize codon expression to suit a particular host (i.e., changing codons in human mRNA to codons preferred by a bacterial host, e.g., E. coli). In some embodiments, the polynucleotide variant includes at least one silent mutation in the non-coding region or coding region of its sequence.

[0258] In some embodiments, polynucleotide variants are generated to modulate or modify the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to decrease the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant has increased expression of the encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, the polynucleotide variant has decreased expression of the encoded polypeptide compared to the parent polynucleotide sequence.

[0259] One or more polynucleotides encoding an antibody or a fragment thereof may be introduced into the host using any suitable vector. Examples of vectors described include replication-deficient retroviral vectors, lentiviral vectors (Kim et al., J. Virol., 72(1):811-816, 1998, Kingsman & Johnson, Scrip Magazine, October, 1998, pp.43-46), parvovirus vectors, such as adeno-associated virus (AAV) vectors (US Patent Nos. 5,474,9351, 5,139,941, 5,622,856, 5,658,776, 5,773,289, 5,789,390, 5,834,441, 5,863,541, 5,851,521, 5,252,479, Gnatenko et al.) al., J. Invest. Med., 45:87-98, 1997), adenovirus (AV) vectors (US Patent Nos. 5,792,453, 5,824,544, 5,707,618, 5,693,509, 5,670,488, 5,585,362, Quantin et al., Proc. Natl. Acad. Sci. USA, 89:2581-2584, 1992, Stratford Perricaudet et al., J. Clin. Invest., 90:626-630, 1992 and Rosenfeld et al.) Examples include, but are not limited to, al., Cell, 68:143-155, 1992), adenovirus adeno-associated virus chimeras (U.S. Patent No. 5,856,152), vaccinia virus vectors or herpesvirus vectors (U.S. Patents No. 5,879,934, 5,849,571, 5,830,727, 5,661,033, and 5,328,688), lipofectin-mediated gene transfer (BRL), liposomal vectors (U.S. Patent No. 5,631,237), and combinations thereof.Any of these expression vectors can be prepared using standard recombinant DNA methods, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994). Optionally, viral vectors can be made replication-deficient by, for example, deleting or disrupting specific genes required for viral replication.

[0260] Other proposed nonviral delivery mechanisms include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990), DEAE-dextran (Gopal, Mol. Cell Biol., 5:1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, 1984), and direct microinjection (Harland and Weintraub, J. Cell Biol., 101:1094-1099, 1985, DNA-filled liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982, Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979, Felgner, Sci Am., 276(6):102-6, 1997, Felgner, Hum Gene Ther., 7(15):1791-3, 1996), cell sonication (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987), gene shock using high-speed microprojectiles (Yang et al., Proc. Natl. Acad. Sci Examples include transfection via receptors (USA, 87:9568-9572, 1990) and transfection via receptors (Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993).

[0261] Vectors (or antibodies or fragments thereof, or nucleic acids, as disclosed herein) may be encapsulated in liposomes. See, for example, Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991), and Radler et al., Science, 275(5301):810-814, (1997). Also intended are various commercial approaches related to “lipofection” technology. In some embodiments, liposomes may be complexed with hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote the entry of liposome-encapsulated DNA into cells (Kaneda et al., Science, 243:375-378, 1989). In some embodiments, liposomes are complexed or utilized in combination with nuclear non-histone chromosome proteins (HMG-1) (Kato et al., J. Biol. Chem., 266:3361-3364, 1991). In some embodiments, liposomes are complexed or utilized in combination with both HVJ and HMG-1. Such expression constructs have been successfully used for the in vitro and in vivo transfer and expression of nucleic acids. In some embodiments, an IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) is included in the liposome, and the liposome is directed to cells expressing IL13Rα2 on its surface (such as cancer cells).

[0262] The cells may contain one or more polynucleotides or one or more vectors, for example, the cells may be transformed or transfected with one or more polynucleotides encoding an IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate), or one or more vectors containing such one or more polynucleotides. In some embodiments, the cells express an IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) containing one or more CDRs (including six) whose identity with CDRs of A22, A33 and / or A52 (see, for example, Tables 1, 2, and / or 3) is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the cells express an IL13Rα2 conjugate (e.g., an antibody) (including a human IL13Rα2 conjugate) containing VH and VL, which are identical to the CDRs of A22, A33 and / or A52 (see, for example, Tables 1, 2, and / or 3). The cells may be prokaryotic cells, e.g., Escherichia coli cells (see, for example, Pluckthun et al., Methods Enzymol., 178:497-515, 1989), or eukaryotic cells, e.g., animal cells (e.g., myeloma cells, Chinese hamster ovary (CHO) cells, or hybridoma cells), yeast (e.g., Saccharomyces cerevisiae), insect cells, or plant cells (e.g., tobacco cells, maize cells, soybean cells, or rice cells). The use of mammalian host cells may result in translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that are desirable to confer optimal biological activity to recombinant expression products. Similarly, polypeptides (e.g., IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates)) may be modified by covalent bonding to include one or more glycans, polyoxyethylene glycol, or polypropylene glycol, and / or to include water-soluble polymers.

[0263] Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Such host cells are also useful for amplifying polynucleotides and expressing polypeptides encoded by those polynucleotides. In this regard, the process of producing IL13Rα2 conjugates (e.g., antibodies) may involve culturing host cells and isolating the IL13Rα2 conjugates. Transferring naked DNA expression constructs into cells can be achieved using particle bombardment, which relies on its ability to rapidly accelerate DNA-coated microprojectiles, allowing them to penetrate the cell membrane and enter cells without killing them (Klein et al., Nature, 327:70-73, 1987). Several devices for accelerating small particles have been developed. One such device relies on high-voltage emission to generate current and, consequently, supply power (Yang et al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990). The microprojectiles used consist of biologically inert materials, such as tungsten or gold beads. Host cells may be isolated and / or purified. Host cells may be cells transformed in vivo to transiently or permanently express their polypeptide in vivo. Host cells may also be isolated cells that have been transformed ex vivo and introduced after transformation, for example, to produce their polypeptide in vivo for therapeutic purposes. Transgenic humans are explicitly excluded from the definition of host cells.

[0264] 5.4. Manufacturing Method Antibodies that bind to IL13Rα2 may be obtained by any suitable method, for example (but not limited to), immunization of whole cells containing IL13Rα2 with antibody aggregates, recombinant methods, or by screening a library of antibodies or antibody fragments using the extracellular domain epitope of IL13Rα2. Monoclonal antibodies can be produced using various known techniques (see, for example, Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991), Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980), Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988), and Picksley et al., “Production of monoclonal antibodies against proteins expressed in E. coli,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). One exemplary technique for generating monoclonal antibodies involves immunizing an animal with the human IL13Rα2 antigen and generating a hybridoma from spleen cells isolated from that animal. The hybridoma may produce a monoclonal antibody or antibody fragment that binds to IL13Rα2.

[0265] In further embodiments, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques described, for example, in Antibody Phage Display: Methods and Protocols, PMO'Brien and R. Aitken, eds., Humana Press, Totawa NJ, 2002. In principle, synthetic antibody clones are selected by screening phage libraries containing phages that display various fragments of the antibody variable region (Fv) fused to the phage coat protein. Such phage libraries are screened against a desired antigen. Clones expressing Fv fragments that can bind to the desired antigen are separated from unbound clones in the library because they adsorb to that antigen. The bound clones can then be eluted from their antigen and further enriched by additional antigen adsorption / elution cycles.

[0266] Variable domains can be functionally presented on phages either as single-stranded Fv(scFv) fragments (VH and VL covalently linked through a short, flexible peptide) or as Fab fragments (VH and VL each fused to a constant domain and interacting noncovalently), as described, for example, in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994).

[0267] The VH and VL gene repertoires can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, after which antigen-binding clones can be examined, as described in the aforementioned literature by Winter et al. Libraries derived from immunized sources yield high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, as described in Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned without any immunization to provide a single source of human antibodies against a wide range of non-self and self-antigens. Finally, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992), naive libraries can also be synthetically constructed by cloning an unrecombined V gene segment from stem cells that encodes the hypervariable CDR3 region and achieves in vitro recombination, and using PCR primers containing random sequences.

