Multi-target drug conjugates and their use in cancer treatment

Abstract

A cancer targeting construct is provided, comprising a first term comprising an IL-13 targeting protein or an IL-13RA2-binding variant or fragment of IL-13, a second term comprising an eA5 targeting protein or an EphA2, EphA3, and EphB2-binding variant or fragment of eA5, and an effector molecule coupled to the first term and / or the second term, wherein the construct is a fusion protein and / or a covalent conjugate. Also provided is a method for treating cancer (e.g., breast cancer) in a subject of need, comprising the step of administering to the subject a treatment-effective amount of a construct or a composition comprising the construct as taught herein.

Description

[Technical Field]

[0001] Related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 499,564, filed 2 May 2023, and U.S. Provisional Patent Application No. 63 / 582,616, filed 14 September 2023, the disclosures thereof, which constitute part of this specification by reference.

[0002] Statement of government support This invention was made with government support under R01 CA256285, R01 CA276233, and 1P01 CA207206-01A1, awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Statement regarding electronic filing of sequence listings An XML sequence listing, created on April 30, 2024, and filed together with this Specification, with the filename 9151-264WO_ST26.xml and size 7,734 bytes, shall constitute part of this Specification by reference as its disclosure. [Background technology]

[0004] Interleukin 13 receptor alpha 2 (IL-13RA2) and erythropoietin-producing human hepatoma (Eph) A2, A3, and B2 receptors are overexpressed in most patients with glioblastoma (GBM), but not in normal brain. Debinski et al. (1999) Clin Cancer Res 5: 985-990; Mintz et al. (2002) Neoplasia 4: 388-399; Debinski (1998) Crit Rev Oncog 9: 255-268; Debinski et al. (2000) Mol Med 6: 440-449. These receptors are also overexpressed in spontaneous canine gliomas, which are the most faithful translational model of human disease. Debinski et al. PLoS One. 2013 Oct 16;8(10):e77719. PMID: 24147065; Candolfi et al. J Neurooncol. 2007 Nov;85(2):133-48; Dickinson et al. (2010) Neuro Oncol 12: 928-940; Dickinson et al. (2008) J Neurosurg 108: 989-998; Rossmeisl et al. Neuro Oncol. 2020 Aug 19, PMID: 32812637.

[0005] Furthermore, IL-13RA2, EphA2, EphA3, and EphB2 are widely present in various compartments of GBM tumors. For example, all four receptors are expressed in core tumor cells and, importantly, in invasive tumor cells, while EphA2 is also overexpressed in tumor neovascularization. Wykosky et al. Clin. Cancer Res. 14, 199-208(2008); Ferluga et al. Oncotarget. 2016 Aug 1. doi: 10.18632 / oncotarget.10978.[Epub ahead of print]PMID: 27494882; Hatano et al. J Transl Med. 2004 Nov 24;2(1):40. PMID: 15563374; Brown et al. PLoS One. 2013 Oct 18;8(10):e77769. eCollection 2013. PMID:24204956.

[0006] Interestingly, IL-13RA2, EphA2, and EphA3 have been associated with the pathobiology of glioma stem cell-like cells (GSCs) and play important roles therein. IL-13RA2 is abundant in cells isolated as GSCs from GBM and contributes to their stemness. EphA2 and EphA3 drive the self-renewal and tumorigenic potential of GSCs. Brown et al. (2013), supra; Brown et al. (2012) Clin Cancer Res 18: 2199-2209; Brown et al. Cancer Res. 69(23):8886-93, 2009; Nguyen et al. (2011) Transl Oncol 4: 390-400; Binda et al. Cancer Cell 22(6):765-80, 2012; Miao et al. Oncogene. 2014 Feb 3. PMID: 24488013; Day et al. Cancer Cell 23(2):238-48, 2013. Finally, the EphA3 receptor can be readily detected in GBM infiltrating cells of monocytic origin, glioma-associated macrophages (GAMs). Ferluga et al. (2016), supra.

[0007] Thus, in summary, IL-13RA2, EphA2, EphA3, and EphB2 are expressed in several GBM compartments that have been demonstrated to be involved in tumor progression and / or resistance to treatment, covering nearly 100% of the tumor microenvironment. Sharma et al. Neuro-Oncology Advances, August 2020 vdaa107, https: / / doi.org / 10.1093 / noajnl / vdaa107; Sharma et al. Pharmaceuticals(Basel). 2020 Apr 23;13(4):77. doi: 10.3390 / ph13040077. PMID: 32340173.

[0008] Despite these advances in our understanding of GBM tumors, treatment for GBM still fails to meet the majority of medical needs. Despite nearly 80 years of effort, the two-year survival rate remains below 15%. Debinski et al. (1999), see above. A more effective approach to GBM treatment is greatly needed. [Overview of the Initiative]

[0009] According to some embodiments, a cancer targeting construct is provided herein, comprising a first terminus comprising an IL-13 targeting protein or an IL-13RA2-binding variant or fragment of IL-13, a second terminus comprising an eA5 targeting protein or an EphA2, EphA3, and EphB2-binding variant or fragment of eA5, and an effector molecule coupled to the first terminus and / or the second terminus, wherein the construct is a fusion protein and / or a covalent conjugate.

[0010] In some embodiments, the construct comprises two IL-13 proteins (e.g., IL-13 dimers) or two first ends having IL-13RA2-binding variants or fragments of IL-13, and two eA5 proteins (e.g., eA5 dimers) or two second ends having EphA2, EphA3, and EphB2-binding variants or fragments of eA5, as well as an effector molecule coupled to the first or second end, wherein the construct is a fusion protein and / or covalent conjugate, and the effector molecule comprises a maytansinoid or maytansinoid analog (e.g., DM1 or DM4).

[0011] In some embodiments, the two eA5 proteins are mutant eA5 proteins. In some embodiments, the two eA5 proteins are fragments of eA5 (e.g., amino acids 21-191 of human eA5).

[0012] In some embodiments, the two IL-13 proteins are mutant IL-13 proteins (e.g., IL-13.E13K).

[0013] In some embodiments, the eA5 and IL-13 proteins are glycosylated.

[0014] In some embodiments, the construct comprises a fusion protein having a protein linker between the first and second ends. In some embodiments, the first end is at the N-terminus of the fusion protein, and the second end is at the C-terminus of the fusion protein. In some embodiments, the protein linker comprises an ADCC and / or CDC activating domain. In some embodiments, the protein linker comprises an Fc fragment of human IgG1.

[0015] In some embodiments, the effector molecule further includes a detectable group.

[0016] Compositions comprising constructs and pharmaceutically acceptable carriers as taught herein are also provided. In some embodiments, the carrier is sterile. In some embodiments, the carrier is a saline solution having a pH of 6 or 6.5 to 7.7 or 8.

[0017] In some embodiments, the composition is suitable for administration to a subject by infusion (e.g., convection-enhanced delivery). In some embodiments, the composition is suitable for administration by continuous infusion at concentrations from 0.1 μg / mL or 0.5 μg / mL to 20 μg / mL, for example, 1 μg / mL, 2 μg / mL, or 3 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, or 15 μg / mL.

[0018] Further details are provided of nucleic acids encoding constructs or their protein or peptide portions as taught herein. Further details are provided of eukaryotic host cells containing such nucleic acids and configured to express the encoding peptide. In some embodiments, the host cell is an insect expression cell.

[0019] Also provided is a method for treating cancer in a subject requiring treatment, comprising the step of administering to the subject a therapeutically effective amount of a construct or a composition containing such construct, as taught herein.

[0020] In some embodiments, the cancer is breast cancer, bladder cancer, pancreatic cancer, colorectal cancer, head and neck cancer, thyroid cancer, prostate cancer, melanoma, or glioma. In some embodiments, the cancer is glioblastoma, prostate cancer, or melanoma. In some embodiments, the cancer is glioblastoma.

[0021] In some embodiments, the administration step includes convection-enhanced delivery (CED). In some embodiments, the construct is administered by continuous infusion from 0.1 μg / mL or 0.5 μg / mL to 20 μg / mL, for example, from 1 μg / mL, 2 μg / mL, or 3 μg / mL to 5 μg / mL, 8 μg / mL, 10 μg / mL, or 15 μg / mL.

[0022] A method for detecting EphA2, EphA3, and / or EphB2 expressing cells is further provided, comprising the steps of administering a construct or a composition comprising such construct to cells or a group of cells, wherein the construct comprises a detectable group, and detecting the detectable group.

[0023] A method for treating breast cancer (e.g., triple-negative breast cancer) in a subject requiring treatment is further provided, comprising the step of administering a construct or a composition containing such construct, as taught herein, to the subject in a treatment-effective amount. In some embodiments, the construct is administered by intravenous injection. In some embodiments, the construct is administered in amounts ranging from about 0.1 mg / kg to about 15 mg / kg, for example, from 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, or 5 mg / kg to 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, or 15 mg / kg.

[0024] Also provided is the use of constructs or compositions containing such constructs, as taught herein, for treating cancer in a subject requiring treatment, or for preparing a medicament for treating cancer in a subject requiring treatment. [Brief explanation of the drawing]

