Methods for treating IGF-1R associated cancers with insulin-like growth factor 1 receptor ligands conjugated to cytotoxic agents

Abstract

The subject matter described herein relates to a method for treating an insulin-like growth factor 1 receptor (IGF-1R) associated cancer in a subject wherein the method comprises administering to the subject a conjugate comprising an IGF-1R ligand, or a portion or variant thereof, and a cytotoxic agent.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 505,345, filed May 31, 2023, and U.S. Provisional Application No. 63 / 550,459, filed February 6, 2024, which are incorporated herein by reference in their entirety. Technical Field

[0003] The currently disclosed topics generally concern methods for treating cancer specifically through the application of IGF-1R ligands conjugated with cytotoxic agents.

[0004] References to sequence lists

[0005] The sequence list written to file name 614073SEQLIST.xml is 16.7 kilobytes long, created on May 23, 2024, and is hereby incorporated by reference. Background Technology

[0006] Insulin-like growth factor-1 receptor (IGF-1R) is widely involved in the regulation of normal immunity and autoimmune diseases. IGF-1 is a 70-amino acid peptide with 40% identity to proinsulin (Daughaday, WH et al., 1989, Endocrine Revs. 10:68). Insulin and IGF-1 exhibit some cross-reactivity with each other's receptors (Soos, MA et al., 1993, Biochem. J. 290:419). IGF-1 is secreted by the liver into the circulatory system and stimulates the growth of many cell types. IGF-1 is also produced by many cell types throughout the body, including in many cancers, for both autocrine and paracrine effects. IGF-1 production is stimulated by growth hormone. (Stewart, CH et al., 1996, Physiol. Revs. 76:1005; Yakar, S. et al., 2002, Endocrine 19:239).

[0007] IGF-1R expression levels are often found to be higher in adult cancer cells than in normal cells of the same tissue type. Increased IGF-1R activity promotes cancer cell proliferation, migration, and invasion, and is associated with tumor metastasis, treatment resistance, poor prognosis, and shortened survival in cancer patients. In addition, epidemiological studies have reported a positive correlation between circulating IGF-1 levels and various primary cancers, such as breast, colorectal, and prostate cancer. A series of studies have shown that high levels of IGF-1 are associated with increased risk of tumors, including prostate cancer, pre- and postmenopausal breast cancer, lung cancer, thyroid cancer, and colorectal cancer (Ma et al., 1999; Renehan et al., 2004; Shi et al., 2001).

[0008] There is a need for treatments for IGF-1R-related cancers. The subject matter described herein addresses this need. SUMMARY

[0009] In certain embodiments, the subject matter described herein relates to a method for treating an insulin-like growth factor 1 receptor (IGF-1R)-related cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent.

[0010] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R-related cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the IGF-1R-related cancer is selected from the group consisting of head and neck cancer, triple negative breast cancer, bladder cancer, gastrointestinal stromal tumor, adenoid cystic carcinoma, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, esophageal cancer, ovarian cancer, kidney cancer, and gastric cancer.

[0011] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R-related cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the IGF-1R-related cancer is selected from the group consisting of head and neck cancer, triple negative breast cancer, bladder cancer, gastrointestinal stromal tumor, and adenoid cystic carcinoma.

[0012] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R associated cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the IGF-1R ligand or a portion or variant thereof comprises a wild-type insulin-like growth factor 1 (IGF-1) (SEQ ID NO: 3), a wild-type insulin (SEQ ID NO: 10 and SEQ ID NO: 11), a wild-type insulin-like growth factor 2 (IGF-2) (SEQ ID NO: 12), a variant of wild-type IGF-1 (SEQ ID NO: 3), a variant of wild-type insulin (SEQ ID NO: 10 and SEQ ID NO: 11), or a variant of wild-type IGF-2 (SEQ ID NO: 12).

[0013] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R associated cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the IGF-1R ligand or a portion or variant thereof comprises a variant of wild-type insulin-like growth factor 1 (IGF-1) (SEQ ID NO: 3).

[0014] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R associated cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the IGF-1R ligand or a portion or variant thereof comprises SEQ ID NO: 2.

[0015] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R associated cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the cytotoxic agent is a chemotherapeutic agent.

[0016] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R associated cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the cytotoxic agent is methotrexate.

[0017] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R associated cancer in a subject, comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the cytotoxic agent comprises a toxin.

[0018] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R associated cancer in a subject, comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the cytotoxic agent comprises a Clostridium perfringens enterotoxin, diphtheria toxin, ricin chain A, Pseudomonas exotoxin, A chain toxin, a ribosome inactivating protein, alpha-sarcin, amanitin, or a ribonuclease.

[0019] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1R associated cancer in a subject, comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent, wherein the IGF-1R ligand or a portion or variant thereof comprises SEQ ID NO: 2, the cytotoxic agent is methotrexate, wherein the methotrexate is covalently bound to a lysine of SEQ ID NO: 2, and the IGF-1R associated cancer is selected from the group consisting of head and neck cancer, triple negative breast cancer, bladder cancer, gastrointestinal stromal tumor, adenoid cystic carcinoma, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, esophageal cancer, ovarian cancer, kidney cancer, and stomach cancer.

[0020] These and other embodiments are described in full detail below. BRIEF DESCRIPTION OF DRAWINGS

[0021] FIGS. 1A and C depict dose response curves and IC50values for the conjugates described herein against head and neck cancer cell lines FaDU (A) and SCC-25 (C). 50 FIGS. 1B and D depict IGF-1R expression levels in FaDU (B) and SCC-25 (D).

[0022] FIGS. 2A and C depict dose response curves and IC50values for the conjugates described herein against triple negative breast cancer cell lines BT-20 (A) and HCC1143 (C). 50 FIGS. 2B and D depict IGF-1R expression levels in BT-20 (B) and HCC1143 (D).

[0023] Figures 3A and 3C depict the dose-response curves and IC50 values ​​of the conjugates described herein against bladder cancer cell lines 5637 (A) and T-24 (C). 50 Figures 3B and D depict the IGF-1R expression levels in 5637 (B) and T-24 (D).

[0024] Figures 4A-C The dose-response curves and IC50 values ​​of the conjugates described herein against lung cancer cell lines A549(A), NCI-H2122(B), and NCI-H526(C) are depicted. 50 .

[0025] Figures 5A-D The dose-response curves and IC50 values ​​of the conjugates described herein against colorectal cancer cell lines COLO 205 (A) and HT-29 (B) and prostate cancer cell lines VCaP (C) and DU 145 (D) are depicted. 50 .

[0026] Figures 6A-D The dose-response curves and IC50 values ​​of the conjugates described herein against pancreatic cancer cell lines Capan-2 (A) and PANC-1 (B) and liver cancer cell lines Hep G2 (C) and Huh-7 (D) are depicted. 50 .

[0027] Figures 7A-D The dose-response curves and IC50 values ​​of the conjugates described herein against esophageal cancer cell lines TE-1 (A) and KYSE-70 (B) and ovarian cancer cell lines OVCAR-8 (C) and Caov-3 (D) are depicted. 50 .

[0028] Figures 8A-D The dose-response curves and IC50 values ​​of the conjugates described herein against renal cell carcinoma lines 786-O (A) and Caki-1 (B), and gastric cell carcinoma lines MKN74 (C) and NUGC-4 (D) are depicted. 50 . Detailed Implementation

[0029] The subject matter described herein relates to methods for treating insulin-like growth factor 1 receptor (IGF-1R)-related cancers using targeted therapies against IGF-1R, wherein the targeted therapy comprises a combination of IGF-1 or a variant thereof with a cytotoxic payload. Although past attempts to inhibit IGF-1R with naked antibodies or small molecules without payloads have shown some clinical activity, no approved therapies have been developed to date.