[0268] The screening of the library can be achieved by various techniques known in the art. For example, IL13Rα2 (e.g., polypeptide, fragment, or epitope of IL13Rα2) can be used to coat wells of an adsorption plate and express on host cells attached to the adsorption plate, or to use in cell sorting, or to conjugate to biotin for capture with streptavidin-coated beads, or to use in any other way for panning a display library. The selection of antibodies with slow dissociation rates (e.g., good binding affinity) can be facilitated by prolonged washing and monovalent phage display (as described in Bass et al., Proteins, 8:309-314 (1990) and WO92 / 09690), as well as the use of antigens with low coating density (as described in Marks et al., Biotechnol., 10:779-783 (1992)).

[0269] IL13Rα2 conjugates (e.g., antibodies) can be obtained by designing an appropriate antigen screening procedure to select a suitable phage clone, and then constructing a full-length IL13Rα2 conjugate (e.g., antibody) clone using the VH sequence and / or VL sequence (e.g., Fv sequence) derived from the suitable phage clone, or various CDR sequences derived from the VH sequence and VL sequence, and a suitable constant region (e.g., Fc) sequence as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0270] Similarly, human antibodies that bind to IL13Rα2 may be produced by any of a number of techniques, including, but are not limited to, transformation of human peripheral blood cells (e.g., cells including B lymphocytes) with Epstein-Barr virus (EBV), in vitro immunization of human B cells, fusion of spleen cells derived from immunized transgenic mice having inserted human immunoglobulin genes, isolation from a human immunoglobulin V region phage library, or other procedures known in the art and based on this disclosure. Methods for obtaining human antibodies from transgenic animals are further described, for example, in Bruggemann et al., Curr. Opin. Biotechnol., 8:455 58, 1997; Jakobovits et al., Ann. NYAcad. Sci., 764:525 35, 1995; Green et al., Nature Genet., 7:13-21, 1994; Lonberg et al., Nature, 368:856-859, 1994; Taylor et al., Int. Immun. 6:579-591, 1994; and U.S. Patent No. 5,877,397.

[0271] For example, a human antibody that binds to IL13Rα2 may be obtained from a transgenic animal that has been engineered to produce a specific human antibody in response to an antigen challenge. For example, WO98 / 24893 discloses a transgenic animal having a human Ig locus that does not produce functional endogenous immunoglobulins due to inactivation of the endogenous heavy and light chain loci. Transgenic mammalian hosts other than primates that can exert an immune response to an immunogen are also described, in which the antibody has a constant and / or variable region of a primate, and the locus encoding its endogenous immunoglobulin is substituted or inactivated. WO96 / 30498 discloses modifying an immunoglobulin locus in a mammal using the Cre / Lox system, for example, by replacing all or part of the constant or variable region to form a modified antibody molecule. WO94 / 02602 discloses a non-human mammalian host having an inactivated endogenous Ig locus and a functional human Ig locus. U.S. Patent No. 5,939,598 discloses a method for producing a transgenic mouse, which lacks an endogenous heavy chain and expresses an exogenous epiglobulin locus containing one or more heterologous constant regions. Transgenic animals, such as those described herein, can be used to induce an immune response against a given antigen molecule, and antibody-producing cells can be removed from the animal and used to generate hybridomas that secrete human-derived monoclonal antibodies. Immunization protocols, adjuvants, etc., are known in the art and have been used in the immunization of transgenic mice, for example, as described in WO96 / 33735. The monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effects of the corresponding protein.

[0272] In some embodiments, the IL13Rα2 conjugates described herein include scaffolds of non-antibody proteins. Non-limiting examples of such non-antibody protein scaffolds include fibronectin scaffolds, antikalin, adonectin, afibody, DARPin, finomers, afitin, affin, avimers, cysteine-rich Nottin peptides, or modified Kunitz-type inhibitors. Methods for generating such non-antibody protein scaffolds are well known in the art, and any one of them can be used to generate an IL13Rα2 conjugate containing a non-antibody protein scaffold (see, for example, Simeon and Chen, Protein Cell, 9(1):3-14 (2018), Yang et al., Annu Rev Anal Chem (Palo Alto Calif). 10(1):293-320 (2017)).

[0273] Various methods for generating antibodies from polynucleotides are generally well known. For example, basic molecular biology procedures are described in Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). In addition, numerous publications describe techniques suitable for DNA manipulation, expression vector preparation, and antibody preparation by appropriate cell transformation and culture (see, for example, Mountain and Adair, Chapter 1 in Biotechnology and Genetic Engineering Reviews, Tombs ed., Intercept, Andover, UK, 1992 and Current Protocols in Molecular Biology, Ausubel ed., Wiley Interscience, New York, 1999).

[0274] IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) are produced by any suitable method, such as isolation from immunized animals, synthesis or recombination, or genetic engineering, including those mentioned above. Antibody fragments derived from antibodies can be obtained, for example, by protein hydrolysis of the antibody. For example, papain digestion of the whole antibody yields a 5S fragment called F(ab')2, or pepsin digestion yields two monovalent Fab and Fc fragments. F(ab')2 can be further cleaved with a thiol reducing agent to produce a 3.5S monovalent Fab fragment. Methods for generating antibody fragments are described, for example, in Edelman et al., Methods in Enzymology, 1: 422 Academic Press (1967); Nisonoff et al., Arch. Biochem. Biophys., 89: 230-244, 1960; Porter, Biochem. J., 73: 119-127, 1959; U.S. Patent No. 4,331,647; and further in Current Protocols in Immunology (Coligan et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1, 2.8.10, and 2.10A.1, 2.10A.5, by Andrews, SM and Titus, JA.

[0275] IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) can be genetically engineered. For example, IL13Rα2 conjugates (e.g., antibodies) (including human IL13Rα2 conjugates) may contain variable region domains generated, for example, by recombinant DNA manipulation. In this regard, the variable region may be arbitrarily modified by insertions, deletions, or changes in the amino acid sequence of the antibody to generate the antibody containing the above-mentioned variable region. The polynucleotide encoding the relevant CDR can be prepared, for example, by synthesizing the variable region using mRNA from antibody-producing cells as a template, using polymerase chain reaction (see, for example, Courtenay Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995), Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley Liss, Inc. 1995), and Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106-110, 1991). Current antibody manipulation techniques enable the construction of a manipulated variable region domain comprising at least one CDR, and optionally, one or more framework amino acids derived from the first antibody and the remainder of the variable region domain derived from the second antibody. Such techniques can be used, for example, to humanize an antibody or to improve its affinity for a binding target. Exemplary methods for generating humanized antibodies are also described in the above chapter.

[0276] 5.5. Pharmaceutical Compositions In one embodiment, the present disclosure further provides a composition, such as a pharmaceutical composition, comprising at least one binder provided in the present invention (e.g., one antibody or antigen-binding fragment provided herein), a nucleic acid provided in the present invention, a vector provided in the present invention, or a cell provided in the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an IL13Rα2 binder provided in the present disclosure (e.g., an antibody or antigen-binding fragment provided in the present disclosure) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a nucleic acid provided in the present disclosure (e.g., a nucleic acid encoding an antibody or antigen-binding fragment provided in the present disclosure) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a vector provided in the present disclosure (e.g., a vector comprising a nucleic acid as disclosed herein and expressing an IL13Rα2 binder as disclosed herein) and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of cells provided in this disclosure (for example, cells containing nucleic acids encoding the antibodies or antigen-binding fragments provided in this disclosure, and / or cells expressing the antibodies or antigen-binding fragments provided in this disclosure) and pharmaceutically acceptable excipients.

[0277] In some embodiments, the pharmaceutical compositions provided in this disclosure are binders, nucleic acids, vectors, or cells provided in this disclosure, which are prepared for storage by mixing a binder, nucleic acid, vector, or cell of a desired purity with any physiologically acceptable excipient (see, for example, Remington, Remington's Pharmaceutical Sciences (18th ed. 1980)) in aqueous solution, lyophilized, or other dry form.