[0025] [Figure 1] Panel A) shows the schema of IL-13M-(Fc)-eA5 (QUAD-DM1) conjugated with DM1-SMCC. Panel B) shows Western blots of the immunoreactivity of all three main components of QUAD: IL-13.E13K, Fc, and ephrin A5. [Figure 2] This figure shows an exceptional response to a targeted cytotoxic cocktail in dogs with spontaneously occurring gliomas. The best four responders out of 18 treated dogs with >95% volume tumor regression in a clinical trial using DT390-IL-13M and eA1M-PE38QQR; 17 animals were enrolled, and no dose-limiting toxicity was observed. Tumor regression was even more pronounced at 16 weeks post-treatment compared to 6 weeks. Dog 141004 developed PD at 16 weeks post-CED. [Figure 3]This figure shows comparative gene expression analysis of canine gliomas before and after treatment with a cocktail of targeted cytotoxicities against IL-13RA2 and EphA2 receptors. Panel A) Ingenuity pathway analysis of top-ranked signaling pathways altered by cytotoxic treatment, based on z-scores. Panel B) Increased expression of Th1 response genes IL2 and IFNG in post-treatment tumors of responder cases. Panel C) Pooled DEG datasets from three dogs were compared using GSEA incorporating cell type signatures for the infiltrating immune cell population. Post-treatment samples showed increased activated helper T lymphocytes (CD4+) and cytotoxic T lymphocytes (CD8+), NK cells, and glioma-infiltrating macrophages and microglia (GIM / Ms). Panel D) Immunofluorescence staining of CD4+ and CD8+ lymphocytes in tumor samples from four dogs. [Figure 4] This figure shows the schemas for QUAD 1.0 and 3.0. The configuration for QUAD 1.0 is eA5-CH2CH3-IL-13M, and the configuration for QUAD 3.0 is IL-13M-CH2CH3-eA5, with an additional cysteine ​​at the C-terminus of the protein chain. The cysteine ​​acts as a specific conjugation site with drugs / toxins. [Figure 5] This figure shows imaging-based tumor responses in dogs after treatment with QUAD-DOX (dog 2; top row) and QUAD-PE38QQR (dog 5; middle and bottom rows). In dog 2, continuous reduction in tumor volume was observed 6 months after treatment, indicating the best post-treatment response. In dog 5, pseudoprogression was observed at 6 weeks, followed by the disappearance of all contrast-enhancing lesions and a 91% reduction in T2 / FLAIR lesion volume by 6 months post-treatment. [Figure 6]Human eA5 interacts with receptors in an interspecies manner. The panel shows flow cytometry of QUAD 3.0 binding to the canine G06-A GBM cell line (Panel A) and potent killing of these cells by QUAD 3.0-PE38QQR (Panel B). The panel shows downregulation of EphA2 and EphA3 receptors by human eA5 (1 g / ml) (R&D Systems) in two canine GBM cell lines, G06-A and SDT-3G (Panel C). These results precisely mirror what we have found in human GBM cells. Panel D) shows activation of phospho-EphA3 by human eA5-Fc in two canine GBM cell lines, G06-A and SDT-3G. Panel E) shows downregulation of EphA2 and Eph3A receptors in mouse glioma GL261 cells (IL-13RA2+). Panel F) shows the ELISA results for the binding of human eA5 to recombinant mice and the human EphA3 receptor. Note that human eA5-Fc binds slightly more strongly to the mouse EphA3 receptor than to the human EphA3 receptor. The concentration values ​​for multiplicative changes are shown in the immunoblot. [Figure 7]Panel A) Figure showing the structure of DM1 (microtubule polymerization inhibitor), which is further thiol-reactive and forms a stable thioether bond with the thiol present at the C-terminal cysteine ​​residue in QUAD; Panel B) Figure showing the SDS-PAGE of QUAD 3.1 (lane 1) and its QUAD-DM1 conjugate (lane 2); Panel C) Figure showing cell viability assays in U-251 MG GBM cells of QUAD 3.1 conjugate, two different batches #1 and #2 of QUAD-DM1 conjugate, QUAD-DTX (deruxtecan) conjugate, and QUAD-WP936 (doxorubicin) conjugate, in addition to the unconjugated DM1-SMCC; Panel D) Figure showing cell viability assays of QUAD-DM1 conjugate in human U-251 and canine G06-A and SDT-3G GBM cells; Panel E) Figure showing cell viability assays in U-251 and T98G GBM cells. T98G is IL-13RA2(-) and a non-responder to IL-13-based cytotoxicity. Panel F) shows that unconjugated QUAD (100 nM) neutralizes the cytotoxic activity of QUAD-DM1 in U-251 GBM cells. Panel G) shows that unconjugated QUAD has some endogenous antitumor activity. Panels H and I) show toxicity studies of QUAD-DM1 in mice conjugated either intracranially (IC; panel H) or intravenously (IV; panel I). Three mice were used per dose. [Figure 8] Panels A-D) These images show pre-treatment T2W MRI of a high-grade oligodendroglioma in the left frontoparietal bone. Panels E-H) These images show intraoperative 3D1W MRI-monitored CED injection of QUAD-DM1 (Vi=837μl; [3.2μg / ml]), demonstrating 92% coverage of T2W lesion load. Panels I-L) These images show T2W MRI 8 weeks post-treatment, exhibiting a partial tumor response characterized by a significant 93% reduction in tumor volume. [Figure 9]Panel A) This figure shows heatmaps of sample-based gene expression for EphA2, EphA3, EphB2, and IL13RA2 in whole or in part from data created by the TCGA Research Network (cancer.gov / tcga).; Panel B) This figure shows Western blots of EphA2, EphA3, EphB2, and β-actin controls in various breast cancer cell lines.; Panel C) This figure shows flow cytometry of QUAD bound to human breast cancer cell lines. [Figure 10] Panels A-D) These figures show immunohistochemical staining for IL-13RA2, EphA3, EphA2, and EphB2 in tissue microarrays of breast cancer, lymph node metastases, and normal breast tissue; the isotype controls (Jackson Immuno) for each stain are as shown in the right panel; rows 1-5 are breast cancer; rows 6-9 are lesion lymph nodes; row 10 is adjacent normal breast tissue. [Figure 11] This figure shows immunohistochemical staining for IL-13RA2, EphA3, EphA2, and EphB2 in primary breast cancer and subsequent brain metastases from the same patient; patient #1 had triple-negative breast cancer, and patient #2 had triple-positive breast cancer. [Figure 12] Panel A) Figure showing cell viability assays of QUAD-DM1 conjugates in seven breast cancer cell lines, including GBM cells (U-251) and two pairs of primary and metastatic tumors (MDA-468 and MDA-231); Panel B) Figure showing flow cytometry data with propidium iodide nuclear staining, indicating the percentage of cells in each growth phase after treatment with 1 nM QUAD-DM1; Panel C) Figure showing MDA-MB-231 tumor volume measurements in nude mice over 24 days after treatment with IV injection of either 12 mg / kg QUAD-DM1 or PBS as a control. [Figure 13]This report presents data on intracranial (IC) treatment of MDA-MB-231-BrM tumors in QUAD-DM1 animals, involving IC transplantation of 200,000 MDA-MB-231-BrM cells per 2 μL. Treatment began 7 days after tumor cell transplantation (day 0). Animals were imaged three times a week using bioluminescence (IVIS) imaging (photons per second). [Figure 14] This figure shows the binding specificity (ELISA assay) of HeK-293 cell-produced QUADs to EphA3, PDL1, and CD80. As expected, the QUADs bound only to the EphA3 receptor. Hek-293 cells are human embryonic kidney cells. The same results were obtained for QUADs produced in Expi-293F cells, which are HEK-293 cells adapted to transient transfection. [Figure 15] This figure shows the killing of MDA-MB-231 BrM cells by QUAD-DM1, read at 48 and 72 hours after the addition of QUAD-DM1. Cell killing by QUAD-DM1 was partially blocked by antibodies that counteract the individual receptor binding ferricity of the QUAD ligands. [Figure 16] This figure shows HC1806-triple-negative breast cancer cells (TNBC) obtained using the same method as the data collection method in Figure 15. [Modes for carrying out the invention]

[0026] The present invention is described in more detail below. This description is not intended to be a detailed list of all different ways in which the present invention may be carried out, or all features that may be added to the present invention. For example, features illustrated in one embodiment may be incorporated into other embodiments, and features illustrated in a particular embodiment may be omitted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein that do not depart from the present invention will be apparent to those skilled in the art in light of this disclosure. Accordingly, the following description is intended to illustrate some specific embodiments of the present invention and not to thoroughly specify all permutations, combinations, and variations thereof.

[0027] All disclosures of U.S. patent references cited herein are incorporated herein by reference to the extent that they are consistent with this disclosure.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. The terminology used in describing the present invention herein is for the purpose of describing specific embodiments only and is not intended to limit the invention.

[0029] Unless otherwise specified, standard methods may be used for cloning genes, amplifying and detecting nucleic acids, etc. Such techniques are known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); and Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0030] When used in the description of the present invention and in the appended claims, the singular forms “a,” “an,” and “the” are intended to also include the plural form unless the context clearly indicates otherwise. Furthermore, as used herein, “and / or” refers to and encompasses one or more of the related enumerated items and any possible combination, in addition, the absence of any combination when interpreted as an option ("or").

[0031] When referring to measurable values ​​such as the amount, dose, time, temperature, enzyme activity, or other biological activity of a polypeptide, the term “about” as used herein is intended to include variations of 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0032] Constructs are provided herein that comprise a first terminus having one or two eA5 proteins or EphA2, EphA3, and EphB2-binding variants or fragments thereof, and a second terminus having one or two IL-13 proteins or IL-13RA2-binding variants or fragments thereof. See, for example, Sharma et al., Neuro-Oncology Advances 2(1), 1-11, 2020; Sharma et al., Pharmaceuticals 13, 77, 2020; and U.S. Patent No. 9,975,942 by Debinski et al., incorporated herein by reference. In some embodiments, the construct comprises human eA5 protein or EphA2, EphA3, and EphB2-binding variants or fragments thereof. In some embodiments, the construct comprises human IL-13 protein or IL-13RA2-binding variants or fragments thereof.

[0033] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to polymers (dipeptides or longer) of any amino acids linked together through peptide bonds.

[0034] As used herein, “recombinant” nucleic acid refers to nucleic acid produced in vitro, for example, by synthesis and / or by combining two or more nucleic acid sequences from different sources (e.g., “heterogeneous” nucleic acid). Recombinant nucleic acid may be provided in the form of a “vector” or “delivery vector” for transforming or transfecting cells to contain that nucleic acid. As used herein, a “vector” or “delivery vector” may be a viral or nonviral vector used to deliver nucleic acid to cells, tissues, or targets.

[0035] Recombinant proteins are proteins produced using recombinant nucleic acids, often in host cells. Nucleic acids may or may not be inserted into the host cell's genome. Nucleic acids can exist, for example, in the form of plasmids in the host cell. Alternatively, recombinant proteins can be produced by in vitro translation of recombinant nucleic acids.

[0036] "Isolated" protein or polypeptide means a protein or polypeptide that is isolated from, or substantially free from, other components of a naturally occurring organism or virus, such as, for example, cellular or viral structural components or other proteins or nucleic acids commonly found in association with that protein. As used herein, "isolated" protein or polypeptide is pure to at least about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% (w / w), or more.

[0037] As used herein, “subjects” generally refers to human subjects, including but not limited to cancer patients. Subjects may be male or female and may be of any race or ethnicity. Subjects may be of any age, including, for example, newborns, neonates, infants, children, adolescents, adults, and the elderly. Subjects may also include animal subjects, particularly mammalian subjects such as canids, felids, bovines, goats, equids, sheep, pigs, rodents (e.g., rats and mice), lagomorphs, and primates (e.g., non-human primates), for purposes such as veterinary medicine or pharmaceutical development.

[0038] The “cancers” that can be detected and / or treated by the constructs, compositions, and methods described herein include, but are not limited to, breast cancer, bladder cancer, pancreatic cancer, colorectal cancer, head and neck cancer, thyroid cancer, prostate cancer, melanoma, and brain cancers such as glioma (e.g., GBM).

[0039] Many cancers overexpress IL-13RA2 (e.g., GBM and other brain cancers, human pediatric brain tumors, brainstem gliomas, renal cell carcinoma, head and neck squamous cell carcinoma, ovarian cell carcinoma, pancreatic cancer, colorectal cancer, and melanoma), EphA2 (e.g., GBM and other brain cancers, breast cancer, prostate cancer, bladder cancer, skin cancer, lung cancer, ovarian cancer, esophageal cancer, kidney cancer, colon cancer, and vulvar cancer), EphA3 (e.g., GBM and other brain cancers, leukemia, lymphoma, lung cancer, skin cancer, and gastric cancer), and / or EphB2 (e.g., GBM and other brain cancers, gastric cancer, colon cancer, neuroblastoma, small cell lung cancer, and melanoma). This expression can coexist in various tumor compartments.

[0040] "Brain cancer" or "brain tumor" can be any stage, grade, histological features, invasiveness, aggressiveness, or degree of malignancy of affected tissue or cell aggregation in any part of the central nervous system (i.e., the brain and spinal cord). In some embodiments, the brain tumor is a glioma. In some embodiments, the tumor is an anaplastic astrocytoma, an anaplastic oligodendrogliocytoma, or an anaplastic oligodendrogliomas, in particular WHO grade II fibrous astrocytoma, WHO grade II oligodendrogliomas, WHO grade III anaplastic astrocytoma, WHO grade III anaplastic oligodendrogliocytoma, or glioblastoma multiforme (see, for example, U.S. Patent Application Publication No. 2010 / 0291590).

[0041] Gliomas are tumors that arise in glial cells, which help support and protect important areas of the brain. Gliomas are the most common type of brain tumor in adults, accounting for approximately 42% of all adult brain tumors. Gliomas are further characterized into categories based on the type of cells they affect: astrocytoma (affecting astrocytic cells), oligodendroglioma (affecting oligodendrogliatic cells), ependymoma (affecting ependymal cells), meningioma (affecting the meninges), acoustic glioma / Schwannoma (affecting Schwann cells), and medulloblastoma (affecting cells in the cerebellum). See also Basile et al. US2013 / 0012452.