[0030] A variety of aggressive cancers with unmet needs have an established link to the IGF-1R pathway, where genetic alterations activate the IGF-1R pathway and / or high IGF-1R expression, which unfortunately is often associated with poor outcomes. A particular conjugate, called LX-101, has shown good tolerability and single agent activity in a previous Phase 1 trial in adult patients with advanced, pretreated cancer. However, there is a need for further studies involving additional types of cancer, bringing improved treatments to patients in need. Disclosed herein are studies showing potent antitumor activity of the conjugate against IGF-1R related multiple cancer cell lines, further supporting the clinical development of the methods described herein to address the unmet need for treating cancers including head and neck cancer, triple negative breast cancer, bladder cancer, gastrointestinal stromal tumor, and adenoid cystic carcinoma.

[0031] The presently disclosed subject matter will now be described more fully hereinafter. However, skilled persons in the art to which the presently disclosed subject matter relates will appreciate that many modifications and other embodiments can be made thereto without departing from the scope of the presently disclosed subject matter as set forth in the appended claims. Accordingly, it is to be understood that the presently disclosed subject matter is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications and equivalents. Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently disclosed subject matter belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict between the definitions in the incorporated literature, patents and similar materials and the definitions set forth herein, the definitions set forth herein control.

[0032] I. Definitions

[0033] As used herein, a patient or subject or the like is any mammal having an IGF-1R related cancer. As used herein, the term "mammal" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs, and sheep. In certain embodiments, the patient or subject is a human, i.e., an adult or a child. In certain embodiments, the methods involve treating a human subject who is about or less than 21 years of age at the time of diagnosis or treatment (i.e., a pediatric subject). The term "pediatric" can be further divided into various subgroups, including: neonates (from birth to the first 28 days of life); infants (from 29 days of age to less than two years of age); children (from two years of age to less than 12 years of age); and adolescents (from 12 years of age to 21 years of age (up to but not including the twenty-second birthday)).

[0034] As used herein, the term "conjugate" refers to a molecule comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent.

[0035] As used herein, the term "insulin-like growth factor 1 receptor (IGF-1R) related cancer" refers to a cancer that afflicts a subject, has an etiology involving overexpression of IGF-1R or in which genetic alterations involve the IGF-1R signaling pathway, such as IGF-1R pathway activation, and is found primarily, but not exclusively, in human adult subjects. That such cancers afflict adult subjects primarily is known to those skilled in the art.

[0036] As used herein, the term "cytotoxic agent" refers to any agent capable of preventing, delaying, reducing and / or reversing the activity, severity and / or progression of a disease when treated according to the methods described herein. Any suitable cytotoxic agent that results in cell killing can be used in the conjugates and methods of treating IGF-1R related cancers.

[0037] As used herein, the term "residue" or "residue of" of a chemical moiety or compound refers to a chemical moiety or compound bound to a molecule, whereby, by virtue of the binding, at least one covalent bond has replaced at least one atom of the original chemical moiety or compound, thereby creating a chemical moiety or compound of the residue of the chemical moiety or compound in the molecule.

[0038] As used herein, a subject is "refractory" to a prior treatment if the subject fails to achieve a response to the therapy such that the therapy is determined to be therapeutically ineffective (e.g., fails to achieve a clinical endpoint, including any response; extends the duration of response; extends disease-free survival, relapse-free survival, and progression-free survival).

[0039] As used herein, "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative ("or").

[0040] As used herein, the term "about," when referring to a measurable value such as an amount of a compound or agent of the current subject matter, dose, time, temperature, and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0041] As used herein, conditional language, such as "can," "could," "might," "may," "e.g.," "for example," "e.g.," "for instance," and the like, unless specifically stated otherwise, are generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements, and / or steps. Thus, such conditional language is not generally intended to imply that features, elements, and / or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements, and / or steps are included or are to be performed in any particular embodiment. The terms "comprising," "including," "having," and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Likewise, the term "or" is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, in a conjunctive manner, it can allow that at least one of the elements linked by the term "or" will be present (e.g., a or b means a will be present, or b will be present, or both a and b will be present for a range of embodiments). Also, the term "based on" is not exclusive and allows for being based on additional factors not mentioned.

[0042] Definitions for additional terms can be set forth below.

[0043] II. Methods of Treatment

[0044] In certain embodiments, the subject matter described herein relates to a method for treating an insulin-like growth factor 1 receptor (IGF-1R)-associated cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion or variant thereof and a cytotoxic agent.

[0045] In certain embodiments, the patient is an adult patient over the age of about 21 years.

[0046] In certain embodiments, the subject is a pediatric subject under the age of about 21 years. In certain embodiments, the patient is from birth to the first 28 days of life, from 29 days of age to under the age of two years, from the age of two years to under the age of 12 years, or from the age of 12 years to under the age of 21 years (up to but not including the twenty-first birthday). In certain embodiments, the patient is from birth to the first 28 days of life, from 29 days of age to under the age of 1 year, from one month of age to under four months of age, from three months of age to under seven months of age, from six months of age to under 1 year of age, from 1 year of age to under 2 years of age, from 2 years of age to under 3 years of age, from 2 years of age to under 7 years of age, from 3 years of age to under 5 years of age, from 5 years of age to under 10 years of age, from 6 years of age to under 13 years of age, from 10 years of age to under 15 years of age, or from 15 years of age to under 21 years of age.

[0047] In certain embodiments, the method treats a subject in need thereof, which can further comprise performing a morphological diagnosis prior to administering the conjugate. The method can further comprise performing a molecular test prior to administering the conjugate. In certain embodiments, the method comprises performing a morphological diagnosis and a molecular test prior to administering the conjugate.

[0048] In certain embodiments, IGF-1R is overexpressed on tumor cells of the IGF-1R- associated cancer. In certain embodiments, IGF-1R is overexpressed on tumor cells relative to non-tumor cells. In other embodiments, overexpression of IGF-1R on tumor cells of the IGF-1R-associated cancer results in poor prognosis. In certain embodiments, overexpression of IGF-1R on tumor cells is measured by flow cytometry or immunohistochemistry.

[0049] In certain embodiments, the IGF-1R-associated cancer has one or more genetic alterations that activate the IGF-1R signaling pathway. In certain embodiments, the genetic alteration is a mutation, gene fusion, gene amplification, or translocation.

[0050] In certain embodiments, the IGF-1R-associated cancer is selected from the group consisting of head and neck cancer, triple negative breast cancer, bladder cancer, gastrointestinal stromal tumor, adenoid cystic carcinoma, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, esophageal cancer, ovarian cancer, kidney cancer, and gastric cancer. In certain embodiments, the IGF-1R-associated cancer is selected from the group consisting of head and neck cancer, triple negative breast cancer, bladder cancer, gastrointestinal stromal tumor, and adenoid cystic carcinoma. In certain embodiments, the IGF-1R-associated cancer is selected from the group consisting of head and neck cancer, triple negative breast cancer, and bladder cancer.

[0051] In some embodiments, the IGF-1R-associated cancer is head and neck cancer or squamous cell carcinoma of the head and neck region (HNSCC). HNSCC is categorized by the presence or absence of HPV. Patients with HNSCC associated with HPV or HPV(+) are generally younger, have a better response to chemotherapy and radiotherapy, and have a better overall prognosis than patients with HPV(-) disease. Of the approximately 50,000 patients diagnosed with HNSCC each year in the United States, it is estimated that 30% have HPV(-) disease. Treatments for HPV(-) HNSCC include chemotherapy, radiotherapy, and surgery. The most commonly used systemic therapies include pembrolizumab, 5-FU, and nivolumab. These treatments have historically resulted in a 5-year overall survival rate of 45% for HPV(-) cases compared to about 80% for disease associated with HPV(+). Despite these efforts, if surgery is not an option, clinical trials using novel treatment modalities are still considered the best option for HPV(-) HNSCC treatment according to the National Comprehensive Cancer Network guidelines. Other targeted therapies currently being tested include tipifarnib, an FTase inhibitor, ficlatuzumab, an HGF inhibitor, and SCT200, an EGFR inhibitor.