[0278] The binders, nucleic acids, vectors, or cells of this disclosure may be formulated in any form suitable for delivery to target cells / tissues, for example, as microcapsules or macroemulsions (Remington, cited above; Park et al., 2005, Molecules 10:146-61; Malik et al., 2007, Curr. Drug. Deliv. 4:141-51), as sustained-release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153-57), or as liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).

[0279] The binders, nucleic acids, vectors, or cells provided in this disclosure may also be encapsulated in microcapsules prepared, for example, by coacervation or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in the aforementioned literature by Remington.

[0280] Various compositions and delivery systems are known and can be used with binders, nucleic acids, vectors, or cells as described herein, including, but not limited to, liposomes, microparticles, microcapsules, encapsulation in recombinant cells, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), and the construction of nucleic acids as part of retroviral vectors or other vectors. In another embodiment, the compositions can be supplied as controlled-release or sustained-release systems. In one embodiment, a pump can be used to achieve controlled or continuous release (see, for example, Langer; Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201-40; Buchwald et al., 1980, Surgery 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. 321:569-74).In another embodiment, polymer materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., antibodies or their antigen-binding fragments as described herein), or compositions provided in this disclosure (e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974), Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984), Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126, Levy et al., 1985, Science 228:190-92, During et al., 1989, Ann. Neurol. 25:351-56, Howard et al. See al., 1989, J. Neurosurg. 71:105-12, U.S. Patents No. 5,679,377, 5,916,597, 5,912,015, 5,989,463 and 5,128,326, WO99 / 15154 and WO99 / 20253. Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of leaching impurities, stable during storage, sterilized, and biodegradable.

[0281] In yet another embodiment, a controlled-release or sustained-release system can be placed in close proximity to a specific target tissue, such as the nasal passages or lungs, requiring only a fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release Vol.2, 115-38 (1984)). Controlled-release systems are discussed, for example, in Langer, 1990, Science 249:1527-33. Sustained-release formulations containing one or more antibodies or antigen-binding fragments as described herein can be produced using any technique known to those skilled in the art (see, for example, U.S. Patent No. 4,526,938, WO91 / 05548 and WO96 / 20698, Ning et al., 1996, Radiotherapy & Oncology 39:179-89, Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60).

[0282] 5.6.How to use In another aspect, the Disclosure provides a method of using the binder or composition provided herein. In a further aspect, the Disclosure provides a binder or composition as disclosed herein, for use as a pharmaceutical. In a further aspect, the Disclosure provides a binder or composition as disclosed herein, for use in treating a disease or condition, for example, a disease or condition disclosed herein. In one aspect, the Disclosure provides a binder or composition as disclosed herein, for use in a manner disclosed herein.

[0283] In some embodiments, the binder binds to IL13Rα2. In some embodiments, the binder binds to the IL13Rα2 epitope. In addition to or instead of this, the binder binds to a complex comprising IL13Rα2 and IL13. In further embodiments, the binder binds to a complex comprising the extracellular domains of IL13Rα2 and IL13. In yet another embodiment, the binder binds to the epitope of a complex comprising the ECD of IL13Rα2 and IL13. In various embodiments, IL13Rα2 is human IL13Rα2. In yet another embodiment, IL13Rα2 also refers to cynoIL13Rα2. In other embodiments, the binder does not bind to cynoIL13Rα2 or a complex comprising cynoIL13Rα2. In yet another embodiment, the binder does not bind to IL13Rα1.

[0284] In some embodiments, methods for identifying cells expressing IL13Rα2 (including cells overexpressing IL13Rα2) are provided herein. In some embodiments, methods for reducing (including, but not limited to, eliminating) the viability or proliferation of IL13Rα2-expressing cells (including IL13Rα2-overexpressing cells) are provided herein. In some embodiments, methods for killing IL13Rα2-expressing cells (including IL13Rα2-overexpressing cells) are provided herein. In further embodiments, the IL13Rα2-expressing cells are cancer cells. Examples of cancer cell types include, but are not limited to, melanoma cells, lung cancer cells (including non-small cell lung cancer), colorectal cancer cells, malignant glioma cells, pancreatic cancer cells, ovarian cancer cells, breast cancer cells, liver cancer cells, head and neck cancer cells, and renal cancer cells.

[0285] In some embodiments, the methods disclosed herein are in vitro or ex vivo methods. In other embodiments, the methods disclosed herein are in vivo methods. In some embodiments, the uses disclosed herein are in vitro or ex vivo uses. In other embodiments, the uses disclosed herein are in vivo uses. In some embodiments, the in vivo methods or in vivo uses disclosed herein include administering a binder as disclosed herein to a subject having cells expressing IL13Rα2, for example, tumor cells expressing IL13Rα2.

[0286] Therapeutic use and treatment methods In some embodiments, the IL13Rα2 binders (e.g., antibodies) described herein are useful in compositions and methods for treating diseases or disorders. Accordingly, in some embodiments, the Disclosure provides a method for treating a disease or disorder in a subject, comprising administering the binder or pharmaceutical composition provided herein to that subject. In other embodiments, the Disclosure provides a use of the binder or pharmaceutical composition provided herein for the treatment of a disease or disorder in a subject. In other embodiments, the Disclosure provides a binder or pharmaceutical composition provided herein for use in the manufacture of a medicament for treating a disease or disorder.

[0287] In some embodiments, the treatments provided in this disclosure include alleviating one or more symptoms associated with a disease or disorder (e.g., cancer or tumor).

[0288] Accordingly, in some embodiments, the Disclosure provides a method for the targeted relief of one or more symptoms associated with cancer or tumors, comprising administering to the subject an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein. In some embodiments, the Disclosure provides an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein for use in the targeted relief of one or more symptoms associated with cancer or tumors. In some embodiments, the Disclosure provides the use of an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein for use in the manufacture of a medicament for the targeted relief of one or more symptoms associated with cancer or tumors.

[0289] In some embodiments, the treatments provided herein involve reducing tumor size in a tumorous subject. Accordingly, in some embodiments, what is described herein is a method for reducing tumor size in a tumorous subject, comprising administering to the subject an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein. In some embodiments, what is provided herein is an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein, for use in reducing tumor size in a tumorous subject. In some embodiments, what is provided herein is the use of an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein, for use in the manufacture of a pharmacopoeia for reducing tumor size in a tumorous subject.

[0290] In some embodiments, the methods described herein are for enhancing tumor cell removal in a tumor-containing subject, comprising administering to the subject an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein. In some embodiments, the disclosure provides an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein for use in enhancing tumor cell removal in a tumor-containing subject. In some embodiments, the disclosure provides the use of an IL13Rα2 conjugate (e.g., an antibody) or a pharmaceutical composition described herein for use in the manufacture of a pharmacopoeia that enhances tumor cell removal in a tumor-containing subject.

[0291] "Enhancing" tumor cell removal includes, but is not mandatory, a 100% enhancement of removal. Any enhancement of the removal rate is intended. Similarly, "regulating" tumor growth refers to reducing tumor size, slowing tumor growth, or inhibiting the growth of an existing tumor. This includes, but is not mandatory, complete disappearance of the tumor, and either reduction in tumor size or slowing of tumor growth constitutes a beneficial biological effect in the subject. In this regard, tumor cell removal may be enhanced by at least about 5%, at least about 10%, or at least about 20% compared to the level of removal observed in the absence of the method of this disclosure (e.g., a biologically compatible control subject or control sample that has not been exposed to the agent of the method). The effect is detected, for example, by reduction in tumor size, decrease or maintenance of tumor marker levels, or decrease or maintenance of the tumor cell population. In some embodiments, tumor cell removal is enhanced by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, and at least about 90% or more (about 100%) compared to the tumor cell removal rate in the absence of the IL13Rα2 conjugate (e.g., antibody) or pharmaceutical composition of the method of the present disclosure.