[0042] Astrocytomas are graded from I to IV according to their rate of progression. Grade I (pilocytic astrocytoma) grows slowly and has little tendency to invade surrounding brain tissue. Grade II (diffuse astrocytoma) grows relatively slowly and has a slight tendency to invade surrounding brain tissue. Grade III (anaplastic / malignant astrocytoma) tumors grow quite rapidly and invade surrounding brain tissue. Grade IV (glioblastoma or "GBM") is extremely invasive and is a fatal form of brain cancer. Unfortunately, it is the most common type of brain tumor in adults, accounting for about 67% of all astrocytomas.

[0043] Oligodendrogliomas, which account for 4% of brain tumors, typically affect people over 45 years of age. Some subtypes of this tumor are particularly sensitive to treatment with radiation therapy and chemotherapy. Half of patients with oligodendrogliomas are still alive after 5 years.

[0044] Ependymomas are rare (about 2% of all brain tumors), but they are the most common brain tumors in children. They generally do not affect healthy brain tissue and do not spread beyond the ependyma. These tumors respond well to surgery, especially those on the spine, but ependymomas cannot always be completely removed. The five-year survival rate for patients over 45 years of age reaches 70%.

[0045] Meningiomas affect the meninges (the tissues that form the protective outer layer of the brain and spine). One-quarter of all brain and spinal tumors are meningiomas, and up to 85% of them are benign.

[0046] Malignant gliomas are fatal diseases with a mean life expectancy of less than one year after diagnosis. The prognosis is very poor for patients with high-grade gliomas, especially for older patients. Of Americans diagnosed with malignant gliomas each year, about half survive one year after diagnosis, and 25% survive two years. Patients with anaplastic astrocytomas survive for about three years. Glioblastomas have the worst prognosis, with a mean life expectancy of less than 9 to 15 months after diagnosis.

[0047] "Breast cancer" or "breast tumor" can be any stage, grade, histological features, invasiveness, aggressiveness, or malignancy of affected tissue or cell aggregates in any part of breast tissue. In some embodiments, breast cancer is inflammatory breast cancer or Paget's disease of the breast. In some embodiments, breast tumor is tubular carcinoma (invasive or non-invasive), lobular carcinoma (invasive or non-invasive), angiosarcoma, adenocarcinoma, and / or phyllodes tumor. In some embodiments, breast cancer is characterized as human epidermal growth factor 2 (HER2) positive (HER2+). In some embodiments, breast cancer is characterized as estrogen receptor negative (ER-), progesterone receptor negative (PR-), and / or HER2 negative (HER2-).

[0048] In some embodiments, breast cancer is ER-, PR-, and HER2- (e.g., triple-negative breast cancer (TNBC)). In some embodiments, breast cancer and / or breast tumors express IL-13RA2, EphA2, EphA3, and / or EphB2. In some embodiments, breast cancer and / or breast tumors overexpress IL-13RA2, EphA2, EphA3, and / or EphB2.

[0049] In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the metastatic breast cancer metastasizes to the bones, lungs, brain, and / or liver. In some embodiments, the metastatic breast cancer metastasizes to more than one site in the subject (e.g., about one, two, three, four, five, or more sites). In some embodiments, the metastatic sites of the metastatic breast cancer are treated in the same way as or differently from the primary tumor (i.e., with the same chemotherapeutic agents and / or methods of administration, or with different chemotherapeutic agents and / or methods of administration).

[0050] As used herein, "effector molecule" includes therapeutic agents, nanoparticles, detectable groups, targeting ligands, and delivery vehicles (e.g., antibodies, lipids, liposomes). See, e.g., U.S. Patent No. 6,630,576.

[0051] As used herein, "therapeutic agent" can be any therapeutic agent, including, but not limited to, genetic material or factors, radionuclides, chemotherapeutic agents, cytotoxic agents (e.g., Sliwkowski, U.S. Patent No. 6,949,245), and amphiphilic antimicrobial peptides. Other exemplary therapeutic agents include, without limitation, radiopharmaceuticals, which can include, for example, Auger electrons, chemotherapeutic agents incorporating radionuclides, and photosensitive substances.

[0052] As described herein, "radionuclide" is 227 Ac, 211 At, 131 Ba, 77 Br, 109 Cd, 51 Cr, 67 Cu, 165 Dy, 155 Eu, 153 Gd, 198 Au, 166 Ho, 113m In, 115m In, 123 I, 125 I, 131 I, 189 Ir, 191 Ir, 192 Ir, 194 Ir, 52 Fe, 55 Fe, 59 Fe, 177 Lu, 109 Pd, 32 P, 226 Ra, 186 Re, 188 Re, 153 Sm, 46 Sc, 47 Sc, 72 Se, 75 Se, 105Ag, 89 Sr, 35 S, 177 Ta, 117 mSn, 121 Sn, 166 Yb, 169 Yb, 90 Y, 212 Bi, 119 Sb, 197 Hg, 97 Ru, 100 Pd, 101m Rh, and 212 This includes, but is not limited to, Pb.

[0053] As used herein, “chemothoracic agent” includes, but is not limited to, methotrexate, daunorubicin, mitomycin C, cisplatin, vincristine, epirubicin, fluorouracil, verapamil, cyclophosphamide, cytosine arabinoside, aminopterin, bleomycin, mitomycin C, demecorsin, etoposide, mitramycin, chlorambucil, melphalan, daunorubicin, doxorubicin, tamoxifen, paclitaxel, vincristine, vinblastine, camptothecin, actinomycin D, and cytarabine. Other examples can be found in U.S. Patent Application Publication No. 2006 / 0121539 (Debinski et al.), which is incorporated herein by reference in its entirety. Examples of chemotherapeutic agents include camptotheci (e.g., topotecan and irinotecan), indenoisoquinolines (e.g., indothecan and indimitecan), and topoisomerase I inhibitors such as deruxtecan (DTX).

[0054] As used herein, “cytotoxic agents” or “toxic agents” include maytansinoids and maytansinoid analogs, taxoids, CC-1065 and CC-1065 analogs, drastatin and drastatin analogs, lysine (or more particularly, lysine A chain), acrasinomycin, diphtheria toxin, monensin, vercarin A, abrin, trichothecenes, and Pseudomonas exotoxin A, taxol, and cytochalasin. B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vinca alkaloids (e.g., vincristine and vinblastine), mitotic inhibitors such as colchicine, anthracyclines such as doxorubicin (including 4'-O-benzylated Dox analogs WP744 and WP769, or Dox analogs WP1244, WP936, and WP1737) and daunorubicin, dihydroxyanthracene This includes, but is not limited to, antibiotics such as ion, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, as well as puromycin and its analogues or homologs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thiotepachlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP)), and, non-limited to, dactinomycin (formerly actinomycin), bleomycin, mitramycin, calichemycin, and anthramycin (AMC).

[0055] In some preferred embodiments, the cytotoxic agent is a microtubule polymerization inhibitor. Specific examples include benzoansamacrolides such as maytansine or their derivatives, or maytansinoids or maytansinoid analogs. Examples of such active ingredients include, but are not limited to, maytansinoid drugs (DMs), such as DM1 and DM4. Protein conjugates of maytansinoids are known; see, for example, U.S. Patent No. 8,624,003 by Kellogg et al., incorporated herein by reference. For example, as shown in Figure 7, Panel C herein, the QUAD with DM1 exhibits significantly higher cytotoxic efficacy compared to the use of other cytotoxic agents.

[0056] In some embodiments, the maytansinoid is conjugated via a disulfide linker (e.g., succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker). See, for example, U.S. Patents 5,208,020, 5,416,064, 6,441,163, and U.S. Patent Publication 2007 / 0048314.

[0057] In some embodiments, the cytotoxic agent includes toxins such as Pseudomonas exotoxin, lysine, abrin, ribonuclease (RNase), DNase I, Staphylococcus enterotoxin-A, pokeweed antiviral protein, geronin, and diphtheria toxin. See, for example, U.S. Patent No. 7,517,964. In some embodiments, Pseudomonas exotoxin or diphtheria toxin is preferred. See, in whole, U.S. Patent No. 5,328,984 by Pastan et al. and U.S. Patent No. 6,296,843 by Debinski. Pseudomonas exotoxin may include, but is not limited to, Pseudomonas exotoxin A (PE). Pseudomonas exotoxin can be modified so that it substantially lacks domain Ia, and in some embodiments, Pseudomonas exotoxin includes PE38QQR and PE4E. Diphtheria toxin may include DT390, which is diphtheria toxin from which the native binding domain has been removed. It will be recognized that in various embodiments, therapeutic agents can be attached, for example, to the amino or carboxyl terminus.

[0058] As used herein, “amphiphilic antimicrobial peptides” include amphiphilic peptides that induce apoptosis in cancer cells, possibly through their ability to depolarize mitochondrial membranes. K. Rege et al., Cancer Res. 67, 6368 (2007). Such peptides are generally 10, 12, or 13 amino acid lengths to 20, 30, or 40 amino acid lengths, or longer, and typically have an amphiphilic alpha-helix structure. Examples include (KLAKLAK)2 (SEQ ID NO: 1); (KLAKKLA)2 (SEQ ID NO: 2); (KAAKKAA)2 (SEQ ID NO: 3); and (KLGKKLG)2 (SEQ ID NO: 4). See, for example, Ruoslahti et al., U.S. Patent Application Publication No. 2001 / 0046498 (November 29, 2001).

[0059] As used herein, “nanoparticles” include particles having a size of about 0.5 to about 1,000 nanometers and may include natural and / or synthetic moieties. In some embodiments, the nanoparticles cross the blood-brain barrier. In some embodiments, the nanoparticles may take up therapeutic agents. See, for example, U.S. Patent No. 8,535,726 by Dai et al.; U.S. Patent No. 8,252,338 by Forte et al.; U.S. Patent No. 8,246,968 by Zale et al.; and U.S. 2013 / 0122056 by Zhang et al. In some embodiments, the nanoparticles include a polymer matrix which may include two or more polymers. One or more polymers in the polymer matrix may include, for example, polyethylene, polycarbonate, polyacid anhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, or a combination thereof. In some embodiments, the polymer matrix comprises one or more polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, or polycyanoacrylates. In some embodiments, at least one polymer is polyalkylene glycol. In some embodiments, the polyalkylene glycol is polyethylene glycol. In some embodiments, at least one polymer is polyester. In some embodiments, the polyester is selected from the group consisting of PLGA, PLA, PGA, and polycaprolactone. In some embodiments, the polyester is PLGA or PLA. In some embodiments, the polymer matrix comprises a copolymer of two or more polymers, for example, a copolymer of polyalkylene glycol and polyester. In some embodiments, the copolymer is a copolymer of PLGA or PLA and PEG.In some embodiments, the polymer matrix comprises PLGA or PLA, and a copolymer of PLGA or PLA and PEG.