[0052] In some embodiments, the IGF-1R associated cancer is muscle-invasive bladder cancer. In the United States, approximately 80,000 new cases of bladder cancer are diagnosed each year. These cancers range from non-invasive, slow progressing tumors to highly invasive, rapidly proliferating malignancies. One of the key defining characteristics of bladder cancer is whether the tumor has invaded the muscle surrounding the bladder. Cancers that stay within the muscle wall of the bladder are considered non-muscle invasive bladder cancer. These patients make up approximately 70% of cases and respond well to current treatment options, but recurrence is common (literature shows anywhere from 30-65%). The other 30% of cases are cancers where the tumor has invaded the muscle surrounding the bladder or metastasized beyond the muscle, which is referred to as muscle-invasive bladder cancer. Muscle-invasive bladder cancer patients have historically responded poorly to available therapies and have poor outcomes. The standard of care for muscle-invasive bladder cancer is currently multi-modal chemotherapy followed by surgical intervention. This treatment regimen has proven to be insufficient as even with successful surgical intervention, 5-year overall survival rates remain as low as 40% for these patients. Importantly, due to disease heterogeneity, muscle-invasive bladder cancer has many other treatment options that target various underlying weaknesses in the malignancy with varying success. Importantly, many of the recent regulatory approvals in muscle-invasive bladder cancer are based on single-arm, non-randomized studies with overall response rate endpoints. Four checkpoint inhibitors, Opdivo, Keytruda, Bavencio, and Imfinzi, were approved in the second-line setting after each conducted a clinical study on approximately 200 patients with ORRs ranging from 13-29% (since then, some have conducted larger scale studies to obtain full approval and / or remove their label restrictions to second-line disease). Additionally, three targeted therapies, Balversa, Trodelvy, and Padcev, were approved in the third-line setting after each conducted a clinical study on approximately 100 patients with ORRs ranging from 28-44%.

[0053] In some embodiments, the IGF-1R-associated cancer is triple-negative breast cancer. Triple-negative breast cancer is a type of breast cancer that has no or little expression of the estrogen receptor, progesterone, and human epidermal growth factor receptor-2. Compared to other breast cancer subtypes, patients with triple-negative breast cancer have more aggressive disease, effective treatment options, and higher frequency of relapse and metastasis, resulting in poorer outcomes and lower survival rates. In the United States, approximately 15-20% of all breast cancers are triple-negative, amounting to an incidence of about 45,000 patients per year. Over the past decade, treatment options for some patients with triple-negative breast cancer have advanced. The main treatment option remains chemotherapy, including taxanes and anthracyclines. Approximately 10-15% of patients with BRCA-mutant disease have had some success with targeted therapies Lynparza and Talzenna, with response rates of 52% and 50% and median PFS of 7.0 and 8.6 months, respectively. In 2020, Tukysa received FDA accelerated approval in pretreated metastatic triple-negative breast cancer based on an ORR of 33.3% and median response duration of 7.7 months in a single-arm study of 108 patients. Other approvals in certain subsets of triple-negative breast cancer include

[0054] In some embodiments, the IGF-1R-associated cancer is gastrointestinal stromal tumor. Gastrointestinal stromal tumors are mesenchymal tumors originating from the gastrointestinal system. Approximately 6,000 patients are diagnosed with gastrointestinal stromal tumors each year, of which 90% of patients have a gain-of-function mutation in the KIT or PDGFRA proto-oncogenes. The remaining 10% of patients without these mutations are considered to have wild-type disease and almost exclusively consist of pediatric patients. Numerous treatments are approved and available to target common mutations in gastrointestinal stromal tumors, such as imatinib, sunitinib, and other chemotherapies, but wild-type disease has historically been more difficult to treat. However, and importantly, patients with disease that do not carry these mutations (i.e., wild-type disease) are often pediatric patients and represent a major unmet medical need as they are generally insensitive to approved treatments, including chemotherapies.

[0055] In some embodiments, the IGF-1R-associated cancer is adenoid cystic carcinoma. Adenoid cystic carcinoma is a malignant disease primarily arising from secretory glands. Adenoid cystic carcinoma is driven by a genetic translocation leading to a MYB-NFIB fusion protein. In the United States, nearly 1,700 patients are diagnosed with adenoid cystic carcinoma each year, with a 5-year and 16-year prevalence pool of 5,000 and 10,500 patients, respectively. Due to the slow but consistent progression of the disease, tumor recurrence is the greatest challenge to the limited therapies currently available. This is evidenced by a decrease in overall survival rate. Due to the high incidence of metastasis and recurrence, the five-year, ten-year, and fifteen-year overall survival rates for adenoid cystic carcinoma are 60%, 50%, and 29%, respectively. Currently, the standard of care for adenoid cystic carcinoma is surgical resection, sometimes combined with radiation therapy. While this surgical resection can help extend survival, more than half of patients have metastatic or recurrent disease, including 30% of patients who have distant metastases after recurrence, for which surgical resection is not an option and treatment is extremely limited. There are no approved systemic therapies for adenoid cystic carcinoma. The most commonly used chemotherapy combination, cisplatin, doxorubicin, and cyclophosphamide, and cisplatin and vinorelbine, produce an overall response rate of between 18% and 31%. Many targeted therapies have been tested in adenoid cystic carcinoma with poor results and no approvals.

[0056] In other embodiments, the IGF-1R-associated cancer is lung cancer, colorectal cancer, prostate cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, or esophageal cancer.

[0057] In embodiments described herein, the conjugate can comprise a conjugate in which the IGF-1R ligand and the cytotoxic agent are chemically linked together, either directly or through a chemical linker. In other embodiments, the conjugate is a genetic recombinant in which the conjugate is expressed as a single polypeptide. When the conjugate is a recombinant conjugate, the translated conjugate preferably comprises a toxin or a portion or variant thereof linked to the IGF-1R ligand by a peptide bond. In certain embodiments, the conjugate is the fusion protein described in U.S. Patent No. 9,675,671, which is hereby incorporated by reference in its entirety.

[0058] Methods for producing the conjugates described herein are known in the art. Nucleotide sequences encoding IGF-1R ligands can be produced by standard recombinant DNA techniques or by protein synthesis techniques, cloned into appropriate expression vectors using standard molecular biology techniques, expressed in bacterial cells, insect cells, or mammalian cells, and purified by any method known in the art for purifying proteins. Conjugates described herein comprising IGF-1R ligands and chemotherapeutic agents can be prepared by standard chemical and protein conjugation techniques, and are described in U.S. Patent No. 7,811,982, U.S. Patent No. 9,675,671, and U.S. Patent No. 9,801,923, each of which is incorporated by reference in its entirety. Conjugates described herein comprising IGF-1R ligands and toxins can be prepared as fusion proteins by standard recombinant DNA techniques, and are described in U.S. Patent No. 8,017,102, which is hereby incorporated by reference in its entirety.

[0059] IGF-1R is a heterotetramer consisting of two extracellular ligand-binding a subunits and two transmembrane b subunits with kinase activity that mediates signal transduction. The natural ligands of IGF-1R are IGF-1, IGF-2, and insulin. IGF-1R has the highest affinity for IGF-1, second highest affinity for IGF-2, and can have an affinity for insulin that is 1 / 100 to 1 / 50. IGF-1R can also form a hybrid receptor through dimerization with the insulin receptor. See Hakuno et al. J Mol Endocrinol. 61(1):T69-T86 (2018).