[0292] Methods for regulating (e.g., inhibiting, reducing, or preventing) tumor growth in a subject are also provided. For example, such methods include administering to the subject a composition comprising an IL13Rα2 conjugate (e.g., an antibody) as disclosed herein, or another composition as disclosed herein, in an amount effective to regulate tumor growth in that subject.

[0293] In some embodiments, the disease or disorder is cancer or a tumor. In further embodiments, the cancer or tumor expresses IL13Rα2. In even further embodiments, the cancer or tumor overexpresses IL13Rα2. In some embodiments, the subject is a human subject.

[0294] As used herein, “tumor” refers to the growth or proliferation of any neoplastic cells, whether malignant or benign, and to all precancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, melanoma, lung cancer (including non-small cell lung cancer), colorectal cancer (CRC), malignant glioma, pancreatic cancer, ovarian cancer, breast cancer, liver cancer, head and neck cancer, and kidney cancer.

[0295] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor or cancer is not a solid tumor. In further embodiments, the cancer is a leukemia. In some embodiments, the tumor or cancer is a recurrent tumor or cancer. In some embodiments, the tumor or cancer is a metastatic tumor or cancer. In some embodiments, the tumor or cancer is a primary tumor or cancer. In some embodiments, the tumor or cancer reaches remission but may recur. In some embodiments, the tumor or cancer is unresectable. In addition to or instead of this, the tumor or cancer is resistant to chemotherapy or other anti-cancer therapies. In further embodiments, the cancer or tumor expresses IL13Rα2. In further embodiments, the cancer or tumor overexpresses IL13Rα2.

[0296] In addition, IL13Rα2 conjugates (e.g., antibodies) may be used to mitigate or reduce cancer-related side effects, such as bone deterioration, spinal collapse, and paralysis. In one embodiment, a subject who has bone metastases or is at risk of developing bone metastases is administered an amount of IL13Rα2 conjugate (e.g., antibody) that reduces the deterioration of the surrounding bone. Thus, in some embodiments, IL13Rα2 conjugates prevent bone deterioration due to bone metastases, and in doing so, reduce or do not reduce the proliferation of tumor cells. In some embodiments, IL13Rα2 conjugates (e.g., antibodies) both prevent bone deterioration due to bone metastases and reduce the proliferation of tumor cells. Generally, the effect on tumor cell proliferation (e.g., inhibition of proliferation or no effect on proliferation) depends on the specific metastatic microenvironment. For example, the spread of metastases in a microenvironment with a substantial amount of type 1 collagen may be inhibited. In contrast, while the spread of metastases in a microenvironment lacking significant amounts of type 1 collagen cannot be inhibited, bone deterioration observed near those metastases can be mitigated or prevented.

[0297] Also provided are methods for treating a disease or disorder (e.g., cancer) by administering, alone or in combination with another agent, an IL13Rα2 conjugate (e.g., an antibody), a human IL13Rα2 conjugate, or a pharmaceutical composition as disclosed herein to a target in need of treatment.

[0298] The methods described herein allow for the administration of one or more additional therapeutic agents in combination with the IL13Rα2 conjugates (e.g., antibodies) or fragments thereof described herein, or with the pharmaceutical compositions described herein. The additional agents may be agents that target tumor cells or cancer cells. The additional agents may also be agents that target immune cells (e.g., NK cells or T cells). In some embodiments, the IL13Rα2 conjugates or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by about 10% to 90% or about 2 to 100 times. In some embodiments, the IL13Rα2 conjugates or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 10%. In some embodiments, the IL13Rα2 conjugates or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 20%. In some embodiments, the IL13Rα2 conjugates or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 30%. In some embodiments, the IL13Rα2 conjugates or pharmaceutical compositions provided herein enhance the therapeutic effect of the additional agent by at least 40%. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided in this disclosure enhances the therapeutic effect of an additional agent by at least 50%. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided in this disclosure enhances the therapeutic effect of an additional agent by at least 60%. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided in this disclosure enhances the therapeutic effect of an additional agent by at least 70%. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided in this disclosure enhances the therapeutic effect of an additional agent by at least 80%. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided in this disclosure enhances the therapeutic effect of an additional agent by at least 90%. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided in this disclosure enhances the therapeutic effect of an additional agent by at least 2 times. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided in this disclosure enhances the therapeutic effect of an additional agent by at least 5 times.In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided herein enhances the therapeutic effect of an additional agent by at least 10 times. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided herein enhances the therapeutic effect of an additional agent by at least 20 times. In some embodiments, the IL13Rα2 binder or pharmaceutical composition provided herein enhances the therapeutic effect of an additional agent by more than 50 times.

[0299] A dosage regimen of an IL13Rα2 conjugate (e.g., an antibody) or pharmaceutical composition as disclosed herein, where a particular dosage regimen for a particular subject will, in part, depend on the drug used, the dosage of the drug, the route of administration, and the cause and severity of any side effects. The amount of drug (e.g., an antibody) administered to the subject (e.g., a mammal such as a human) must be sufficient to produce the desired response over a reasonable time frame. Thus, in some embodiments, the amount of the IL13Rα2 conjugate (e.g., an antibody) or pharmaceutical composition described herein administered to the subject is an effective amount.

[0300] Suitable routes for administering the IL13Rα2 conjugates (e.g., antibodies), such as human IL13Rα2 conjugates (e.g., antibodies), or compositions described herein are well known in the art and include, for example, intravenous injection (e.g., intravenous infusion), intratumoral injection, or injection adjacent to a tumor or cancer. More than one route can be used to administer a drug (e.g., antibody), but certain routes may yield a faster and more effective response than others.

[0301] Gene therapy and cell therapy In some embodiments, a composition for use in accordance with the Disclosure comprises one or more nucleic acids encoding an IL13Rα2 conjugate (e.g., an antibody or a fragment thereof) provided in the Disclosure, or a nucleic acid complementary thereto. In specific embodiments, the nucleic acid is administered to a subject by gene therapy for use in the manner provided in the Disclosure, for example, to prevent, manage, treat and / or improve a disease or disorder (e.g., cancer, e.g., cancer expressing IL13Rα2). Such therapies include therapies carried out by administering the expressed nucleic acid or a nucleic acid that can be expressed to a subject. In embodiments, the nucleic acid produces an antibody encoded by the nucleic acid, which mediates a preventive or therapeutic effect.

[0302] Any method available in the art for recombinant gene expression (or gene therapy) may be used.

[0303] For a general overview of gene therapy methods, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods of recombinant DNA technology that are generally known in the art and can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993) and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0304] In specific embodiments, the composition comprises a nucleic acid encoding the antibody or fusion protein provided in this disclosure, the nucleic acid being part of an expression vector that expresses the antibody or fusion protein, or its heavy or light chain, in a suitable host. In particular, such nucleic acid has a promoter functionally linked to the coding region, for example, a heterologous promoter, the promoter being inducible or constitutive, and optionally tissue-specific and / or tumor-specific / cancer-specific. In another specific embodiment, a nucleic acid molecule is used in which the antibody sequence and any other desired sequence are flanked by regions that promote homologous recombination at desired sites in the genome, thereby achieving intrachromosomal expression of the nucleic acid encoding the antibody or fusion protein (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935, Zijlstra et al., 1989, Nature 342:435-438).

[0305] The nucleic acid can be delivered either directly (in which case the target is directly exposed to the nucleic acid or nucleic acid-carrying vector) or indirectly (in which case cells are first transformed with the nucleic acid in vitro before being transplanted into the target). These two approaches are known as in vivo gene therapy and ex vivo gene therapy, respectively.