[0060] As used herein, “detectable group” or “label” means a radioactive label (e.g., 35 S, 125 I, 32 P, 3 H, 14 C, 131 I) Enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), gold beads, chemiluminescent labels, ligands (e.g., biotin, digoxin) and / or fluorescent labels (e.g., rhodamine, phycoerythrin, fluorescein, fluorescent proteins), fluorescent proteins, e.g., non-limited, green fluorescent protein or one of its many variants, nucleic acid segments by known techniques, and energy absorbers and energy emitters, including but not limited to these. Thus, as used herein, “label” or “detectable group” can be any suitable label or detectable group that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means, e.g., non-limited, biotin, fluorophores, antigens, porphyrins, and radioisotopes. Useful labels in the present invention include biotin for staining with labeled avidin or streptavidin conjugate, magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate [FITC], Texas Red, rhodamine, green fluorescent protein, highly sensitive green fluorescent protein, lysamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX [Amersham], SyBR Green I & II [Molecular Probes], etc.), and radiolabeling (e.g., 3 H, 35 S, 14 C, or 32Examples include P), enzymes (e.g., hydrolases, especially phosphatases such as alkaline phosphatase, esterases, and glycosidases, or oxidoreductases, especially peroxidases such as horseradish peroxidase, and others of the same kind), substrates, cofactors, inhibitors, chemiluminescent groups, pigment-producing substances, and colorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0061] As used herein, “to treat,” “to treat,” or “treatment” means any type of treatment that benefits a subject suffering from a disease, including, for example, improvement of the subject’s condition (e.g., one or more symptoms), delaying the progression of the disease, and including reduction of tumor volume or invasiveness, or extension of life expectancy.

[0062] As used herein, “pharmaceutically acceptable” means that the construct or composition is suitable for administration to a subject to achieve the treatment described herein without excessive adverse side effects, taking into account the severity of the disease and the need for treatment.

[0063] In some embodiments, the construct is a fusion protein and / or covalent conjugate construct comprising a first end having two eA5 proteins (eA5 dimers) or EphA2, EphA3, and EphB2-binding variants or fragments thereof, and a second end having two IL-13 proteins (IL-13 dimers) or IL-13RA2-binding variants or fragments thereof. The protein dimers may be formed using methods known in the art, for example, using Fc fusion proteins or hinge linkers. See, for example, Roopenian et al. US2010 / 0209424 and Yan et al. US2012 / 0039880, incorporated herein by reference.

[0064] In some embodiments, the construct comprises eA5, its variants, or glycosylated forms of its fragments. See Ferluga et al., J Biol Chem 288(25):18448-18457(2013). In some embodiments, the construct comprises IL-13, its variants, or glycosylated forms of its fragments.

[0065] In some embodiments, the eA5 variant is a GH loop variant, for example, eA5 118 FQLFTPFSLGFEFRPG 133 (Sequence ID 5) (UniProtKB / Swiss-Prot accession number P52803.1). See Lema Tome et al., J Biol Chem 287:14012-14022 (2012). In some embodiments, the mutation is in amino acids 119(Q), 123(P), 125(S), 127(G), 132(P) of eA5, or any combination thereof. In some embodiments, the mutation is in P123, S125, G127, or P132 of eA5, where its wild-type amino acid is substituted with A. In some embodiments, the eA5 mutant may have more than one, two, or even three or more amino acid substitutions. In some embodiments, the eA5 mutant has enhanced binding affinity to EphA2, EphA3, and / or EphB2 compared to the corresponding wild-type eA5 binding affinity.

[0066] As a non-limiting example of an eA5 fragment that can be used as an EphA2, EphA3, and EphB2-binding fragment according to the present invention, the eA5 fragment may consist of amino acids 21-191 of human eA5, and as shown in SEQ ID NO: 6, the terminal cysteine ​​(underlined) enables conjugation of the active substance: [ka]

[0067] A non-restrictive example of an optimized DNA sequence that could be used to encode the eA5 fragment provided as SEQ ID NO: 6 is the DNA sequence of SEQ ID NO: 7: [ka]

[0068] The targeting peptide of the present invention can be coupled or conjugated with one or more effector molecules, cytoplasmic localization elements, and / or intracellular compartment localization signaling elements by any suitable technique, the suitable technique including techniques for forming the construct, which can be used for therapeutic and / or diagnostic purposes.

[0069] Recombinant IL-13 is commercially available from several suppliers (e.g., R&D Systems, Minneapolis, MN, and Sanofi Bio-Industries, Inc., Tervose, PA). Alternatively, the gene or cDNA encoding IL-13 can be cloned into a plasmid or other expression vector according to methods well known to those skilled in the art and expressed in any of several expression systems. Methods for cloning and expressing IL-13, and the nucleic acid sequence of IL-13, are well known (see, e.g., Minty et al. (1993) and McKenzie (1987)). Certain IL-13 variants are also known and are described in U.S. Patent No. 6,630,576 (Debinski) and No. 6,884,603 (Debinski et al.), which are incorporated herein by reference. In some embodiments, the IL-13 variant is IL-13.E13K, in which the amino acid residue at position 13 is substituted with lysine. Other useful IL-13 variants in this invention include, but are not limited to, IL-13.R66D, IL-13.S69D, and IL-13.K105R. See Van Nguyen et al., Neuro-Oncology 14(10):1239-1253 (2012). Any variant or combination of variants may be used.

[0070] Those skilled in the art will recognize that analogues or fragments of IL-13 or IL-13 variants can also specifically bind to IL-13RA2. For example, conservative substitutions of residues containing native IL-13 (e.g., serine instead of alanine, or aspartic acid instead of glutamic acid) can similarly provide IL-13 analogues that specifically bind to the IL-13 receptor. Thus, the terms “IL-13” or “IL-13 variant” as used in reference to targeting molecules also include fragments, analogues, or peptide mimetic compounds of IL-13 or IL-13 variants that similarly bind specifically to the IL-13 receptor. Further discussion of IL-13 as envisioned by the present invention can be found in U.S. Patents No. 5,328,984 (Pastan et al.), No. 5,614,191 (Puri et al.), No. 5,919,456 (Puri et al.), No. 6,296,843 (Debinski), No. 6,428,788 (Debinski et al.), No. 6,518,061 (Puri et al.), No. 6,576,232 (Debinski et al.), No. 6,630,576 (Debinski), No. 6,884,603 (Debinski et al.), and No. 8,362,207 (Debinski et al.).

[0071] The protein portion of the construct can be produced using methods known in the art, such as bacterial expression, prokaryotic or eukaryotic expression. See, for example, U.S. Patent No. 7,381,408 by Mezo et al.; U.S. Patent No. 7,655,413 by Butt et al.; and U.S. Patent No. 8,603,807 by Reed. In some embodiments, the protein component can be produced by bacterial expression and / or eukaryotic expression. For example, glycosylated IL-13.E13K-Fc-eA5-Cys can be produced using a baculovirus expression system in insect cells. In some embodiments, the nucleic acid for the expression of the protein component is codon-optimized for that expression system (e.g., codons optimized for insect cells).

[0072] Targeting proteins as described herein may be coupled or conjugated with linkers, other targeting proteins, and / or effector molecules such as diagnostic and / or therapeutic agents, according to any of the various techniques used for the production of immunoconjugates. See, for example, Sliwkowski, U.S. Patent No. 6,949,245.

[0073] In some embodiments, constructs are incorporated internally in response to target protein binding. For example, EphA2 is overexpressed in the majority of patients with GBM, and its ligand induces receptor-mediated internalization when it binds to its receptor (Walker-Daniels et al. (2002) Mol. Cancer Res. 1:79-87). The ligand can be used, for example, to enable recombinant bacterial toxin-containing cytotoxicities to exert antitumor effects (Debinski (2002) Molecular "Targeting of Brain Tumors with Cytotoxin," In: Chimeric Toxins (Lorberboum-Galski & Lazarovici, eds., Harwood Academic Publishers) pp. 222-246; Debinski (2002) Cancer Invest. 20:801-809). In addition, the IL-13RA2 receptor ligand is taken up internally through receptor-mediated endocytosis. See also Debinski et al., U.S. Patent No. 8,362,207.

[0074] The above are examples of chemotherapeutic agents useful as effectors. Small molecule toxins such as calichemycin, meitansine (see U.S. Patent No. 5,208,020), trichothecenes, and CC 1065 are also intended herein as effectors. In some embodiments, Pseudomonas exotoxins are used as effectors (see Pastan et al., U.S. Patent No. 5,328,984). Enzymatic toxins and their fragments that can be used as effectors include the diphtheria A chain, an unbound active fragment of diphtheria toxin; the exotoxin A chain (derived from Pseudomonas aeruginosa); the lysine A chain; the abrin A chain (derived from Corrybacterium typhimuriae); the modesin A chain; alpha-sarcin; Aleurites fordii protein; dianthin protein; Phytolacca americana protein (PAPI, PAPII, and PAP-S); bitter melon (momordica charantia) inhibitors; curcin; crotin; soapwort (Saponaria officinalis) inhibitors; geronin; mitogenin; restrictosin; phenomycin; enomycin; and trichothecenes. See, for example, WO93 / 21232.

[0075] The effectors may also include compounds with nucleic acid degradation activity (e.g., ribonucleases or deoxyribonucleases; DNA endonucleases such as DNase).

[0076] Various radioisotopes or radionuclides can be used to create the radioactive conjugate structures described above.

[0077] Linkers may or may not be peptides. Non-peptide linkers may include aliphatic hydrocarbon linkers such as alkyl, alkenyl, or alkynyl linkers, which optionally contain one or more functional groups suitable for covalent attachment of ligands, localization elements, and / or effectors.

[0078] In some embodiments, the conjugate of the targeting protein, linker, and / or therapeutic agent or detectable group is N-succinimidyl-3-(2-pyridyldithiol)propyronate (SPDP), succinidimyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), imide ester (e.g., dimethyl HCl adipimidoate), active ester (e.g., disuccinimidyl suberate), aldehyde Difunctional protein coupling agents can be used to prepare these conjugates, such as difunctional derivatives of bis-azide compounds (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., torylene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine conjugates can be prepared as described in Vitetta et al. (1987) Science 238:1098. Carbon-14 labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionuclides to targeting peptides. See WO94 / 11026. The linker may be a "cleavable linker" that facilitates the release of cytotoxic drugs in cells. For example, acid-unstable linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al. (1992) Cancer Res. 52:127-131) may be used.

[0079] In some embodiments, the linker may include an Fc domain or fragment thereof that attaches directly or indirectly (e.g., through a chemical spacer) to the ligand, localization element, and / or effector molecule. The terms “Fc,” “Fc domain,” or “Fc fragment” encompass both native and variant forms of polypeptides derived from the Fc region of an antibody bound by an Fc receptor. In some embodiments, the Fc domain is derived from a human antibody (i.e., “human” Fc). The Fc domain typically has at least two heavy chain constant region domains (CH2 and CH3).

[0080] This also includes types of Fc domains that contain a hinge region that promotes dimerization. One suitable Fc fragment described in PCT applications WO2005 / 047334 A1 and WO2004 / 074455 A2 is a single-stranded polypeptide extending from the N-terminal hinge region to the natural C-terminus.

[0081] For example, variants of the Fc fragment that exhibit improved serum half-life, altered effector function, or altered spatial orientation are also being considered. These variants of the Fc fragment can be achieved using any genetic engineering technique known in the art. In some embodiments, the Fc domain is linked to more than one, for example, two, three, or four effector molecules.

[0082] Conjugation of the Fc domain can be carried out using methods known in the art, for example, see US2010 / 0209424 by Roopenian et al. and US2012 / 0039880 by Yan et al., which are incorporated herein by reference. For example, fusion proteins comprising a ligand, a localization signaling element, and / or an effector may be prepared by recombinant techniques or peptide synthesis, and they may also subsequently involve covalent coupling of polypeptides, linkers, and / or non-peptide effectors.

[0083] In some embodiments, the Fc domain of the Fc fusion protein is a human Fc domain. The Fc domain may be derived from immunoglobulin G (IgG), IgA, IgE, or IgM. In one embodiment, the Fc domain is IgG-derived and may be derived from any of the subclasses of IgG. For example, in humans, there are four subclasses of IgG: IgG1; IgG2; IgG3; and IgG4. In some embodiments, the Fc domain is from human IgG1.