[0060] In certain embodiments, the IGF-1R ligand in the conjugate comprises wild-type IGF-1 (SEQ ID NO: 3), wild-type insulin (SEQ ID NO: 10 and SEQ ID NO: 11; the mature insulin consists of two chains linked by disulfide bonds (chain A corresponding to SEQ ID NO: 10 and chain B corresponding to SEQ ID NO: 11), thus two SEQ ID NOs are recited), or wild-type IGF-2 (SEQ ID NO: 12). In other embodiments, the IGF-1R ligand in the conjugate comprises a variant of wild-type IGF-1 (SEQ ID NO: 3), a variant of wild-type insulin (SEQ ID NO: 10 and SEQ ID NO: 11), or a variant of wild-type IGF-2 (SEQ ID NO: 12). In particular embodiments, the variant of wild-type IGF-1 is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to IGF-1 (SEQ ID NO: 3), the variant of wild-type insulin is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to insulin (SEQ ID NO: 10 and SEQ ID NO: 11), or the variant of wild-type IGF-2 is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to IGF-2 (SEQ ID NO: 12).

[0061] In certain embodiments, the IGF-1R ligand in the conjugate comprises a variant of IGF-1 that has reduced binding affinity to an IGFBP compared to the binding affinity of wild-type IGF-1 (SEQ ID NO: 3) to the IGFBP or a variant of IGF-2 that has reduced binding affinity to an IGFBP compared to the binding affinity of wild-type IGF-2 (SEQ ID NO: 12) to the IGFBP. IGFBPs belong to a family of at least six proteins that bind IGF-1 and IGF-2 with high affinity. IGFBPs bind to most of the IGFs in circulation, increasing their half-life, modulating their bioavailability, and generally inhibiting their ability to bind to IGF receptors. See Baxter, Am J Physiol Endocrinol Metab., 278(6):E967-76 (2000) and Allard et al., Front Endocrinol (Lausanne). 9;9:117 (2018). Thus, variants of IGF-1 or IGF-2 that have reduced binding to IGFBPs are more bioactive in vivo.

[0062] IGF-1 variants with reduced binding affinity for IGFBP are known in the art and include IGF132 (disclosed in U.S. Patent No. 4,876,242), in which the first 17 amino acids of the B chain of insulin (SEQ ID NO: 11) replace the first 16 amino acids of human IGF-1 (SEQ ID NO: 3); R3-IGF-1 (SEQ ID NO: 6), in which the glutamic acid in position 3 of native human IGF-1 (SEQ ID NO: 3) is replaced with an arginine; and des(l-3)IGF-1 (SEQ ID NO: 7), which lacks the first three amino acids of human IGF-1 (SEQ ID NO: 3). R3-IGF-1 and des(l-3)IGF-1 are described in Francis et al., J. Mol. Endocrinol. 8(3):213-23 (1992). In certain embodiments, the conjugate comprises IGF132 (SEQ ID NO: 4), R3-IGF-1 (SEQ ID NO: 6), or des(l-3)-IGF-1 (SEQ ID NO: 7).

[0063] In certain embodiments, the variant of IGF-1 has a higher affinity for IGF-1R than wild-type IGF-1 (SEQ ID NO: 3) or the variant of IGF-2 has a higher affinity for IGF-1R than wild-type IGF-2 (SEQ ID NO: 12).

[0064] In certain embodiments, the IGF-1R ligand in the conjugate comprises 765IGF (SEQ ID NO: 2), long-R3-IGF-1 (SEQ ID NO: 5), long-IGF-1 (SEQ ID NO: 8), or long-G3-IGF-1 (SEQ ID NO: 9). 765IGF, long-R3-IGF-1, long-IGF-1, and long-G3-IGF-1 have an N-terminal leader sequence that facilitates protein purification and provides a site for conjugation of a cytotoxic agent as described above. 765IGF (SEQ ID NO: 2) comprises SEQ ID NO: 1 followed by R3-IGF-1 (SEQ ID NO: 6); long-R3-IGF-1 (SEQ ID NO: 5) comprises the first 11 amino acids of methionyl porcine growth hormone followed by a Val-Asn dipeptide followed by R3-IGF-1 (SEQ ID NO: 6); long-IGF-1 (SEQ ID NO: 8) comprises the first 11 amino acids of methionyl porcine growth hormone followed by a Val-Asn dipeptide followed by human IGF-1 (SEQ ID NO: 3); and long-G3-IGF-1 comprises the first 11 amino acids of methionyl porcine growth hormone followed by a Val-Asn dipeptide followed by a variant of human IGF-1 in which the glutamic acid in position 3 of native human IGF-1 (SEQ ID NO: 3) is replaced by a glycine.

[0065] In certain embodiments, the IGF-1R ligand comprises wild-type insulin-like growth factor 1, wild-type insulin, or wild-type insulin-like growth factor 2 (IGF-2). In certain aspects, the wild-type insulin-like growth factor 1 (IGF-1) comprises SEQ ID NO: 3, wherein the wild-type insulin comprises SEQ ID NO: 10 or 11, and wherein the wild-type insulin-like growth factor 2 (IGF-2) comprises SEQ ID NO: 12.

[0066] In certain embodiments, the IGF-1R ligand comprises a variant of wild-type IGF-1, a variant of wild-type insulin, or a variant of wild-type IGF-2. In certain aspects, the variant of wild-type IGF-1 is at least 90% identical to SEQ ID NO: 3, the variant of wild-type insulin is at least 90% identical to SEQ ID NO: 10 or 11, and the variant of wild-type IGF-2 is at least 90% identical to SEQ ID NO: 12.12. In certain aspects: (i) the variant of wild-type IGF-1 has reduced binding affinity for an insulin-like growth factor binding protein (IGFBP) compared to the binding affinity of wild-type IGF-1 for the IGFBP, or the variant of wild-type IGF-2 has reduced binding affinity for an IGFBP compared to the binding affinity of wild-type IGF-2 for the IGFBP, and / or (ii) the variant of wild-type IGF-1 has increased affinity for IGF-1R compared to the affinity of wild-type IGF-1 for IGF-1R, or the variant of wild-type IGF-2 has increased affinity for IGF-1R compared to the affinity of wild-type IGF-2 for IGF-1R.

[0067] In certain embodiments, the IGF-1R ligand or portion or variant thereof comprises a leader sequence. In certain aspects, the leader sequence comprises SEQ ID NO: 1.

[0068] In certain embodiments, the IGF-1R ligand comprises 765IGF (SEQ ID NO: 2), IGF-132 (SEQ ID NO: 4), long-R3-IGF-1 (SEQ ID NO: 5), R3-IGF-1 (SEQ ID NO: 6), des(1-3)-IGF-1 (SEQ ID NO: 7), long-IGF-1 (SEQ ID NO: 8), or long-G3-IGF-1 (SEQ ID NO: 9).

[0069] In certain embodiments, the IGF-1R ligand comprises 765IGF (SEQ ID NO: 2).

[0070] In certain embodiments, the IGF-1R ligand or portion or variant thereof is covalently bound to a cytotoxic agent.

[0071] In certain embodiments, the cytotoxic agent comprises a chemotherapeutic agent. In certain aspects, the chemotherapeutic agent is amsacrine, azacytidine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, dacarbazine, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine, hexamethylmelamine, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin C, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, plicamycin, procarbazine, raltitrexed, semustine, streptozocin, temozolamide, teniposide, thioguanine, thiotepa, topotecan, trimetrexate, valrubicin, vincristine, vinblastine, vindesine, or vinorelbine. In certain aspects, the chemotherapeutic agent is methotrexate.In certain aspects, the chemotherapeutic agent is covalently bound to a lysine residue in the leader sequence. In certain aspects, the chemotherapeutic agent is one or more methotrexate residues covalently bound to any available lysine residue in the leader sequence.