[0306] In a specific embodiment, the nucleic acid sequence is administered directly in vivo, and the sequence is expressed to produce the encoded product. This can be achieved by any of the many methods known in the art, for example, by constructing the nucleic acid sequence as part of a suitable nucleic acid expression vector and administering the vector so that the sequence is located within the cell; by infection with a deficient or attenuated retroviral vector or other viral vector (see U.S. Patent No. 4,980,286); by direct injection of naked DNA; by the use of microparticle bombardment (e.g., a gene gun (Biolistic, Dupont)); by coating with lipids, cell surface receptors or transfection agents; by encapsulation in liposomes, microparticles or microcapsules; by administering the nucleic acid sequence as a conjugate to a peptide known to enter the nucleus; or by administering the nucleic acid sequence as a conjugate to a ligand that undergoes receptor-mediated endocytosis (e.g., see Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types that specifically express the receptor). In another embodiment, a complex can be formed between the nucleic acid and a ligand, the ligand containing a fusion-inducing viral peptide that disrupts endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be guided in vivo for cell-specific uptake and expression by targeting a specific receptor (see, e.g., WO92 / 06180, WO92 / 22635, WO92 / 20316, WO93 / 14188, WO93 / 20221). Alternatively, the nucleic acid can be introduced into cells for expression by homologous recombination and integrated into host cell DNA (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935 and Zijlstra et al., 1989, Nature 342:435-438).

[0307] In specific embodiments, a viral vector containing a nucleic acid sequence encoding an antibody is used. For example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for the precise packaging of the viral genome and its integration into host cell DNA. The nucleic acid sequence encoding the antibody used in gene therapy can be cloned into one or more vectors, thereby facilitating the delivery of the gene to the target. Further details on retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the MDR1 gene to hematopoietic stem cells in order to make those stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include Clowes et al., 1994, J. Clin. Invest. 93:644-651, Klein et al., 1994, Blood 83:1467-1473, Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141, and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

[0308] Adenoviruses are another viral vector that can be used in recombinant antibody production. Adenoviruses are particularly attractive vehicles for delivering genes to the airway epithelium. Adenoviruses infect the airway epithelium in nature, and in that airway epithelium, adenoviruses cause mild illness. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, provides an overview of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10, demonstrates the use of adenovirus vectors to transfer genes into the airway epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234, International Publication W094 / 12649; and Wang et al., 1995, Gene Therapy 2:775-783. In specific embodiments, adenovirus vectors are used.

[0309] Adeno-associated virus (AAV) can also be used (Walsh et al., 1993, Proc.Soc.Exp.Biol.Med.204:289-300 and U.S. Patents 5,436,146). In specific embodiments, an AAV vector is used to express an anti-IL13Rα2 antibody, such as those provided in this disclosure. In certain embodiments, the AAV contains a nucleic acid encoding the VH domain. In other embodiments, the AAV contains a nucleic acid encoding the VL domain. In certain embodiments, the AAV contains nucleic acids encoding both the VH and VL domains. In some embodiments of the methods provided in this disclosure, the subject is administered an AAV containing a nucleic acid encoding the VH domain and an AAV containing a nucleic acid encoding the VL domain. In other embodiments, the subject is administered an AAV containing nucleic acids encoding both the VH and VL domains. In certain embodiments, the VH and VL domains are overexpressed.

[0310] In some embodiments, oncolytic viruses may be used for the recombinant production of the antibodies provided in this disclosure. Oncolytic viruses may preferentially infect and kill cancer cells. Once infected cancer cells are destroyed by oncolysis, they may release new infectious viral particles or viral virions to help destroy the remaining tumor. In specific embodiments, the oncolytic virus is a virus that, when injected into a tumor, results in tumor regression. In another specific embodiment, the oncolytic virus is a virus that selectively replicates in cancer cells, kills the cancer cells, and spreads within the tumor. In yet another specific embodiment, the oncolytic virus is a virus that selectively replicates in cancer cells, kills the cancer cells, and spreads within the tumor without causing any significant damage to normal tissue. In some embodiments, the selectivity of the virus to replicate in cancer cells is determined by using in vitro or ex vivo assays known to those skilled in the art, compared to non-cancer cells (e.g., healthy cells). In one embodiment, a virus selectively replicates in cancer cells if, after incubation with the virus, a statistically significant increase in the number of viral particles is detected in cancer cells compared to the number of viral particles detected in non-cancer cells (e.g., healthy cells) in an in vitro or ex vivo assay. In another embodiment, a virus selectively kills cancer cells if, in an in vitro or ex vivo assay, a statistically significant amount of cancer cells are killed compared to the amount of non-cancer cells (e.g., healthy cells) killed in the same assay. In one embodiment, an oncolytic virus preferentially replicates in cancer cells in nature and is non-pathogenic in humans. Oncolytic viruses can be non-pathogenic in humans due to increased sensitivity to natural antiviral signals or reliance on carcinogenic signaling pathways. In some embodiments, the oncolytic virus is a parvovirus (e.g., autonomic parvovirus), myxoma virus, triparamyxovirus (e.g., Newcastle disease virus), reovirus, or Seneca Valley virus.In one embodiment, the oncolytic virus is wild-type parvovirus H1 (ParvOryx). In another embodiment, the oncolytic virus is vesicular stomatitis virus. In yet another embodiment, the oncolytic virus is triparamyxovirus. In some embodiments, the oncolytic virus is genetically modified influenza virus, measles virus, poliovirus, vaccinia virus, poxvirus, picornavirus, alphavirus, retrovirus, rhabdovirus, reovirus, adenovirus, herpes simplex virus, or vesicular stomatitis virus. In some embodiments, such viruses are attenuated.

[0311] Another approach to gene therapy and cell therapy involves transferring genes into cells during tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, this transfer method involves transferring a selectable marker into the cells. These cells are then selectively targeted to incorporate the transferred gene, isolating cells that express that gene. These cells are then delivered to the target.

[0312] In this embodiment, nucleic acids are introduced into cells, and the resulting recombinant cells are administered in vivo. Such introduction can be carried out by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral vector or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, and spheroplast fusion. Numerous techniques are known in the art for introducing foreign genes into cells (see, for example, Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618, Cohen et al., 1993, Meth. Enzymol. 217:618-644, Clin. Pharma. Ther. 29:69-92 (1985)), and these techniques can be used in accordance with the methods provided herein, provided that the necessary development and physiological functions of the recipient cells are not disrupted. This technique ensures that nucleic acids are stably transferred into cells, and as a result, these nucleic acids become expressible by those cells, and for example, become heritable and expressible by the offspring of those cells.

[0313] The resulting recombinant cells can be delivered to the target by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem cells or progenitor cells) can be administered intravenously. The amount of cells to be used depends on the desired effect, the patient's condition, etc., and can be determined by those skilled in the art.

[0314] Cells into which nucleic acids can be introduced for the purpose of gene therapy and / or cell therapy include any desired available cell types, such as epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells, e.g., T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, and various stem cells or progenitor cells, in particular stem cells or progenitor cells obtained from, for example, bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

[0315] In specific embodiments, the cells used in gene therapy and / or cell therapy are autologous to the subject. In other embodiments, the cells used in cell therapy and / or gene therapy are allogeneic to the subject.

[0316] In embodiments of gene and / or cell therapy using recombinant cells, an antibody-encoding nucleic acid sequence is introduced into the cells so that the nucleic acid sequence can be expressed by the cells or their offspring, and then the recombinant cells are administered in vivo to obtain a therapeutic effect. In specific embodiments, stem cells or progenitor cells are used. Any stem cells and / or progenitor cells that can be isolated and maintained in vitro can potentially be used according to this embodiment of the method provided in this disclosure (see, for example, WO94 / 08598, Stemple and Anderson, 1992, Cell 7 1:973-985, Rheinwald, 1980, Meth. Cell Bio. 21A:229, and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

[0317] In specific embodiments, the nucleic acid introduced for the purpose of gene therapy and / or cell therapy includes an inducible promoter functionally linked to the coding region, thereby allowing the expression of the nucleic acid to be controlled by controlling the presence or absence of appropriate transcription inducers.

[0318] Diagnostic use and detection methods Labeled conjugating molecules, such as labeled antibodies and derivatives, and their analogues, which bind immunospecifically to antigens, can be used for diagnostic purposes to detect, diagnose, or monitor diseases.

[0319] The antibodies provided herein can be used to assay antigen levels in biological samples using classical immunohistochemical methods described herein or known to those skilled in the art (see, for example, Jalkanen et al., 1985, J. Cell. Biol. 101:976-985 and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, e.g., enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Suitable antibody assay labels are known in the art, and such labels include enzyme labels, e.g., glucose oxidase, radioisotopes, e.g., iodine. 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) and technetium ( 99 Examples include luminescent labels such as luminol, fluorescent labels such as fluorescein and rhodamine, and biotin.