[0084] In some embodiments, the Fc domain may include an antibody-dependent cytotoxicity (ADCC) activation domain and / or a complement-dependent cytotoxicity (CDC) activation domain. Such domains may be useful in activating immune cells to bind to and / or attack target cancer cells, thereby increasing their cytotoxic efficacy. See, for example, Di Gaetano et al., Complement Activation Determines the Therapeutic Activity of Rituximab In Vivo, J Immunol 171: 1581-87, 2003; see U.S. Patent No. 7,829,084 by Ledbetter et al.

[0085] In some embodiments, the effector molecule may be a Pseudomonas exotoxin or a diphtheria toxin (see U.S. Patent No. 5,328,984 by Pastan et al. and U.S. Patent No. 6,296,843 by Debinski). The Pseudomonas exotoxin includes, but is not limited to, Pseudomonas exotoxin A (PE). The Pseudomonas exotoxin can be modified to substantially lack domain Ia, and further examples of Pseudomonas exotoxins include PE38QQR and PE4E. The diphtheria toxin includes DT390, which is a diphtheria toxin from which the natural binding domain has been removed.

[0086] It is likely that targeting proteins and / or effector molecules can be linked to either the amino or carboxyl terminus of a polypeptide linker, in addition to their internal amino acids (e.g., cysteine).

[0087] Pharmaceutical preparations and methods The constructs, conjugates, and / or compositions described herein may be formulated for administration in a pharmaceutical carrier according to known techniques. See, for example, Remington, The Science and Practice of Pharmacy (9th Ed. 1995). In the preparation of a pharmaceutical formulation according to the present invention, the construct (including its physiologically acceptable salt) is typically mixed with an acceptable carrier, among other things. The carrier must, of course, be acceptable in the sense that it is compatible with any other components in the formulation and must not be excessively harmful to the patient. The carrier may be solid or liquid or both and is preferably formulated with the construct as a unit-dose formulation, e.g., a tablet, which may contain 0.01% or 0.5% to 95% or 99% by weight of the active construct. One or more active constructs may be incorporated into a formulation of the present invention, which may be prepared by any known technique of pharmacy, including mixing its components, and may optionally contain one or more minor components.

[0088] The formulations of the present invention include those suitable for oral, rectal, topical, buccal (e.g., sublingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both the skin surface and mucosal surfaces, including the airway surface), and transdermal administration, but the most appropriate route in any given case depends on the nature and severity of the condition to be treated, as well as the nature of the specific active construct to be used.

[0089] Specific routes of parenteral administration include intraarachnoid injection (including brain tumors that have spread locally into the meninges), which may involve direct injection into the tumor or tumor resection cavity, and intraventricular injection into the ventricles.

[0090] The constructs and compositions may be administered by intratumor injection (including tumors in any region, such as breast and / or brain tumors).

[0091] Specific routes of parenteral administration for breast cancer treatment include intravenous administration, such as injection into breast tissue, i.e., into a vein or venous catheter leading to breast tissue. In some embodiments, the active substance or construct is administered directly to the breast tissue of the subject and / or other areas where the breast cancer has metastasized. In some embodiments, the construct is administered to the subject through a central venous catheter (CVC; e.g., a central venous line or central venous access device). In some embodiments, the CVC is a peripherally inserted central catheter (PICC line) or a tunnel-type CVC. In some embodiments, the construct is administered to the subject through a subcutaneously implanted port. In some embodiments, intravenous administration may occur over a period of about 1 minute to about 60 minutes (e.g., about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or about 60 minutes).

[0092] Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injectable solutions of the active construct, the preparations preferably isotonic with the blood of the intended recipient. These preparations may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may contain suspending agents and thickeners. Formulations may be provided in unit-dose or multi-dose containers, e.g., sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, e.g., saline or water for injection, immediately before use. Instantaneous injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets of the types described above. For example, in one embodiment of the present invention, an injectable or injectable, stable sterile composition is provided, comprising the active construct or composition in unit dosage form in a sealed container. The construct or composition is provided in the form of a lyophilized product that can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for administration to its target. The unit dosage form typically contains about 10 mg to about 10 g of the construct or composition. If the construct or composition is substantially water-insoluble, a sufficient amount of physiologically acceptable emulsifier may be used in an amount sufficient to emulsify the construct or composition in an aqueous carrier. One such useful emulsifier is phosphatidylcholine.

[0093] Furthermore, the present invention provides liposome formulations of the constructs and compositions disclosed herein. Technologies for forming liposome suspensions are well known in the art. If the construct or composition is a water-soluble composition, it can be incorporated into lipid vesicles using conventional liposome technology. In such cases, due to the water solubility of the construct or composition, it is substantially incorporated into the hydrophilic center or core of the liposome. The lipid layer used can be of any conventional composition and may or may not contain cholesterol. If the construct or composition of interest is water-insoluble, it can also be substantially incorporated into the hydrophobic lipid bilayer that forms the structure of the liposome using conventional liposome formation technology, as before. In either case, the resulting liposomes can be reduced in size, for example, by using standard sonication and homogenization techniques.

[0094] Liposome formulations containing the constructs or compositions thereof disclosed herein (e.g., polyvalent conjugates) may be lyophilized to produce lyophilized products which may be reconstituted with a pharmaceutically acceptable carrier such as water to regenerate liposome suspensions. Examples of usable liposome formulations include the neutral lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DPOC). See, for example, Landen Jr. et al. (2005) Cancer Res. 65:6910-6918.

[0095] Other pharmaceutical compositions, such as aqueous emulsions, may be prepared from the water-insoluble constructs or compositions disclosed herein. In such cases, the composition contains a pharmaceutically acceptable emulsifier in an amount sufficient to emulsify a desired amount of the construct or composition. Particularly useful emulsifiers include phosphatidylcholine and lecithin.

[0096] The pharmaceutical composition may contain other additives in addition to the construct, such as pH-adjusting additives. Particularly useful pH adjusters include acids such as hydrochloric acid, bases, or buffers such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Furthermore, the composition may contain antimicrobial preservatives. Useful antimicrobial preservatives include methylparaben, propylparaben, and benzyl alcohol. Antimicrobial preservatives are typically used when the formulation is encapsulated in vials designed for multiple doses. Of course, as shown, the pharmaceutical composition of the present invention may be freeze-dried using techniques well known in the art.

[0097] The therapeutically effective dosage may vary slightly depending on the construct and the patient, and depends on factors such as the patient's age and condition, as well as the route of delivery. Such dosages can be determined according to routine pharmaceutical procedures known to those skilled in the art.

[0098] Certain pharmaceutical compositions containing constructs of the present invention suitable for administration may include a pH-neutral (e.g., pH 6 or 6.5 to pH 7.5 or 8) low-molar saline solution, such as phosphate-buffered saline (PBS) (e.g., Dulbecco's PBS pH 7.2).

[0099] As a general suggestion, the initial pharmacokinetic effective dose of the parenterally administered active construct ranges from approximately 0.1 to 50 mg per kg of patient body weight per day, with a typical initial range of 0.3 to 20 mg / kg / day, more preferably 0.3 to 15 mg / kg / day. The desired dosage can be delivered by a single bolus, multiple bolus, or continuous infusion of the active construct, depending on the pharmacokinetic decay pattern that the physician wishes to achieve.

[0100] The construct may be administered to the patient, appropriately, in a single dose or over a series of treatments. Depending on the type and severity of the disease, the initial candidate dose for administration to the patient is approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 to 20 mg / kg) of the active construct, whether by a single dose, multiple separate doses, or continuous infusion. Typical daily doses may range from approximately 0.1 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, or 100 μg / kg to 100 mg / kg, 200 mg / kg, or 500 mg / kg or higher, depending on the factors mentioned above. For repeated administrations over several days or more, the treatment is continued, depending on the patient's condition, until the desired suppression of disease symptoms occurs. More specific dosages of the active construct range from approximately 0.05 mg / kg to approximately 20 mg / kg, for example, from approximately 0.05 mg / kg, 0.25 mg / kg, 0.5 mg / kg, or 1.0 mg / kg to approximately 5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg (or any combination thereof) may be administered to a patient. Such doses may be administered intermittently, for example, weekly or every three weeks. A higher initial loading dose may be administered, followed by one or more lower doses. An exemplary dosing regimen involves an initial loading dose of approximately 0.5–15 mg / kg of the active construct, followed by weekly maintenance doses of approximately 0.5–5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. However, other dosing regimens may be useful. The course of this treatment is readily monitored using standard techniques and assays.

[0101] Subjects treated by the method of the present invention may also be administered one or more additional therapeutic agents. See U.S. Patent No. 5,677,178. Chemotherapy agents may be administered by methods well known to physicians, for example, systemically by direct injection into the cancer, or by localization at the site of the cancer by linking the desired chemotherapeutic agent with an appropriate sustained-release material or intra-arterial perfusion of the tumor. The preferred dose may be selected by the physician based on the nature of the cancer to be treated and other factors routinely considered in administration. See, for example, U.S. Patent No. 7,078,030.

[0102] In some embodiments, the active substance or construct is administered directly to the target brain (i.e., within the blood-brain barrier) and / or other parts of the central nervous system. In some embodiments, the active substance is administered intracerebral infusion. In some embodiments, the active substance is administered to the target by intracerebral infusion. In some embodiments, the active substance is administered by subarachnoid delivery. In some embodiments, the active substance is administered by convective-enhanced delivery.

[0103] Convection-enhanced delivery (CED) is a continuous infusion of a therapeutic agent under positive pressure. In the central nervous system (CNS), this delivery technique bypasses the blood-brain barrier in delivering the active ingredient. See, for example, Strauss et al. US2005 / 0002918; Haider et al. US2012 / 0041394; Bankiewicz et al. US2012 / 0209110. CED diffuses the substance into the extracellular space using a flow gradient and bulk flow established at the tip of the infusion catheter. CED allows for further diffusion of the extracellular infusion material through rhythmic vasoconstriction, which acts as an efficient transport force for the perivascular space and the infusion fluid. As a result, higher concentrations of the drug can be distributed more evenly over a larger area of ​​target tissue than is seen with simple infusion. CEDs have been clinically tested in the fields of neurodegenerative diseases and neuro-oncology and are useful in a wide range of applications, including the delivery of small molecules, macromolecules, viral particles, magnetic nanoparticles, and liposomes. In some embodiments, CEDs include the use of anti-reflux catheters. In some embodiments, CEDs include real-time MRI monitoring.

[0104] With regard to administration by continuous infusion such as CED, the dosage of the construct for administration may be, for example, from 0.1 μg / mL or 0.5 μg / mL to 20 μg / mL, for example, from 1 μg / mL, 2 μg / mL, or 3 μg / mL to 5 μg / mL, 8 μg / mL, 10 μg / mL, or 15 μg / mL.

[0105] In some embodiments, the construct is administered in combination with radiotherapy. In some embodiments, the construct is administered in combination with surgery to remove at least a portion of the cancerous tissue. In some embodiments, the construct is administered in combination with another different chemotherapeutic agent.

[0106] Radiation therapy may include, for example, external beam radiotherapy, which can be any appropriate dose (e.g., 20-70 Gy or more per tumor, typically delivered by a fractional schedule).

[0107] A pharmaceutical composition containing an effector-free targeting construct may be administered to a subject as a blocking reagent in a similar manner to that described by Abrams et al., U.S. Patent No. RE38,008, along with the administration of a targeting construct coupled with an effector such as a therapeutic agent.

[0108] Targeting constructs coupled with detectable groups can also be used in vitro as histological reagents in tissue samples; for example, binding to EphA2, EphA3, EphB2, or IL-13RA2 receptors indicates cancerous tissue in the tissue sample.