[0072] In certain embodiments, the conjugate comprises more than one cytotoxic agent that binds to an IGF-1R ligand. In certain aspects, the conjugate can comprise one to twelve cytotoxic agents, or six to ten cytotoxic agents, or about eight cytotoxic agents. In certain aspects, the conjugate can comprise one to twelve covalently bound cytotoxic agents, or six to ten covalently bound cytotoxic agents, or about eight covalently bound cytotoxic agents. In certain aspects, the chemotherapeutic agent is covalently bound to any available position on the IGF-1R ligand. In certain aspects, the chemotherapeutic agent is covalently bound to any available lysine residue. In certain aspects, the chemotherapeutic agent is covalently bound to any available lysine in the leader sequence, if present.

[0073] The leader sequence can incorporate a tag, such as a polyhistidine tag, which serves to facilitate protein purification as well as to provide a site for cytotoxic agent conjugation. In particular embodiments, the leader sequence comprises SEQ ID NO: 1.

[0074] In certain embodiments, the cytotoxic agent comprises a toxin. In certain aspects, the toxin comprises Clostridium perfringens enterotoxin, diphtheria toxin, ricin chain A, Pseudomonas exotoxin, A chain toxin, ribosome inactivating protein, alpha-sarcin, amanitin, or ribonuclease. In certain aspects, the toxin comprises Clostridium perfringens enterotoxin or a portion or variant thereof. In certain aspects, the toxin comprises SEQ ID NO: 14 or SEQ ID NO: 15. In certain aspects, the toxin comprises diphtheria toxin or a portion or variant thereof. In certain aspects, the toxin comprises SEQ ID NO: 13 or SEQ ID NO: 16.

[0075] In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1 R-associated cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1 R ligand or a portion or variant thereof and a cytotoxic agent, wherein the IGF-1 R ligand or a portion or variant thereof comprises SEQ ID NO: 2, the cytotoxic agent is methotrexate, wherein the methotrexate is covalently bound to a lysine of SEQ ID NO: 2, and the IGF-1 R-associated cancer is selected from the group consisting of head and neck cancer, triple negative breast cancer, and bladder cancer. In certain aspects of these embodiments, the conjugate is LX-101 (a conjugate as described above, wherein the IGF-1 R ligand is SEQ ID NO: 2, the cytotoxic agent is methotrexate, and wherein there are at least 6 and at most 10, or at least 6 and at most 9, or at least 7 and at most 9, or at least 8 and at most 9 methotrexate for each IGF-1 R ligand). The methotrexate can be covalently bound to the IGF-1 R ligand, specifically to a lysine residue of SEQ ID NO: 2. The average number of methotrexate residues per SEQ ID NO: 2 is 8. In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1 R-associated cancer in a subject, the method comprising administering to the subject LX-101, wherein the IGF-1 R-associated cancer is head and neck cancer. In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1 R-associated cancer in a subject, the method comprising administering to the subject LX-101, wherein the IGF-1 R-associated cancer is triple negative breast cancer. In certain embodiments, the subject matter described herein relates to a method for treating an IGF-1 R-associated cancer in a subject, the method comprising administering to the subject LX-101, wherein the IGF-1 R-associated cancer is bladder cancer. In all embodiments, the number of methotrexate residues per conjugate is 6, 7, 8, 9, or 10. In all embodiments, the average number of methotrexate residues per conjugate in the composition is 6, 7, 8, 9, or 10.

[0076] In some embodiments, the subject (i) has not previously received treatment for IGF-1R-related cancer; (ii) has previously received treatment for IGF-1R-related cancer; (iii) has relapsed from previous treatment for IGF-1R-related cancer; (iv) is refractory to previous treatment for IGF-1R-related cancer; or (v) is susceptible to adverse reactions caused by other treatments for IGF-1R-related cancer. Therefore, in some embodiments, the IGF-1R-related cancer is recurrent, meaning the patient has relapsed after previous treatment. In some embodiments, the IGF-1R-related cancer is stage II, III, or IV. In some embodiments, the IGF-1R-related cancer has progressed to stage II, III, or IV during or after initial therapy.

[0077] In some embodiments, the treatment resulted in a reduction in the growth of tumor cells in the subject. In some embodiments, the reduction was caused by the killing of tumor cells expressing IGF-1R.

[0078] Table 1 provides a list of the sequences cited in this paper.

[0079] Table 1.

[0080]

[0081]

[0082]

[0083] In some embodiments, the methods described herein are part of a combination therapy. Specifically, the methods described herein can be used alone or in combination with standard care treatment options for each type of cancer. Typically, standard care options include surgery, systemic chemotherapy (pre- or post-operative), and / or radiation therapy.

[0084] In some embodiments, subjects with IGF-1R-related cancer treated according to the methods described herein have not previously received treatment for IGF-1R-related cancer.

[0085] In some embodiments, subjects with IGF-1R-related cancer treated according to the methods described herein have previously received treatment for IGF-1R-related cancer.

[0086] In some embodiments, the subject had relapsed from previous treatment for IGF-1R-related cancers.

[0087] In some embodiments, the subject has refractory history of prior treatment for IGF-1R-related cancer.

[0088] In all embodiments, the conjugate is administered at a dose and frequency appropriate for the subject and the IGF-1R associated cancer being treated, as determined by the practitioner.