[0320] In this field, it will be understood that the amount of imaging required to produce a diagnostic image is determined by the size of the target and the imaging system used. In the case of radioactive isotopes, for human subjects, the amount of radioactivity injected is usually about 5 to 20 millicuries. 99This would be within the Tc range. Subsequently, the labeled antibody accumulates at the location of cells containing specific proteins. In vivo tumor imaging is described in SW Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes, eds., Masson Publishing Inc. (1982)).

[0321] The time interval after administration, during which the labeled antibody becomes concentrated at the site in the target and the unbound labeled antibody disappears to background levels, is 6 to 48 hours, 6 to 24 hours, or 6 to 12 hours, depending on several variable factors (including the type of label used and the method of administration). In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days.

[0322] The presence of labeled molecules can be detected in a subject using methods known in the art for in vivo scanning. These methods depend on the type of label used. Those skilled in the art will be able to determine an appropriate method for detecting a particular label. Methods and apparatus that may be used in the diagnostic methods provided herein include, but are not limited to, computed tomography (CT), whole-body scans, such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound.

[0323] In specific embodiments, the molecule is labeled with a radioisotope and detected in the patient using a radioresponsive surgical device (U.S. Patent No. 5,441,050 by Thurston et al.). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescence-responsive scanning device. In yet another embodiment, the molecule is labeled with a positron-emitting metal and detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

[0324] 5.7. Kit Furthermore, the Disclosure also provides IL13Rα2 binders (e.g., antibodies) or compositions (e.g., pharmaceutical compositions) provided herein, including kits containing the binders or compositions packaged in appropriate packaging materials. The kits optionally include a label or accompanying leaflet containing a description of its components, or instructions for using the components of the kit in vitro, in vivo, or ex vivo.

[0325] The term "packaging" refers to the physical structure that contains the components of the kit. Packaging can be made from materials that can keep the components sterile and are commonly used for this purpose (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0326] The kits provided in this disclosure may include labels or accompanying documents. These labels or accompanying documents may include “printed materials,” such as paper or cardboard, which may be separate, attached to components, kits, or packaging materials (e.g., boxes), or attached to ampoules, tubes, or vials containing kit components. In addition, the labels or accompanying documents may include computer-readable media, such as disks (e.g., hard disks, cards, memory disks), optical discs, such as CD-ROMs or DVD-ROM / RAM, DVDs, MP3s, magnetic tapes, electrical storage media, such as RAM and ROM, or hybrids thereof, such as magneto / optical storage media, FLASH® media, or memory type cards. The labels or accompanying documents may include manufacturer information, lot numbers, and information identifying the manufacturer’s location and date.

[0327] In addition, the kits provided in this disclosure may include other components. Each component of the kit may be placed in an individual container, and all of these various containers may be contained within a single package. The kits may also be designed for refrigerated storage. The kits may be further designed to contain cells containing the antibodies provided in this disclosure, or nucleic acids encoding the antibodies provided in this disclosure. The cells in the kits may be maintained under appropriate storage conditions until ready for use.

[0328] Furthermore, the Disclosure also provides a panel of antibodies that bind immunospecifically to the IL13Rα2 antigen. In specific embodiments, the Disclosure provides a panel of antibodies that have different association rate constants, different dissociation rate constants, different affinities to the IL13Rα2 antigen, and / or different specificities to the IL13Rα2 antigen. In particular embodiments, the Disclosure provides a panel of about 10, preferably about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 or more antibodies. Antibody panels can be used for assays such as ELISA, for example, in 96-well or 384-well plates.

[0329] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In practicing or testing the present invention, similar or equivalent methods and materials may be used, but only suitable methods and materials are described herein.

[0330] Where used herein, numerical values ​​are often expressed in range form throughout this specification. The use of range form is for convenience and simplification only and should not be construed as an immutable limitation to the scope of the invention unless clearly indicated otherwise in the context. Accordingly, unless clearly indicated otherwise in the context, the use of range expressly includes all possible partial ranges, all individual numerical values ​​within those ranges, and all numerical or numerical ranges (including integers within such ranges, and values ​​or parts of integers within those ranges). This interpretation applies regardless of the width of the range and in all contexts throughout this patent document. Thus, for example, where the range 90-100% is referred to, it includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so on. When referring to the range of 90-100%, this includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., and 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc.

[0331] In addition, when referring to the ranges 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, and 225-250, this includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In further examples, when referring to ranges such as 25-250, 250-500, 500-1,000, 1,000-2,500, 2,500-5,000, 5,000-25,000, and 25,000-50,000, it includes any numbers or ranges within or included in such values, such as 25, 26, 27, 28, 29…250, 251, 252, 253, 254…500, 501, 502, 503, 504… and so on.

[0332] Furthermore, as used herein, a set of ranges is disclosed throughout this document. The use of a set of ranges includes combinations of upper and lower ranges to produce different ranges. This interpretation applies regardless of the width of the range and in all contexts throughout this patent document. Therefore, for example, when referring to a set of ranges, e.g., 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, this includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, 10-30, 10-40, 10-50, 10-75, 10-100, 10-150 and 20-40, 20-50, 20-75, 20-100, 20-150, etc.

[0333] Modifications that do not substantially affect the activity of the various embodiments described herein are also made within the definition of subject matter as described herein. Accordingly, the following examples illustrate the disclosure but are not intended to limit it.

[0334] 6. Embodiments This disclosure includes the following non-limiting embodiments:

[0335] 1. An antibody or fragment thereof that binds to IL13Rα2, (i) VH CDR1, VH CDR2, and VH CDR3 shown in VH containing the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3 shown in VL containing the amino acid sequence of SEQ ID NO: 26, or (ii) VH CDR1, VH CDR2, and VH CDR3 shown in VH containing the amino acid sequence of SEQ ID NO: 48, and VL CDR1, VL CDR2, and VL CDR3 shown in VL containing the amino acid sequence of SEQ ID NO: 49, or (iii) VH CDR1, VH CDR2, and VH CDR3 shown in VH containing the amino acid sequence of SEQ ID NO: 73, and VL CDR1, VL CDR2, and VL CDR3 shown in VL containing the amino acid sequence of SEQ ID NO: 74, The antibody or a fragment thereof, comprising one or more of (i) to (iii).

[0336] 2. (a) (1) VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 5, (2) VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, and 10, and (3) A VH region comprising VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14, (b) (1) VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 15, 16, 17, and 18, (2) VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21, and (3) A VL region comprising VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 24, The antibody or fragment thereof according to Embodiment 1, comprising:

[0337] 3. (i) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing the amino acid sequence of SEQ ID NO: 6, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22. (ii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 2, VH CDR2 containing the amino acid sequence of SEQ ID NO: 7, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 12, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, (iii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 3, VH CDR2 containing the amino acid sequence of SEQ ID NO: 6, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, (iv) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 4, VH CDR2 containing the amino acid sequence of SEQ ID NO: 8, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 13, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 17, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 23, (v) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 5, VH CDR2 containing the amino acid sequence of SEQ ID NO: 9, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 14, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 18, VL CDR2 containing the amino acid sequence of SEQ ID NO: 21, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 24, or (vi) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing the amino acid sequence of SEQ ID NO: 10, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, The antibody or fragment thereof according to Embodiment 1, comprising one or more of (i) to (vi).

[0338] 4. (a) (1) VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 27, 28, 29, 30, and 31, (2) VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 32, 33, 34, 35, and 36, and (3) A VH region comprising VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40, (b) (1) VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, and 44, (2) VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21, and (3) A VL region comprising VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 45, 46, and 47, The antibody or fragment thereof according to Embodiment 1, comprising:

[0339] 5. (i) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 45, (ii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 28, VH CDR2 containing the amino acid sequence of SEQ ID NO: 33, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 38, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 42, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 45, (iii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 29, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 45, (iv) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 30, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 39, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 43, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 46, (v) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 35, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 44, VL CDR2 containing the amino acid sequence of SEQ ID NO: 21, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 47, or (vi) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 45, The antibody or fragment thereof according to Embodiment 1, comprising one or more of (i) to (vi).