[0109] The present invention has been described above, but will be explained in more detail in the following examples, which are included herein for illustrative purposes only and are not intended to limit the present invention. [Examples]

[0110] [Example 1] Development of QUAD targeting constructs In a pioneering approach, the inventors conducted a Phase I clinical trial in dogs with gliomas using a cocktail of cytotoxic agents targeting IL-13RA2 and EphA2 receptors as a superior clinical alternative to human diseases. The cocktail was administered locally via convection-enhanced delivery (CED) using an anti-reflux catheter and real-time MRI monitoring of drug distribution. The inventors were able to inject the cocktail into both contrast-enhancing and non-contrast-enhancing tumors containing invasive tumor cells. In this dose-determination study, the inventors observed significant antitumor responses, including several near-complete regressions, extended survival, and improved quality of life, but did not reach dose-limiting toxicity. The inventors had already observed 4 out of 17 dogs with >95% tumor regression and several dogs with long-term survival, but the majority of dogs responded with >50% tumor regression (Figure 2). See also Rossmeisl et al., Phase I trial of convection-enhanced delivery of IL13RA2 and EPHA2 receptor targeted cytotoxins in dogs with spontaneous intracranial gliomas. Neuro Oncol. 23(3): 422-434 (2021).

[0111] Using the Canine Cancer Immunology Panel (Nanostring IO, Seattle, WA, USA), the inventors performed preliminary comparative gene expression analyses on pre- and post-treatment canine glioma samples from three dogs (Rossmeisl et al., dogs 2, 4, and 10 above) treated in the inventors' Phase I cytotoxic cocktail trial. The treatment resulted in activation of injury-related molecular pattern recognition pathways (Figure 3A), upregulation of genes related to cellular immunity and cytokine and chemokine signaling networks (Figure 3B), and infiltration of immune effector cells into tumors (Figures 3C, D). Increased Th1-responsive cytokine gene expression was most robust in responder dogs, and cytokine gene expression changes paralleled serum cytokine analysis reported in the Phase I trial. Taken together, these data further support the inventors' hypothesis that cytotoxic treatment induces immunogenic cell death and in-situ tumor vaccination-type effects.

[0112] Encouraged by these exceptional results, the inventors pursued the idea of ​​targeting all four receptors with a single pharmaceutical compound in their next step. To achieve this objective, the inventors designed, fabricated, and tested a multivalent ligand, named QUAD, that binds to all four receptors of interest, including mouse and canine receptors. The QUAD1 molecule (Figure 4, Panel A) consists of (i) ephrin A5 (eA) that binds to EphA2, EphA3, and EphB2 receptors, (ii) the Fc region of IgG1, and (iii) a mutant interleukin 13, IL-13.E13K, that binds to IL-13RA2 [eA5-Fc-IL-13.E13K]. This protein chain was frequently obtained as a doublet on SDS-PAGE, but this problem was solved by altering the order of ligands in the molecule (QUAD2). In fact, the IL-13.E13K-Fc-eA5 construct was reproducibly generated in high yield as a single band of the expected size of approximately 150 kDa QUAD (e.g., Figure 7B) (see Sharma et al., Multi-receptor targeting of glioblastoma. Neuro-Oncology Advances 2(1),1-11, 2020; Sharma et al., Drug Conjugates for Targeting Eph Receptors in Glioblastoma. Pharmaceuticals(Basel).2020 Apr 23;13(4):77).

[0113] The inventors also initiated with High Five cells possessing the ability to produce higher yields of recombinant protein. High Five (H5) cells in suspension culture produced up to 4.2 mg of recombinant protein per liter of medium. Insect cell-produced proteins are approved for both human and veterinary use for clinical applications. QUAD can be uniformly purified, and ELISA assays have shown that all four target receptors, IL-13RA2, EphhA2, EphA3, and EphB2, bind to the thus constructed QUAD, which is not an unremarkable result for a polyvalent recombinant protein. In addition, the inventors also constructed another version of QUAD, QUAD-Cys or QUAD 3, IL-13.E13K-Fc-eA5-Cys, intended to have free thiol groups available for conjugation with drugs / labels (Figure 4, Panel B). This construct can be efficiently fabricated and purified as a single-band protein, and QUAD-Cys also efficiently binds to all four target receptors. Thus, we have established and improved the conditions for QUAD fabrication that fully preserves the function of the ligand and has further conjugation with drugs / labels.

[0114] Next, the inventors created a conjugate of QUAD 3.0 and PE38QQR that potently killed GBM cells (Sharma et al., Multi-receptor targeting of glioblastoma. Neuro-Oncology Advances 2(1),1-11,2020). For example, IC 50The concentration was close to 1.0 nM in U-251 and G48a cells, and 10 nM in BTCOE4795 GBM cells. These results directly demonstrated the feasibility and utility of the QUAD-drug approach. In addition, normal C57BL / 6 mice were intracranially injected with bolus doses of 0.1 μg, 0.5 μg, and 1.0 μg of QUAD 3.0-PE38QQR per mouse without any signs of toxicity. Next, QUAD was conjugated with various derivatives of doxorubicin (Dox), such as WP1244, WP936, and WP1737, to create single drug conjugates. The inventors have characterized the conjugate well by maintaining its binding affinity to the target receptor while exhibiting excellent killing activity against GBM cells (Sharma et al., Drug Conjugates for Targeting Eph Receptors in Glioblastoma. Pharmaceuticals (Basel). 2020 Apr 23;13(4):77). However, the QUAD 3.0-WP1244 conjugate had the narrowest therapeutic window of the three conjugates tested and was excluded from further experiments. As expected, greedy internal migration of QUAD 3.0-WP936 was observed, and cells showed readily detectable signals for the two individual components of the conjugate, QUAD and WP936 (a fluorescent derivative of Dox). Thus, QUAD 3.0-WP936 not only efficiently binds to the target receptor in vitro but is also recognized by living cells and internally migrated by them. Most importantly, QUAD 3.0-WP936 and QUAD 3.0-PE38QQR have already demonstrated dramatic, long-lasting (>6 months) antitumor effects in dogs with spontaneous gliomas, without toxicity, at a dose of 1.6 μg / ml, which is half the highest dose used in the cocktail trial (Figure 5). These studies represent proof in principle of our hypothesis that QUAD proteins may be universal vectors for effective drug delivery.In addition, the modified human-derived IL-13 and eA5 ligands included in our constructs were sufficiently reactive with canine and mouse receptors, as previously reported for IL-13RA2 (Debinski et al., New agents for targeting of IL-13RA2 expressed in primary human and canine brain tumors. PLoS One. 2013 Oct 16;8(10):e77719), and the canine / mouse Eph receptor interaction with human eA5 was further demonstrated in flow cytometry, cytotoxicity assays, and functional assays (Figure 6). This confirms the human relevance of all our animal studies, which is a strength of our experimental methods.

[0115] Our approach offers current treatment modalities an unparalleled opportunity to gain increased access to highly resistant or poorly utilized tumor compartments. It also addresses the problems of high GBM tumor heterogeneity and immunosuppressive microenvironment.

[0116] [Example 2] Evaluation of drug conjugates QUAD was conjugated with various derivatives of doxorubicin to create single pharmaceutically acceptable compounds. These drug conjugates retained their binding affinity to target receptors while exhibiting excellent killing activity against GBM cells. One of the QUAD-doxorubicin conjugates, and a conjugate with the bacterial toxin derivative PE38QQR, has already shown a remarkable antitumor response without toxicity in dogs with spontaneously occurring canine gliomas. We have also found the first evidence of immune system activation in dogs treated with targeted cytotoxicity, based on blood and tissue analysis.

[0117] In further exploration of the best and most active QUAD-based drug conjugates, the inventors decided to test other chemotherapeutic agents for conjugation with QUAD 3.0 or 3.1 (the 3.1 version has codons optimized for insect cells). The inventors selected two drugs with very different modes of action. One is deruxtecan (DTX), a topoisomerase I inhibitor. The other is DM1, a microtubule polymerization inhibitor (Figure 7, Panel A). This agent has been used in conjugation with Herceptin to produce Kadcyla, a highly effective anticancer drug (Park et al., I-SPY 2 Investigators. N Engl J Med. 2016 Jul 7;375(1):11-22). The inventors have successfully conjugated the QUAD 3.0 protein with these two chemotherapeutic agents (e.g., Figure 1, Panel A; DM1 conjugate shown). Chemical conjugation of QUAD 3.0 / 3.1 with DM1 is carried out as previously described (Debinski et al., An immunotoxin with increased activity and homogeneity produced by reducing the number of lysine residues in recombinant Pseudomonas exotoxin. Bioconj. Chem. 5:40-46, 1994; Debinski et al., (1995) Recombinant C242 F(ab') - Pseudomonas exotoxin, but not the whole antibody-based immunotoxin, causes regression of a human colorectal carcinoma xenograft. Clin. Cancer Res. 1:1015-1022). QUAD 3.0 / 3.1 has a cysteine ​​at the C-terminus that provides a reactive thiol group for conjugation.Derivatized DM1 (DM1-SMCC, Figure 7, Panel A) and DTX form a stable thioether bond through the cysteine ​​residue in QUAD 3. The conjugate is purified from the unconjugated counterpart using size exclusion chromatography on a HiPrep® 16 / 60 Sephacryl® S-200 HR (GE, Boston, MA) column.

[0118] Both conjugates were highly potent in killing U-251 MG GBM cells (Figure 7, Panel C). However, IC for the QUAD-DM1 conjugate was... 50 The low phentomole range is quite surprising with respect to drug conjugates. Similar phentomole ranges were observed in A-172, BTCOE-4525, and BTCOE-4975 GBM cells. 50The following was observed (not shown). As shown in Figure 7, Panel C, the activity of the QUAD-DM1 conjugate was more than 1000-fold superior in concentration to that of the non-conjugated drug. QUAD-DM1 was approximately 50-fold more potent than all other QUAD-based drug conjugates tested, QUAD-WP936, QUAD-DTX, and also QUAD-PE38QQR (not shown). The QUAD-DM1 conjugate was active not only in human GBM cells but also in canine GBM cells (Figure 7, Panel D). Furthermore, T98G GBM cells that do not express IL-13RA25 and are completely unresponsive to single-targeted IL-13-based cytotoxicity were clearly affected by QUAD-DM1 (Figure 7, Panel E). This is because T98G cells express some EphB2 and EphA3 receptors (not shown). Furthermore, the killing effect of QUAD-DM1 was neutralized by an excess of unconjugated QUAD, which showed competition at the binding site between unconjugated QUAD and its DM1 conjugate (Figure 7, Panel F). The same effect was observed in BTCOE4795 cells. Perhaps not surprisingly, as eA5 has been previously shown to have tumor suppressor effects, the QUAD protein affected the cell viability of U-251 GBM cells in vitro (Figure 7, Panel G). This demonstrated the potential dual antitumor effect of the QUAD conjugate. Finally, the QUAD-DM1 conjugate was administered either intracranially or intravenously to C57 / BL6 non-tumor-bearing mice (Figure 7, Panels H, I).The highest doses used in these experiments, equivalent to the IC-given doses for other QUAD 3.0 conjugates (Sharma et al., Multi-receptor targeting of glioblastoma. Neuro-Oncology Advances 2(1),1-11,2020; Sharma et al., Drug Conjugates for Targeting Eph Receptors in Glioblastoma. Pharmaceuticals(Basel).2020 Apr 23;13(4):77), did not produce any signs of toxicity such as changes in body weight, neurological symptoms, or changes in intracranial heat capacity (IHC). In our most recent research, we have established that 4.0 μg of QUAD-DM1 per mouse is a DLT for intracranial bolus injection. Thus, we have created an extremely potent and safe drug conjugate, QUAD-DM1.