[0089] In certain aspects, the conjugate is administered at a dose of about 0.05 μEq / kg body weight, 0.10 μEq / kg body weight, 0.20 μEq / kg body weight, 0.40 μEq / kg body weight, 0.80 μEq / kg body weight, 1.0 μEq / kg body weight, 1.5 μEq / kg body weight, 1.6 μEq / kg body weight, 2.0 μEq / kg body weight, 2.5 μEq / kg body weight, 3.0 μEq / kg body weight, 3.5 μEq / kg body weight, 4.0 μEq / kg body weight, 4.5 μEq / kg body weight, 5.0 μEq / kg body weight, 5.5 μEq / kg body weight, 6.0 μEq / kg body weight, 6.5 μEq / kg body weight, 7.0 μEq / kg body weight, 7.5 μEq / kg body weight, 8.0 μEq / kg body weight, 8.5 μEq / kg body weight, 9.0 μEq / kg body weight, 9.5 μEq / kg body weight, or 10.0 μEq / kg body weight; or at a dose ranging from about 0.05-10.0 μEq / kg body weight, 0.1-8.0 μEq / kg body weight, 0.2-4.0 μEq / kg body weight, 0.3-3 μEq / kg body weight, 0.4-2.5 μEq / kg body weight, 0.05-0.5 μEq / kg body weight, 0.5-1.0 μEq / kg body weight, 1.0-1.5 μEq / kg body weight, 1.5-2.0 μEq / kg body weight, 2.0-2.5 μEq / kg body weight, 2.5-3.0 μEq / kg body weight, 3.0-3.5 μEq / kg body weight, 3.5-4.0 μEq / kg body weight, 4.0-4.5 μEq / kg body weight, 4.5-5.0 μEq / kg body weight, 5.0-5.5 μEq / kg body weight, 5.5-6.0 μEq / kg body weight, 6.0-6.5 μEq / kg body weight, 6.5-7.0 μEq / kg body weight, 7.0-7.5 μEq / kg body weight, 7.5-8.0 μEq / kg body weight, 8.0-8.5 μEq / kg body weight, 8.5-9.0 μEq / kg body weight, 9.0-9.5 μEq / kg body weight, or 9.5-10.0 μEq / kg body weight. In certain aspects, the conjugate is administered at about 0.05 μEq / kg body weight, 0.10 μEq / kg body weight, 0.20 μEq / kg body weight, 0.40 μEq / kg body weight, 0.80 μEq / kg body weight, 1.6 μEq / kg body weight, or 2.5 μEq / kg body weight. One μEq is equivalent to one μmol of chemotherapeutic agent group conjugated to an IGF-1 R ligand. In certain aspects, the conjugate is administered at a dose of about 0.05 mg / kg body weight, 0.10 mg / kg body weight, 0.15 mg / kg body weight, 0.20 mg / kg body weight, 0.25 mg / kg body weight, 0.30 mg / kg body weight, 0.35 mg / kg body weight, 0.40 mg / kg body weight, 0.45 mg / kg body weight, 0.50 mg / kg body weight, 0.55 mg / kg body weight, 0.60 mg / kg body weight, 0.65 mg / kg body weight, 0.70 mg / kg body weight, 0.75 mg / kg body weight, 0.80 mg / kg body weight, 0.85 mg / kg body weight, 0.90 mg / kg body weight, 0.95 mg / kg body weight, 1.0 mg / kg body weight, 1.1 mg / kg body weight, 1.2 mg / kg body weight, 1.3 mg / kg body weight, 1.4 mg / kg body weight, 1.5 mg / kg body weight, 1.6 mg / kg body weight, 1.7 mg / kg body weight, 1.8 mg / kg body weight, 1.9 mg / kg body weight, 2.0 mg / kg body weight, 2.1 mg / kg body weight, 2.2 mg / kg body weight, 2.3 mg / kg body weight, 2.4 mg / kg body weight, 2.5 mg / kg body weight, 2.6 mg / kg body weight, 2.7 mg / kg body weight, 2.8 mg / kg body weight, 2.9 mg / kg body weight, 3.0 mg / kg body weight, 3.1 mg / kg body weight, 3.2 mg / kg body weight, 3.3 mg / kg body weight, 3.4 mg / kg body weight, 3.5 mg / kg body weight, 3.6 mg / kg body weight, 3.7 mg / kg body weight, 3.8 mg / kg body weight, 3.9 mg / kg body weight, 4.0 mg / kg body weight, 4.1 mg / kg body weight, 4.2 mg / kg body weight, 4.3 mg / kg body weight, 4.4 mg / kg body weight, 4.5 mg / kg body weight, 4.6 mg / kg body weight, 4.7 mg / kg body weight, 4.8 mg / kg body weight, 4.9 mg / kg body weight, 5.0 mg / kg body weight, 5.1 mg / kg body weight, 5.2 mg / kg body weight, 5.3 mg / kg body weight, 5.4 mg / kg body weight, 5.5 mg / kg body weight, 5.6 mg / kg body weight, 5.7 mg / kg body weight, 5.8 mg / kg body weight, 5.9 mg / kg body weight, 6.0 mg / kg body weight, 6.1 mg / kg body weight, 6.2 mg / kg body weight, 6.3 mg / kg body weight, 6.4 mg / kg body weight, 6.5 mg / kg body weight, 6.6 mg / kg body weight, 6.7 mg / kg body weight, 6.8 mg / kg body weight, 6.9 mg / kg body weight, 7.0 mg / kg body weight, 7.1 mg / kg body weight, 7.2 mg / kg body weight, 7.3 mg / kg body weight, 7.4 mg / kg body weight, 7.5 mg / kg body weight, 7.6 mg / kg body weight, 7.7 mg / kg body weight, 7.8 mg / kg body weight, 7.9 mg / kg body weight, 8.0 mg / kg body weight, 8.1 mg / kg body weight, 8.2 mg / kg body weight, 8.3 mg / kg body weight, 8.4 mg / kg body weight, 8.5 mg / kg body weight, 8.6 mg / kg body weight, 8.7 mg / kg body weight, 8.8 mg / kg body weight, 8.9 mg / kg body weight, 9.0 mg / kg body weight, 9.1 mg / kg body weight, 9.2 mg / kg body weight, 9.3 mg / kg body weight, 9.4 mg / kg body weight, 9.5 mg / kg body weight, 9.6 mg / kg body weight, 9.7 mg / kg body weight, 9.8 mg / kg body weight, 9.9 mg / kg body weight, 10.0 mg / kg body weight, 10.1 mg / kg body weight, 10.2 mg / kg body weight, 10.3 mg / kg body weight, 10.4 mg / kg body weight, 10.5 mg / kg body weight, 10.6 mg / kg body weight, 10.7 mg / kg body weight, 10.8 mg / kg body weight, 10.9 mg / kg body weight, 11.0 mg / kg body weight, 11.1 mg / kg body weight, 11.2 mg / kg body weight, 11.3 mg / kg body weight, 11.4 mg / kg body weight, 11.5 mg / kg body weight, 11.6 mg / kg body weight, 11.7 mg / kg body weight, 11.8 mg / kg body weight, 11.9 mg / kg body weight, 12.0 mg / kg body weight, 12.1 mg / kg body weight, 12.2 mg / kg body weight, 12.3 mg / kg body weight, 12.4 mg / kg body weight, 12.5 mg / kg body weight, 12.6 mg / kg body weight, 12.7 mg / kg body weight, 12.8 mg / kg body weight, 12.9 mg / kg body weight, 13.0 mg / kg body weight, 13.1 mg / kg body weight, 13.2 mg / kg body weight, 13.3 mg / kg body weight, 13.4 mg / kg body weight, 13.5 mg / kg body weight, 13.6 mg / kg body weight, 13.7 mg / kg body weight, 13.8 mg / kg body weight, 13.9 mg / kg body weight, 14.0 mg / kg body weight, 14.1 mg / kg body weight, 14.2 mg / kg body weight, 14.3 mg / kg body weight, 14.4 mg / kg body weight, 14.5 mg / kg body weight, 14.6 mg / kg body weight, 14.7 mg / kg body weight, 14.8 mg / kg body weight, 14.9 mg / kg body weight, 15.0 mg / kg body weight, 15.1 mg / kg body weight, 15.2 mg / kg body weight, 15.3 mg / kg body weight, 15.4 mg / kg body weight, 15.5 mg / kg body weight, 15.6 mg / kg body weight, 15.7 mg / kg body weight, 15.8 mg / kg body weight, 15.9 mg / kg body weight, 16.0 mg / kg body weight, 16.1 mg / kg body weight, 16.2 mg / kg body weight, 16.3 mg / kg body weight, 16.4 mg / kg body weight, or 16.5 mg / kg body weight; or in a dose range of about 0.05-0.5 mg / kg body weight, 0.5-1.0 mg / kg body weight, 1.0-1.5 mg / kg body weight, 1.5-2.0 mg / kg body weight, 2.0-2.5 mg / kg body weight, 2.5-3.0 mg / kg body weight, 3.0-3.5 mg / kg body weight, 3.5-4.0 mg / kg body weight, 4.0-4.5 mg / kg body weight, 4.5-5.0 mg / kg body weight, 5.0-5.5 mg / kg body weight, 5.5-6.0 mg / kg body weight, 6.0-6.5 mg / kg body weight, 6.5-7.0 mg / kg body weight, 7.0-7.5 mg / kg body weight, 7.5-8.0 mg / kg body weight, 8.0-8.5 mg / kg body weight, 8.5-9.0 mg / kg body weight, 9.0-9.5 mg / kg body weight, 9.5-10.0 mg / kg body weight, 10.0-10.5 mg / kg body weight, 10.5-11.0 mg / kg body weight, 11.0-11.5 mg / kg body weight, 11.5-12.0 mg / kg body weight, 12.0-12.5 mg / kg body weight, 12.5-13.0 mg / kg body weight, 13.0-13.5 mg / kg body weight, 13.5-14.0 mg / kg body weight, 14.0-14.5 mg / kg body weight, 14.5-15.0 mg / kg body weight, 15.0-15.5 mg / kg body weight, 15.5-16.0 mg / kg body weight, or 16.0-16.5 mg / kg body weight, where mg refers to the amount of IGF-1R ligand present in the conjugate.

[0090] In certain embodiments, the conjugate is administered daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, or once every three months.