[0340] 6. (a) (1) VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 50, 51, 52, 53, and 54, (2) VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 55, 56, 57, 58, and 59, and (3) A VH region comprising VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 60, 61, 62, and 63, (b) (1) VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 64, 65, 66, and 67, (2) VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 68, 20, and 69, and (3) A VL region comprising a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 71, and 72, The antibody or fragment thereof according to Embodiment 1, comprising:

[0341] 7. (i) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 50, VH CDR2 containing the amino acid sequence of SEQ ID NO: 55, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 70, (ii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 51, VH CDR2 containing the amino acid sequence of SEQ ID NO: 56, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 61, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 70, (iii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 52, VH CDR2 containing the amino acid sequence of SEQ ID NO: 55, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 70, (iv) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 53, VH CDR2 containing the amino acid sequence of SEQ ID NO: 57, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 62, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 66, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 71, (v) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 54, VH CDR2 containing the amino acid sequence of SEQ ID NO: 58, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 63, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of SEQ ID NO: 69, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 72, or (vi) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 50, VH CDR2 containing the amino acid sequence of SEQ ID NO: 59, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 70, The antibody or fragment thereof according to Embodiment 1, comprising one or more of (i) to (vi).

[0342] 8. An antibody or fragment thereof according to any one of Embodiments 1 to 7, further comprising the sequences of Framework 1 (FR1), Framework 2 (FR2), Framework 3 (FR3), and / or Framework 4 (FR4).

[0343] 9. An antibody or fragment thereof according to any one of Embodiments 1 to 8, further comprising, optionally, the human framework sequence shown in any one of SEQ ID NOs: 25, 26, 48, 49, 73, and 74, and optionally, the framework sequence 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4).

[0344] 10. (i) VH containing the amino acid sequence of SEQ ID NO: 25, and VL containing the amino acid sequence of SEQ ID NO: 26, or (ii) VH containing the amino acid sequence of SEQ ID NO: 48, and VL containing the amino acid sequence of SEQ ID NO: 49, (iii) VH containing the amino acid sequence of SEQ ID NO: 73, and VL containing the amino acid sequence of SEQ ID NO: 74, An antibody or fragment thereof according to any one of Embodiments 1 to 9, including the antibody or fragment thereof.

[0345] 11. (i) A heavy chain containing the amino acid sequence of SEQ ID NO: 75, and a light chain containing the amino acid sequence of SEQ ID NO: 76, or (ii) A heavy chain containing the amino acid sequence of SEQ ID NO: 79, and a light chain containing the amino acid sequence of SEQ ID NO: 80, or (iii) A heavy chain containing the amino acid sequence of SEQ ID NO: 81, and a light chain containing the amino acid sequence of SEQ ID NO: 82, An antibody or fragment thereof as described in any one of Embodiments 1 to 10, including the antibody or fragment thereof.

[0346] 12. The antibody or fragment thereof according to any one of Embodiments 1 to 11, wherein the antibody is a monoclonal antibody.

[0347] 13. The antibody or fragment thereof according to any one of Embodiments 1 to 12, wherein the antibody is a humanized antibody, a human antibody, or a chimeric antibody.

[0348] 14. An antibody or fragment thereof according to any one of Embodiments 1 to 13, which is one of Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a bivariate region antibody, a single variable region antibody, a linear antibody, a V region, or a polyspecific antibody formed from an antibody fragment.

[0349] 15. An antibody or fragment thereof according to any one of Embodiments 1 to 14, which is conjugated to or fused by recombination to a diagnostic agent, detectable agent or therapeutic agent.

[0350] 16. The antibody or fragment thereof according to Embodiment 15, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxin, or a drug.

[0351] 17. The antibody or fragment thereof according to any one of Embodiments 1 to 16, wherein the antibody is a polyspecific antibody.

[0352] 18. The antibody or fragment according to Embodiment 17, wherein the polyspecific antibody is a bispecific antibody.

[0353] 19. A binder that binds to essentially the same epitope as the antibody or fragment described in any one of Embodiments 1 to 18.

[0354] 20. The binder according to Embodiment 19, which is an antibody or a fragment thereof.

[0355] 21. The binder according to Embodiment 20, wherein the antibody is a polyspecific antibody.

[0356] 22. A binder that competes with the antibody or fragment described in any one of Embodiments 1 to 18 for binding to human IL13Rα2.

[0357] 23. The binder according to Embodiment 22, which is an antibody or a fragment thereof.

[0358] 24. The binder according to Embodiment 23, wherein the antibody is a polyspecific antibody.

[0359] 25. A polynucleotide encoding an antibody or fragment thereof as described in any one of Embodiments 1 to 14 and 17 to 18, or a binder as described in any one of Embodiments 19 to 24.

[0360] 26. One or more vectors comprising one or more polynucleotides described in Embodiment 25, or polynucleotides complementary thereto.

[0361] 27. A cell comprising one or more of the antibodies or fragments thereof described in any one of Embodiments 1 to 18, the binder described in any one of Embodiments 19 to 24, the polynucleotide described in Embodiment 25, or one or more vectors described in Embodiment 26.

[0362] 28. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more of the antibodies or fragments thereof described in any one of Embodiments 1 to 18, the binders described in any one of Embodiments 19 to 24, the polynucleotide described in Embodiment 25, one or more vectors described in Embodiment 26, or the cells described in Embodiment 27.

[0363] 29. A method for identifying IL13Rα2-expressing cells, comprising contacting the cells with an antibody or fragment thereof described in any one of Embodiments 1 to 18, a binder described in any one of Embodiments 19 to 24, or a pharmaceutical composition described in Embodiment 28.

[0364] 30. A method for reducing the viability or proliferation of IL13Rα2-expressing cells, comprising contacting the cells with an antibody or fragment thereof described in any one of Embodiments 1 to 18, a binder described in any one of Embodiments 19 to 24, or a pharmaceutical composition described in Embodiment 28.

[0365] 31. A method for killing IL13Rα2-expressing cells, comprising contacting the cells with an antibody or fragment thereof described in any one of Embodiments 1 to 18, a binder described in any one of Embodiments 19 to 24, or a pharmaceutical composition described in Embodiment 28.

[0366] 32. The method according to any one of Embodiments 29 to 31, wherein the IL13Rα2-expressing cells are cancer cells.

[0367] 33. The method according to Embodiment 32, wherein the cancer cells overexpress IL13Rα2.

[0368] 34. The method according to Embodiment 32, wherein the cancer cells are melanoma cells, lung cancer cells, or colorectal cancer cells.

[0369] 35. A method for treating a target disease or disorder, co...

Claims

1. An antibody or fragment thereof that binds to IL13Rα2, (i) VH CDR1, VH CDR2, and VH CDR3 shown in VH containing the amino acid sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2, and VL CDR3 shown in VL containing the amino acid sequence of SEQ ID NO: 26, or (ii) VH CDR1, VH CDR2, and VH CDR3 shown in VH containing the amino acid sequence of SEQ ID NO: 48, and VL CDR1, VL CDR2, and VL CDR3 shown in VL containing the amino acid sequence of SEQ ID NO: 49, or (iii) VH CDR1, VH CDR2, and VH CDR3 shown in VH containing the amino acid sequence of SEQ ID NO: 73, and VL CDR1, VL CDR2, and VL CDR3 shown in VL containing the amino acid sequence of SEQ ID NO: 74, The antibody or a fragment thereof, comprising one or more of (i) to (iii).