[0119] The multivalently targeted cytotoxic drug conjugate, QUAD-DM1 (Figure 1), is engineered to simultaneously target four receptors [interleukin-13 receptor alpha-2 (IL-13RA2), EphA2, EphA3, and EphB2 receptors] that are overexpressed in patients with glioblastoma (GBM), the most frequently occurring primary brain tumor with a poor prognosis. The cytotoxic drug conjugate consists of a targeting moiety and DM1, a microtubule inhibitor used in antibody-drug conjugates named Kadcyla. The targeting moiety consists of IL-13 (IL-13M) and ephrin A5 (eA5), modified for more specific binding to the tumor-associated receptor IL-13RA2; both are located at the N-terminus and C-terminus of the IgG1 scaffold (Fc), respectively, forming the multivalent protein, QUAD (Figure 1). The recombinant product is produced in a single eukaryotic host cell (insect or mammal, e.g., FS9 insect cells or HEK293 mammalian cells). QUAD-DM1 is a chemical conjugate of QUAD with DM1-SMCC. QUAD-DM1 is delivered using a non-reflux catheter under real-time MRI monitoring of convection-enhanced delivery (CED) (CLEAR POINT Neuro, formerly known as MRI Interventions; e.g., IND #117959 or NCT02858895).

[0120] This innovative approach to treating GBM simultaneously targets tumor cells, glioma stem cell-like cells (GSCs), angiogenic and invasive tumor cells, and TAMs (all expressing IL-13RA2, EphA2, EphA3, and EphB2 receptors) with a single pharmaceutical compound, QUAD-DM1. The four receptors targeted are present in large quantities in virtually all GBM patients, meaning that molecular pre-screening of patients may not be necessary for its proposed monotherapy. Furthermore, QUAD-DM1 is delivered via convection-enhanced delivery (CED), bypassing the limitations imposed by the blood-brain barrier (BBB) ​​or blood-brain tumor barrier (BBTB).

[0121] QUAD preparation and purification High Five cells (derived from ovarian cells of the nettle moth; Thermo Fisher, Waltham, MA) are removed from a 10 cm dish by sloughing. Live cells are counted using a hemocytometer and trypsin blue exclusion. 5 × 10 5 Plate cells / well in a 6-well dish containing serum-free SFX insect cell culture medium in a volume of 2 ml and incubate overnight at 27°C without CO2. The next day, remove the medium and add 1.8 ml of fresh medium to each well. Prepare 1-3 μg of QUAD plasmid (pMIB V5 His A, optimized for insect cells and synthesized by Genewiz) for transfection with Cellfectin according to the manufacturer's instructions. Briefly, add the DNA to 100 μl of medium. In a separate tube, add 4 μl of selectin to 100 μl of medium. Gently mix the DNA tube and the selectin tube and allow to conjugate at room temperature for 30 minutes. Slowly add the mixture to the cells and incubate overnight at 27°C without CO2. The next day, change the medium (Cytiva, Marlborough, MA Thermo Fisher, Waltham, MA) and allow the cells to recover overnight. Positively transfected cells are selected with 500 μg / ml blastosidine. Positive selection is maintained with 50 μg / ml blastosidine. Cells are divided into 3 × 10⁻⁶ cells. 5 cells / cm 2 The cells are grown in a suspension culture at the specified density. After 3-4 days of culture, the suspension cells are centrifuged at 1,000xg for 5 minutes, and the culture medium containing the secreted QUAD protein is collected.

[0122] Before purification using a HiTrap Protein G HP (Cytiva) column with an AKTA Pure FPLC system (Cytiva, Marlborough, MA), the QUAD-containing medium is filtered through a 0.22 μm filter. First, the column is equilibrated with 10 column volumes (CV) of 20 mM sodium phosphate, pH 7.0 before loading the QUAD-containing medium. After washing 10 CV with 20 mM sodium phosphate, pH 7.0, the protein is eluted from a 1 ml fraction with 0.1 M glycine-HCl, pH 2.7. The pH of the eluate is immediately adjusted to 7.0 by adding 200 μl of 1 M TRIS-HCl, pH 8.0. The positive fraction (based on SDS-PAGE) is pooled, concentrated using a 30K MWCO centrifuge filter (Sartorius Vivaspin), and buffered with Dulbecco's phosphate-buffered saline containing 5 mM EDTA (D-PBS / EDTA), pH 7.2. Dilute QUAD to a concentration of 1 mg / ml and store at -80°C until use. Verify purity by SDS-PAGE.

[0123] QUAD conjugation and conjugate purification Conjugate QUAD with DM1 at a 16x molar ratio. Add 10.85 μl of 5 mM DM1 in DMSO to 189.15 μl of D-PBS / EDTA. Slowly add this mixture dropwise to 200 μg of QUAD (in 200 μl) while stirring (maximum 130 rpm). Incubate the mixture at room temperature for 60 minutes while stirring. Continue the reaction overnight at 4°C. The next day, centrifuge the mixture at 10,000xg for 5 minutes to remove any precipitate, if present.

[0124] At least 1 mg of conjugated QUAD is concentrated using a 30K MWCO centrifuge filter before injection into a HiPrep 16 / 60 Sephacryl S-300 HR column (Cytiva). The column is flowed at a rate of 0.5 ml / min, and a 0.4 ml fraction is collected. The positive fraction is pooled, concentrated, and stored at -80°C. The identity and purity of the conjugate are confirmed by Western blotting (Figure 1B).

[0125] The conjugate is diluted to the final concentration with pharmaceutical buffer and sterile filtered into a final product container / closure (e.g., a pharmaceutical-grade 1.0 mL glass serum vial with a rubber serum stopper and aluminum crimp seal).

[0126] Initial QUAD-DM1 Conjugate Stability Test Method: QUAD-DM1 is incubated in 100 μl volumes in plasma from GBM patients, plasma from non-tumor patients (normal), or PBS at -80°C, -20°C, 40°C, 27°C (room temperature), and 37°C. At the specified time points, PBS containing 0.1% BSA is added, and the mixture is filtered through a 0.22 μm filter. In the MTT cell viability assay, QUADcyla is serially diluted and added to U-251 GBM cells.

[0127] Results: QUAD-DM1 maintained its cytotoxic activity for at least 8 weeks after incubation in PBS, GBM patient serum, and normal patient serum at -80°C, -20°C, and also 4°C. When incubated at RT, QUAD-DM1 began to lose its cytotoxic activity after 1 week for samples incubated in plasma. QUAD-DM1 incubated at RT in PBS also retained most of its cytotoxic efficiency up to 8 weeks. After 4 weeks of incubation at 37°C, plasma QUAD-DM1 lost 3 log of cytotoxic activity, while samples incubated in PBS lost only 1 log of activity. After 8 weeks at 37°C, all cytotoxic activity was lost under all conditions.

[0128] [Example 3] Preclinical research Chemical conjugation of QUAD with DM1 is carried out as previously described. QUAD has a cysteine ​​at its C-terminus that provides a reactive thiol group for conjugation. Derivatized DM1 (DM1-SMCC) forms a stable thioether bond through the cysteine ​​residue in QUAD. The conjugate is purified from the unconjugated counterpart using size exclusion chromatography on a HiPrep™ 16 / 60 Sephacryl® S-200 HR (GE, Boston, MA) column.

[0129] In Figure 8, the inventors demonstrate a dramatic response to non-toxic QUAD-DM1 in a dog with spontaneously occurring high-grade glioma. This was somewhat expected by the inventors, given the favorable properties described for QUAD-DM1. Since residual contrast-enhancing lesions on MRI after cytotoxic treatment typically undergo further lysis over time, it is possible that this case will result in a complete response (CR).

[0130] [Example 4] Evaluation of QUAD treatment in breast cancer and breast cancer brain metastases Because the pharmaceutical needs for triple-negative breast cancer (TNBC) remain unmet, we investigated the presence of four QUAD-targeted receptors in microarrays of breast cancer cells and tissues, including lesion lymph nodes, spanning two molecular subtypes of breast cancer, TNBC and HER2+, as well as in primary tumor-brain metastasis pairs. We found that these four target receptors were expressed in almost all breast cancer specimens and associated brain metastasis specimens, based on expression levels of the genes and gene products (Figure 9, Panel A). We also identified several established breast cancer cell lines expressing EphA3, EphA2, and EphB2 by Western blotting (Figure 9, Panel B). Cell lysates were collected from subconfluent cultures in RIPA buffer (Sigma) containing protease inhibitors and phosphatase inhibitors. Proteins were separated by 10% SDS-PAGE under reducing conditions and transcribed onto PVDF membranes. After blocking nonspecific interactions, the primary antibody was added and incubated overnight at 4°C. The antibodies used were EphA2 (Wykosky, et al. Mol. Cancer Ther. 6(12): 3208-3218 (2007)), EphA3 (MyBiosource), and EphB2 (R&D Systems). After multiple washes with PBS, the secondary antibody (Sigma) was added. Chemiluminescence detection was performed, and the bands were detected using an Amersham RGB600 imager. Equal protein loading was verified by probing for β-actin (Sigma).

[0131] Flow cytometry was performed to analyze the binding of QUAD to human breast cancer cell lines (Figure 9, Panel C). Cells were desorbed with Barzen's solution and washed once with PBS. 200,000 cells in PBS / 1% BSA were dispensed into tubes and incubated on ice for 1 hour to block nonspecific sites. 2 μg of QUAD protein or human Fc control was added to the tubes and incubated on ice for 2 hours, with occasional mixing. Cells were washed with PBS / 1% BSA and a secondary antibody (anti-human Alexa fluor 647, Invitrogen) was added. After 1 hour incubation on ice, cells were washed and fixed with formalin. Detection was performed using an Accuri6 flow cytometer (BD Biosciences), and data were analyzed using FCS Express (DeNovo Software).

[0132] Immunohistochemical (IHC) data from tissue microarrays (TMAs) derived from human breast cancer, lymph node metastases, and normal breast tissue were stained for the presence of IL-13RA2, EphA3, EphA2, and EphB2 proteins (Figure 10). Staining was performed on US Biomax slides deparaffinized with xylene and rehydrated with ethanol. Antigens were restored by quenching endogenous peroxidase and heating in sodium citrate, pH 6.0. Nonspecific binding was blocked with Superblock (Scytek), and the slides were stained with IL-13RA2 (0.5 μg / ml; clone 1E10B9, see Debinski et al., PLoS One (2013)), EphA3 (1:200; Genetex N1N3), EphA2 (2.5 μg / ml; NovusBio), and EphB2 (2 μg / ml; R&D Systems). After overnight incubation at 4°C, the slides were washed with PBS before applying the secondary antibody (anti-rabbit polymer HRP, Vector Labs). Detection was performed with Nova Red (Vector Labs). The slides were counterstained with hematoxylin, dehydrated, cleared with xylene, and mounted with Permount. The slides were digitally scanned in the Virtual Microscope Core Lab at WFU Comprehensive Cancer Center. These data showed that IL-13RA2, EphA3, EphA2, and EphB2 proteins were frequently observed in breast cancer tissue and lymph node metastases compared to normal tissue and isotype-stained controls. Similar IHC staining in primary breast cancer and brain metastases from two patients yielded the same results, indicating the presence of these proteins in both tissues (Figure 11). Histological scoring of IL-13RA2, EphA3, EphA2, and EphB2, estrogen receptor (ER), progesterone receptor (PR), and HER2 in 10 patient-matched breast cancers and their brain metastases, obtained from initial disease reports of patients after surgery, is shown in Table 1 below.