[0091] In certain embodiments, the conjugate can be administered at a lower dose and / or frequency at which the cytotoxic agent would be effective when administered as a single agent.

[0092] In some embodiments, the conjugate is administered at a maximum tolerated dose (MTD). As used herein, "MTD" refers to the highest dose of a pharmaceutical agent that can be tolerated by an individual patient as determined by the practitioner. In other words, side effects in a given patient can determine the MTD.

[0093] In certain embodiments, the method does not cause significant or unacceptable hyperglycemia in the subject. In certain aspects, the method does not cause unacceptable hyperglycemia in the subject. Hyperglycemia is another term for high blood glucose, and can occur when there is insufficient insulin in the body or the body cannot properly use insulin. Unacceptable hyperglycemia refers to a side effect of Grade 3 or higher as determined by the treating physician, and / or a side effect that cannot be controlled with diabetes medications and results in termination of treatment with the conjugate.

[0094] In certain embodiments, the method does not cause significant or unacceptable hyperglycemia in the subject. In certain aspects, the method does not cause unacceptable hyperglycemia in the subject. Hyperglycemia is another term for high blood glucose, and can occur when there is insufficient insulin in the body or the body cannot properly use insulin. Unacceptable hyperglycemia refers to a side effect of Grade 3 or higher as determined by the treating physician, and / or a side effect that cannot be controlled with diabetes medications and results in termination of treatment with the conjugate.

[0095] The conjugates described herein can be formulated in a pharmaceutical composition for use in the methods described herein. In some embodiments, the pharmaceutical composition comprises an effective amount of the conjugate and a pharmaceutically acceptable carrier or vehicle. Such pharmaceutical compositions can be formulated to be suitable for administration to a subject, and can be in any form that allows the composition to be administered to a subject.

[0096] The materials used to make the pharmaceutical compositions can be nontoxic as used in amounts. It will be clear to those of ordinary skill in the art that the optimal dose of active ingredient in a pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of subject (e.g., human), the overall health level of the subject, the type of condition from which the subject is suffering, the use of the composition as part of a multi-drug regimen, the particular form of the composition, and the mode of administration. The pharmaceutical composition comprises an effective amount of the composition such that an appropriate dose will be obtained.

[0097] The term "carrier" refers to a diluent, adjuvant, or vehicle with which the composition comprising the conjugate is administered. Any auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, the composition and pharmaceutically acceptable carrier are sterile when administered to a subject. Water can be the carrier when the composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The composition of the application can further contain minor amounts of pH buffering agents, if desired.

[0098] Liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, can further include one or more of the following: sterile diluents such as water for injection, saline solution, physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; agents for the adjustment of pH such as hydrochloric acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral composition can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass, plastic or other material. In some embodiments, physiological saline is an adjuvant. The injectable composition can be sterile.

[0099] The compositions of the present application can take the form of solutions, suspensions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences.

[0100] In some embodiments, the composition is formulated according to conventional procedures to be a pharmaceutical composition suitable for intravenous administration to human subjects. Typically, the carrier or mediator for intravenous administration is a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer. The composition for intravenous administration may optionally contain a local anesthetic, such as lidocaine for relieving injection site pain. Typically, the components are provided separately or mixed together in unit dosage forms, for example, as a lyophilized powder or anhydrous concentrate in a hermetically sealed container (such as an ampoule or capsule indicating the amount of active agent). When the composition is to be administered by infusion, it may be dispensed, for example, using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is to be administered by injection, ampoules of sterile water for injection or saline may be provided, allowing the components to be mixed prior to administration.

[0101] Pharmaceutical compositions can be prepared using methods well-known in the pharmaceutical field. For example, a composition intended for injection can be prepared by combining the composition with water to form a solution. Surfactants can be added to promote the formation of homogeneous solutions or suspensions. Surfactants are complexes that can non-covalently interact with the composition to promote its dissolution or homogeneous suspension in an aqueous delivery system.

[0102] The conjugates described herein can be administered via any convenient route, such as by infusion or bolus injection, or by absorption through the epithelial or mucosal lining of the skin (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, such as microparticles, microcapsules, capsules, etc., and can be used to administer compositions containing the conjugates. Administration methods can include, but are not limited to, oral and parenteral administration; parenteral administration includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, intravaginal, transdermal, rectal, inhalation, or local application to the ear, nose, eye, or skin. The mode of administration is determined by the practitioner and will depend in part on the site of the medical condition. In some embodiments, the conjugates are administered intravenously, subcutaneously, or intramuscularly.

[0103] In some embodiments, the conjugate is administered parenterally. In some embodiments, the conjugate is administered intravenously. In some embodiments, the conjugate is administered via continuous infusion. In some embodiments, the conjugate is administered via infusion over a continuous period of 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours.

[0104] In certain embodiments, it can be desirable to administer the conjugate locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In certain embodiments, the conjugate can be injected intraperitoneally.

[0105] In certain embodiments, the conjugate can be delivered in a controlled release system.

[0106] The following examples are provided in an illustrative, rather than a restrictive, sense.

[0107] Example

[0108] Example 1 - IGF-IR expression assay

[0109] Table 2. Abbreviations used in this study

[0110]

[0111]

[0112] The IGF-1R expression of the cell lines shown in Table 3 was determined by FACS. The data results are depicted in Figures 1-3. The MCF7 cell line was used as a positive control.

[0113] Table 3. Cell lines

[0114]

[0115] First, 1 million cells were resuspended in 100 μΐ^of PBS with 2.5 μg of human Fc-block (BD Biosciences) and incubated for 10 minutes at room temperature in the dark to block. For staining, PE CD221 (IGF-1R) antibody (clone 1H7, BD Biosciences) and eFluor780 live / dead dye (eBiosciences) were added to each sample and incubated for 30 minutes at 4°C in the dark. PE Mouse IgGl, kappa Isotype Control (BD Biosciences) was used to set the isotype control for each cell line. Then, 2 mL of PBS was added to each sample, the cells were gently resuspended and centrifuged at 500 x g for 5 minutes and the supernatant was discarded. The cells were washed twice more in PBS, then resuspended in 200 μΐ^of IC Fixation Buffer (eBioscience) and incubated for 30 minutes at room temperature in the dark. The cells were washed with 2 mL of PBS and resuspended in 250 μΐ^of PBS for acquisition. Data was acquired using a CytoFLEX S flow cytometer (Beckman Coulter) and analyzed using Kaluza 2.0 (Beckman Coulter).

[0116] The resulting data is depicted in Figures 1-3. Greater fluorescence intensity of cells stained with PE CD221 antibody compared to cells stained with isotype control indicates that cell lines FADU (Figure IB), SCC-25 (Figure ID), BT-20 (Figure 2B), HCC1143 (Figure 2D), 5637 (Figure 3B), and T-24 (Figure 3D) express IGF-1R on the cell surface.

[0117] Example 2 - Cytotoxicity assay in specific types of cancer cells

[0118] LX-101-induced cytotoxicity was evaluated on specific types of cancer cells. Cell lines in Table 6 were treated with 9 concentrations of LX-101 using 2.5-fold dilutions and cell viability was evaluated.

[0119] Table 5. Abbreviations used in this protocol

[0120]

[0121] Approximately 7 days prior to treatment, the frozen cell vials were thawed by transferring them to a water bath on dry ice. The contents of each vial were slowly transferred to a 15 mL tube containing 10 mL of media as shown in Table 6. The cells were centrifuged at 125 x g for 5 minutes at room temperature. The cell pellets were resuspended in pre-warmed media in a T-25 or T-75 flask and incubated at 37°C with 5% C02. When the cell cultures reached approximately 80% confluence, the cells were passaged at a 1 :3 to 1 :6 split ratio using TrypLE.

[0122] Table 6. Cell lines

[0123]

[0124] IGF-1R expression was not tested for cell lines 101-119.