2. (a) (1) VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 5, (2) VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, and 10, and (3) VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14, The VH region, which includes, (b) (1) VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18, (2) VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21, and (3) VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 24, The VL region including, The antibody or fragment thereof according to claim 1, comprising:

3. (i) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing the amino acid sequence of SEQ ID NO: 6, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, (ii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 2, VH CDR2 containing the amino acid sequence of SEQ ID NO: 7, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 12, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 16, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, (iii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 3, VH CDR2 containing the amino acid sequence of SEQ ID NO: 6, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, (iv) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 4, VH CDR2 containing the amino acid sequence of SEQ ID NO: 8, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 13, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 17, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 23, (v) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 5, VH CDR2 containing the amino acid sequence of SEQ ID NO: 9, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 14, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 18, VL CDR2 containing the amino acid sequence of SEQ ID NO: 21, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 24, or (vi) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing the amino acid sequence of SEQ ID NO: 10, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 11, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 22, The antibody or fragment thereof according to claim 1, comprising one or more of (i) to (vi).

4. (a) (1) VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, 30, and 31, (2) VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, and 36, and (3) VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40, The VH region, which includes, (b) (1) VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, and 44, (2) VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21, and (3) VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 46, and 47, The VL region including, The antibody or fragment thereof according to claim 1, comprising:

5. (i) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 45, (ii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 28, VH CDR2 containing the amino acid sequence of SEQ ID NO: 33, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 38, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 42, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 45, (iii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 29, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 45, (iv) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 30, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 39, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 43, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 46, (v) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 35, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 44, VL CDR2 containing the amino acid sequence of SEQ ID NO: 21, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 47, or (vi) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 27, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 41, VL CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 45, The antibody or fragment thereof according to claim 1, comprising one or more of (i) to (vi).

6. (a) (1) VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, and 54, (2) VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 56, 57, 58, and 59, and (3) VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 61, 62, and 63, The VH region, which includes, (b) (1) VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66, and 67, (2) VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 20, and 69, and (3) VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 71, and 72, The VL region including, The antibody or fragment thereof according to claim 1, comprising:

7. (i) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 50, VH CDR2 containing the amino acid sequence of SEQ ID NO: 55, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 70, (ii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 51, VH CDR2 containing the amino acid sequence of SEQ ID NO: 56, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 61, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 70, (iii) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 52, VH CDR2 containing the amino acid sequence of SEQ ID NO: 55, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 70, (iv) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 53, VH CDR2 containing the amino acid sequence of SEQ ID NO: 57, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 62, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 66, VL CDR2 containing the amino acid sequence of SEQ ID NO: 20, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 71, (v) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 54, VH CDR2 containing the amino acid sequence of SEQ ID NO: 58, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 63, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of SEQ ID NO: 69, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 72, or (vi) A VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO: 50, VH CDR2 containing the amino acid sequence of SEQ ID NO: 59, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 60, and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO: 64, VL CDR2 containing the amino acid sequence of SEQ ID NO: 68, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 70, The antibody or fragment thereof according to claim 1, comprising one or more of (i) to (vi).

8. An antibody or fragment thereof according to any one of claims 1 to 7, further comprising the sequences of Framework 1 (FR1), Framework 2 (FR2), Framework 3 (FR3), and / or Framework 4 (FR4).

9. An antibody or fragment thereof according to any one of claims 1 to 8, further comprising, optionally, the human framework sequence shown in any one of sequence numbers 25, 26, 48, 49, 73, and 74, and the sequences of framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4).

10. (i) VH containing the amino acid sequence of SEQ ID NO: 25, and VL containing the amino acid sequence of SEQ ID NO: 26, (ii) VH containing the amino acid sequence of SEQ ID NO: 48, and VL containing the amino acid sequence of SEQ ID NO: 49, or (iii) VH containing the amino acid sequence of SEQ ID NO: 73, and VL containing the amino acid sequence of SEQ ID NO: 74, An antibody or fragment thereof according to any one of claims 1 to 9, comprising:

11. (i) A heavy chain containing the amino acid sequence of SEQ ID NO: 75, and a light chain containing the amino acid sequence of SEQ ID NO: 76, or (ii) A heavy chain containing the amino acid sequence of SEQ ID NO: 79, and a light chain containing the amino acid sequence of SEQ ID NO: 80, or (iii) A heavy chain containing the amino acid sequence of SEQ ID NO: 81, and a light chain containing the amino acid sequence of SEQ ID NO: 82, The antibody or fragment thereof according to any one of claims 1 to 10, including the antibody or fragment thereof.

12. The antibody or a fragment thereof according to any one of claims 1 to 11, wherein the antibody is a monoclonal antibody.

13. The antibody or fragment thereof according to any one of claims 1 to 12, wherein the antibody is a humanized antibody, a human antibody, or a chimeric antibody.

14. Fab, Fab', F(ab') ２ , Fv, scFv, (scFv) ２ The antibody or fragment thereof according to any one of claims 1 to 13, which is one of a single-chain antibody molecule, a bivariate region antibody, a single variable region antibody, a linear antibody, a V region, or a polyspecific antibody formed from an antibody fragment.

15. An antibody or fragment thereof according to any one of claims 1 to 14, which is conjugated to or fused by recombination to a diagnostic agent, detectable agent or therapeutic agent.

16. The antibody or fragment thereof according to claim 15, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxin, or a drug.

17. The antibody or a fragment thereof according to any one of claims 1 to 16, wherein the antibody is a polyspecific antibody.

18. The antibody or fragment according to claim 17, wherein the polyspecific antibody is a bispecific antibody.

19. A binder that binds to essentially the same epitope as the antibody or fragment described in any one of claims 1 to 18.

20. The binder according to claim 19, wherein the antibody or a fragment thereof.

21. The binder according to claim 20, wherein the antibody is a polyspecific antibody.

22. A binder that competes with the antibody or fragment described in any one of claims 1 to 18 for binding to human IL-13Rα2.

23. The binder according to claim 22, wherein the antibody or a fragment thereof.

24. The binder according to claim 23, wherein the antibody is a polyspecific antibody.

25. A polynucleotide encoding an antibody or fragment thereof according to any one of claims 1 to 14 and 17 to 18, or a binder according to any one of claims 19 to 24.

26. One or more vectors comprising one or more polynucleotides according to claim 25, or one or more polynucleotides complementary thereto.

27. A cell comprising one or more of the antibodies or fragments thereof according to any one of claims 1 to 18, the binder according to any one of claims 19 to 24, the polynucleotide according to claim 25, or one or more vectors according to claim 26.

28. A pharmaceutical composition comprising a pharmaceutically acceptable excipient, one or more antibodies or fragments thereof as described in any one of claims 1 to 18, a binder as described in any one of claims 19 to 24, a polynucleotide as described in claim 25, one or more vectors as described in claim 26, or one or more cells as described in claim 27.

29. A method for identifying IL13Rα2-expressing cells, comprising contacting the cells with an antibody or fragment thereof according to any one of claims 1 to 18, a binder according to any one of claims 19 to 24, or a pharmaceutical composition according to claim 28.

30. A method for reducing the viability or proliferation of IL13Rα2-expressing cells, comprising contacting the cells with an antibody or fragment thereof according to any one of claims 1 to 18, a binder according to any one of claims 19 to 24, or a pharmaceutical composition according to claim 28.

31. A method for killing IL13Rα2-expressing cells, comprising contacting the cells with an antibody or fragment thereof according to any one of claims 1 to 18, a binder according to any one of claims 19 to 24, or a pharmaceutical composition according to claim 28.

32. The method according to any one of claims 29 to 31, wherein the IL13Rα2-expressing cells are cancer cells.

33. The method according to claim 32, wherein the cancer cells overexpress IL13Rα2.

34. The method according to claim 32, wherein the cancer cells are melanoma cells.

35. A method for treating a target disease or disorder, comprising administering to the target an antibody or fragment thereof according to any one of claims 1 to 18, a binder according to any one of claims 19 to 24, or a pharmaceutical composition according to claim 28.

36. The method according to claim 35, wherein the disease or disorder is cancer.

37. The method according to claim 36, wherein the cancer expresses IL13Rα2.

38. The method according to claim 36, wherein the cancer overexpresses IL13Rα2.

39. The method according to claim 36, wherein the cancer is melanoma.

40. The method according to any one of claims 35 to 39, wherein the subject is a human subject.

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