[0133] [Table 1]

[0134] The inventors tested QUAD-DM1 in several human breast cancer cell lines and found that they were highly responsive to treatment (Figure 12, Panel A). Specifically, breast cancer cell lines such as HC1806 (acantholytic squamous cell carcinoma), MDA-MB-468 (metastatic adenocarcinoma), MDA-MB-231 (adenocarcinoma), BT549 (ductal carcinoma), and the metastatic mammary gland cancer cell line MDA-MB-231-BRM cells were killed at sub-picomolecal concentrations of QUAD-DM1. Further cell staining and flow cytometry analysis showed that QUAD-DM1 induced cell cycle arrest in the G2 phase (Figure 12, Panel B). Logarithmic phase MDA-MB-231 cells were serum-deficient for 24 hours, and then applied to normal growth medium containing 1 nM QUAD or human Fc (fake). After another 24 hours, cells were collected and fixed with cold ethanol. The nuclei were stained with propidium iodide containing RNAse A (BD Biosciences). Cell cycle data were collected using a Canto II flow cytometer (BD Bioscience). The data were analyzed using FCS Express with multiple DNA analyses and graphed in Prism GraphPad. Non-conjugate QUAD (100 nM) neutralized the cytotoxic activity of QUAD-DM1.

[0135] MDA-MB-231 tumors growing in the mammary fat pads of thymus-deficient mice responded significantly to intravenously administered QUAD-DM1 (Figure 12, Panel C), demonstrating that QUAD recognizes mouse receptors similarly to humans. Nude mice were injected with 500,000 MDA-MB-231 cells in a 100 μL volume into the fourth mammary fat pad. Tumors were measured twice weekly using calipers, and the volume was calculated using the formula V = (W² × L) / 2. Tumors were allowed to grow until they reached 100 cubic mm. Mice were randomized into two groups and intravenously injected either 240 μg of QUAD-DM-1 (12 mg / kg) or PBS in a 100 μL volume into the tail vein. Injections were continued weekly. Mice were euthanized on day 24. This data supports QUAD-DM1 as a novel type of multivalent drug conjugate for IV treatment of breast cancer and its brain metastases, applicable to TNBC but not exclusively.

[0136] [Example 5] Further evaluation of QUAD treatment Figure 13 shows data for intracranial (IC) treatment of MDA-MB-231-BrM tumors with QUAD-DM1, involving IC transplantation of 200,000 MDA-MB-231-BrM cells in 2 μL. Treatment began 7 days after tumor cell transplantation (day 0). Animals were imaged three times a week by IVIS imaging using bioluminescence (photons per second). The mean percentage change in response to treatment is shown in Figure 13. These data demonstrate that QUAD-DM1 produced a significant antitumor effect in mice with intracranial tumors originating from breast cancer brain metastases.

[0137] Figure 14 shows data on the binding specificity (ELISA assay) of HeK-293 cell-produced QUADs to EphA3, PDL1, and CD80. As expected, the QUADs bound only to the EphA3 receptor. Hek-293 cells are human embryonic kidney cells. The same results were obtained for QUADs produced in Expi-293F cells, which are HEK-293 cells adapted to transient transfection. These data indicate that QUAD-DM1 binding is specific to the target receptor.

[0138] Figure 15 shows data on MDA-MB-231 BrM cell killing by QUAD-DM1, read at 48 and 72 hours after QUAD-DM1 addition. Cell killing by QUAD-DM1 was partially blocked by antibodies that counteracted the individual receptor binding capabilities of the QUAD ligands. These data indicate that the killing effect depends on binding to the Eph receptor via eA5, eA1, and eB1 ephrin binding activity.

[0139] Figure 16 shows data for HC1806-triple-negative breast cancer cells (TNBC) using the same method as in Figure 15.

[0140] Bioluminescence imaging using IVIS. Intracranial tumors: Nu / nu mice were intracranially injected with 200,000 MDA-MB-231-BrM-luc-RFP cells via stereotactic injection. Seven days after injection, after verifying tumor growth, the mice were intracranially injected once with QUAD-DM1 or non-conjugated QUAD+DM1. Three times a week, the mice were intraperitoneally injected with 150 mg / kg D-luciferin (Gold Biotechnology, St Louis MO), and the tumors were imaged using an in vivo imaging system (Perkin Elmer, Shelton, CT).

[0141] Using a non-optimized dose of the conjugate, the inventors observed significant tumor growth inhibition in visual recordings of treatment of mice with IC tumors (data not presented). These results confirm that the set of IL-13RA2, EphA3, EphA2, and EphB2 receptors represents an excellent tetramolecule of targetable receptors in peripheral and brain metastatic breast cancer.

[0142] In experiments involving MDA-MB-231 BrM treated with QUAD-DM1, it was found that QUAD-DM1 was taken up by the treated cells (data not presented), resulting in potent cell killing.

[0143] The embodiments described above are examples of the present invention and should not be construed as limitations of the invention. While the present invention has been described in detail with reference to preferred embodiments, variations and modifications are within the scope and spirit of the invention as described and defined in the following claims.

Claims

1. A cancer targeting construct including a first end and a second end, The first terminus has two IL-13 proteins (e.g., IL-13 dimers), or two IL-13RA2-binding variants or fragments of IL-13, The second terminus has two eA5 proteins (e.g., eA5 dimers) or two eA5 EphA2, EphA3, and EphB2-binding variants or fragments, An effector molecule is coupled to the first or second end, A construct wherein the construct is a fusion protein and / or a covalent conjugate, and the effector molecule comprises a maytansinoid or a maytansinoid analog (e.g., DM1 or DM4).

2. The construct according to claim 1, wherein the two eA5 proteins are mutant eA5 proteins.

3. The construct according to claim 1 or 2, wherein the two eA5 proteins are fragments of eA5 (for example, amino acids 21 to 191 of human eA5).

4. The construct according to any one of claims 1 to 3, wherein the two IL-13 proteins are mutant IL-13 proteins (e.g., IL-13.E13K).

5. The construct according to any one of claims 1 to 4, wherein the eA5 and IL-13 proteins are glycosylated.

6. The construct according to any one of claims 1 to 5, comprising a fusion protein having a protein linker between the first and second ends.

7. The construct according to claim 6, wherein the first end is at the N-terminus of the fusion protein and the second end is at the C-terminus of the fusion protein.

8. The construct according to claim 6 or 7, wherein the protein linker comprises an ADCC and / or CDC activating domain.

9. The construct according to any one of claims 6 to 8, wherein the protein linker comprises a human IgG1 Fc fragment.

10. The construct according to any one of claims 1 to 9, wherein the effector molecule further comprises a detectable group.

11. A composition comprising the construct according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. The composition according to claim 11, wherein the carrier is sterile.

13. The composition according to claim 11 or 12, wherein the carrier is a salt solution having a pH of 6 or 6.5 to 7.7 or 8.

14. The composition according to any one of claims 11 to 13, which is suitable for administration to a subject by infusion (e.g., convection-enhanced delivery).

15. The composition according to claim 14, suitable for administration by continuous infusion at concentrations from 0.1 μg / mL or 0.5 μg / mL to 20 μg / mL, for example, 1 μg / mL, 2 μg / mL, or 3 μg / mL, or 5 μg / mL, 8 μg / mL, 10 μg / mL, or 15 μg / mL.

16. A nucleic acid encoding the construct or its protein or peptide portion according to any one of claims 1 to 10.

17. A eukaryotic host cell comprising the nucleic acid described in claim 16 and configured to express the encoding peptide thereof.

18. The eukaryotic host cell according to claim 17, wherein the host cell is an insect expression cell.

19. A method for treating cancer in a subject requiring treatment, comprising the step of administering to the subject an effective treatment amount of a construct according to any one of claims 1 to 10 or a composition according to any one of claims 11 to 15.

20. The method according to claim 19, wherein the cancer is breast cancer, bladder cancer, pancreatic cancer, colorectal cancer, head and neck cancer, thyroid cancer, prostate cancer, melanoma, or glioma.

21. The method according to claim 19, wherein the cancer is glioblastoma, prostate cancer, or melanoma.

22. The method according to claim 19, wherein the cancer is glioblastoma.

23. The method according to any one of claims 19 to 22, wherein the administration step includes convection-enhanced delivery (CED).

24. The method according to any one of claims 19 to 23, wherein the construct is administered to the subject by continuous infusion at a concentration of 0.1 μg / mL or 0.5 μg / mL to 20 μg / mL, for example, 1 μg / mL, 2 μg / mL, or 3 μg / mL to 5 μg / mL, 8 μg / mL, 10 μg / mL, or 15 μg / mL.

25. A method for detecting EphA2, EphA3, and / or EphB2 expressing cells, comprising the steps of administering a construct according to any one of claims 1 to 10 or a composition according to any one of claims 11 to 15 to cells or a group of cells, wherein the construct comprises a detectable group, and the step of detecting the detectable group.

26. Use of the construct according to any one of claims 1 to 10 or the composition according to any one of claims 11 to 15 for treating cancer in a subject that needs it, or for preparing a pharmacopoeia for treating cancer in a subject that needs it.

27. The use according to claim 26, wherein the cancer is breast cancer, bladder cancer, pancreatic cancer, colorectal cancer, head and neck cancer, thyroid cancer, prostate cancer, melanoma, or glioma.

28. The use according to claim 26, wherein the cancer is glioblastoma, prostate cancer, or melanoma.

29. The use according to claim 26, wherein the cancer is glioblastoma.

30. The use according to any one of claims 26 to 29, including convection-enhanced delivery (CED).

31. The use according to any one of claims 26 to 30, comprising continuous infusion of the construct from 0.1 μg / mL or 0.5 μg / mL to 20 μg / mL, for example, 1 μg / mL, 2 μg / mL, or 3 μg / mL to 5 μg / mL, 8 μg / mL, 10 μg / mL, or 15 μg / mL.

32. The use according to claim 26, wherein the cancer is breast cancer (e.g., squamous cell carcinoma, adenocarcinoma, or ductal carcinoma) (e.g., triple-negative breast cancer).

33. The use according to claim 32, including intravenous injection.

34. The use according to claim 33, wherein the administration step includes intravenous injection through a central venous catheter.

35. The use according to claim 33 or 34, wherein the intravenous injection is administered to the subject over a period of approximately 1 minute to approximately 60 minutes.

36. The use according to any one of claims 32 to 35, wherein the administration step includes administering the construct in an amount ranging from about 0.1 mg / kg to about 20 mg / kg.

37. A method for treating breast cancer in a subject in need, comprising the step of administering to the subject an effective amount of a construct according to any one of claims 1 to 10 or a composition according to any one of claims 11 to 15.

38. The method according to claim 37, wherein the breast cancer includes squamous cell carcinoma, adenocarcinoma, or ductal carcinoma.

39. The method according to claim 37 or 38, wherein the breast cancer is triple-negative breast cancer.

40. The method according to any one of claims 37 to 39, wherein the breast cancer is metastatic breast cancer.

41. The method according to claim 40, wherein the metastatic breast cancer has metastasized to the bone, lungs, brain, and / or liver.

42. The method according to claim 40 or 41, wherein the metastatic breast cancer has metastasized to one or more sites within the subject.

43. The method according to any one of claims 37 to 42, wherein the administration step includes intravenous injection.

44. The method according to claim 43, wherein the administration step includes intravenous injection through a central venous catheter.

45. The method according to claim 43 or 44, wherein the intravenous injection is performed over a period of approximately 1 minute to approximately 60 minutes.

46. The method according to any one of claims 37 to 45, wherein the administration step includes administering the construct in an amount ranging from about 0.1 mg / kg to about 15 mg / kg.

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