[0125] The day prior to LX-101 treatment, cells were collected during log growth and counted using TrypLE. The cell numbers shown in Table 7 were seeded in 100 μL per well of a 96-well plate according to the plate layout shown below. The plates were incubated overnight at 37°C, 5% C02.

[0126] Table 7. Cell Seeding

[0127]

[0128] Plate Layout (one cell line per plate)

[0129]

[0130]

[0131] Compound

[0132]

[0133] A 1 / 10 working stock solution of LX-101 was prepared in 1 mM HC1 such that the working stock solution had a concentration of 0.4 μEq / ml drug in 1.9 mM HC1. Sterile Eppendorf tubes were prepared containing 3 times the final well concentration in media for each cell line (Table 6). The first dilution was prepared using the 1 / 10 working stock solution diluted in media. Subsequent 2.5 times dilutions were prepared using media. Visual inspection for signs of precipitation was performed as the compound was diluted in media.

[0134] For vehicle control, a 10 mM HC1 stock solution was prepared by diluting HC1 in cell culture grade water and confirmed that the pH was between 1 and 3. Next, a working stock solution was prepared with a final concentration of 1.9 mM HC1. In an Eppendorf, 75 μΐ, of working stock solution was added to 425 μΐ, of media to make a 3-fold final concentration of 285 μΜ HC1.

[0135] As shown in Table 8, 50 μΐ, of LX-101 or prepared dilution of HC1 was added to the appropriate well to provide a total volume of 150 μΐ, per well. Unused wells were filled with 150 μΐ, of PBS.

[0136] Table 8. LX-101 titration

[0137]

[0138]

[0139] After treatment, the plates were incubated at 37°C, 5% C02. Four days later, the plates were removed and equilibrated to room temperature for 30 minutes. Black tape was placed at the bottom of the plates to block light. Then, 75 μΐ, of CellTiter-Glo 2.0 reagent (Promega) was added to each well and the contents were mixed on an orbital shaker for 2 minutes. The plates were incubated at room temperature for an additional 10 minutes to stabilize the luminescent signal. Luminescence was recorded using an Envision 2104 Multilabel Microplate Reader (PerkinElmer) using an integration time of 0.25-1 second per well.

[0140] LX-101 concentrations based on IGF-1 variant protein content were derived by dividing drug concentrations based on methotrexate content by 8, the average number of methotrexate groups per IGF-1 protein as determined by MALDI-TOF. IC 50 values were calculated using GraphPad PRISM software

[0141] Viability (% relative to control) = (Lum 测试制品 -Lum 空白对照 ) / (Lum 媒剂 -Lum 空白对照 ) x 100%.

[0142] Results

[0143] Cell toxicity of lot LIR00223 of LX-101 was tested on MCF7 breast cancer cells as a reference. As shown in Table 9, the average IC 50 was found to be 31 nM for lot LIR0023 over 3 independent experiments.

[0144] Table 9. Cytotoxicity of LX-101 (Lot LIR0023) on MCF7 breast cancer

[0145]

[0146]

[0147] Cytotoxicity of LX-101 (Lot LIR0023) on the cell lines shown in Table 10 was determined following the same protocol. LX-101 exhibited an absolute IC50 less than 70 nM in certain cell lines of pharyngeal, triple negative breast, bladder, lung, colorectal, prostate, pancreatic, liver, and esophageal, ovarian, renal, and gastric cancer cell lines, indicating a significant anti-cancer property. In particular, LX-101 was highly cytotoxic to pharyngeal FaDU, bladder 5637, prostate DU 145, liver Hep G2, esophageal KYSE-70, and renal 786-O, with very low absolute IC50 values less than 10 nM (based on the concentration of IGF-1R ligand, including approximately 8 covalently bound methotrexate molecules). 50 50 Values less than 10 nM.

[0148] Table 10. Cytotoxicity of LX-101 (Lot LIR0023) on cancer cell lines

[0149]

[0150]

[0151] While efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) used in the examples, some experimental errors and deviations should be accounted for.

[0152] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the subject matter described herein. The present disclosure is in no way limited to the methods and materials described.

[0153] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs.

[0154] Throughout this specification and claims, the word "comprise," and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not used as a listing of elements or members. The term "consisting of" is used in the claims to refer exclusively to the specified components.​

[0155] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are thus contemplates where nothing else is specifically excluded, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included extremes are also included.

[0156] Many modifications and other embodiments of the set forth herein will come to mind to one skilled in the art to which the subject matter pertains having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it is to be understood that the subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. A method for treating a subject with insulin-like growth factor 1 receptor (IGF-1R)-related cancer, the method comprising administering a conjugate to the subject, the conjugate comprising an IGF-1R ligand or a portion thereof or a variant thereof and a cytotoxic agent.

2. The method according to claim 1, wherein the IGF-1R-related cancer is selected from the group consisting of: head and neck cancer, triple-negative breast cancer, bladder cancer, gastrointestinal stromal tumor, adenoid cystic carcinoma, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, esophageal cancer, ovarian cancer, kidney cancer, and stomach cancer.

3. The method of claim 2, wherein the IGF-1R-related cancer is selected from the group consisting of: head and neck cancer, triple-negative breast cancer, bladder cancer, gastrointestinal stromal tumor, and adenoid cystic carcinoma.

4. The method of claim 3, wherein the IGF-1R-related cancer is selected from the group consisting of head and neck cancer, triple-negative breast cancer, and bladder cancer.

5. The method according to any one of claims 1 to 4, wherein IGF-1R is overexpressed in the tumor cells of the IGF-1R-related cancer.

6. The method according to any one of claims 1 to 5, wherein the IGF-1R-related cancer has one or more genetic alterations that activate the IGF-1R signaling pathway.

7. The method according to claim 6, wherein the genetic alteration is a mutation, gene fusion, gene amplification, or translocation.

8. The method according to any one of claims 1 to 7, wherein the IGF-1R ligand comprises wild-type insulin-like growth factor 1, wild-type insulin, or wild-type insulin-like growth factor 2 (IGF-2).

9. The method of claim 8, wherein the wild-type insulin-like growth factor 1 (IGF-1) comprises SEQ ID NO: 3, wherein the wild-type insulin comprises SEQ ID NO: 10 or 11, and wherein the wild-type insulin-like growth factor 2 (IGF-2) comprises SEQ ID NO:

12.

10. The method according to any one of claims 1 to 7, wherein the IGF-1R ligand comprises a variant of wild-type IGF-1, a variant of wild-type insulin, or a variant of wild-type IGF-2.

11. The method of claim 10, wherein the variant of wild-type IGF-1 is at least 90% identical to SEQ ID NO: 3, the variant of wild-type insulin is at least 90% identical to SEQ ID NO: 10 or 11, and the variant of wild-type IGF-2 is at least 90% identical to SEQ ID NO:

12.

12. The method according to claim 10 or 11, wherein: (i) the wild-type IGF-1 variant has a lower affinity for insulin-like growth factor binding protein (IGFBP) than wild-type IGF-1, or the wild-type IGF-2 variant has a lower affinity for IGFBP than wild-type IGF-2, and / or (ii) the wild-type IGF-1 variant has an increased affinity for IGF-1R than wild-type IGF-1, or the wild-type IGF-2 variant has an increased affinity for IGF-1R than wild-type IGF-2.

13. The method according to any one of claims 1 to 11, wherein the IGF-1R ligand or a portion or variant thereof comprises a leader sequence.

14. The method of claim 13, wherein the leader sequence comprises SEQ ID NO:

1.

15. The method according to any one of claims 1 to 7 and 10 to 14, wherein the IGF-1R ligand comprises 765IGF (SEQ ID NO: 2), IGF-132 (SEQ ID NO: 4), long-R3-IGF-1 (SEQ ID NO: 5), R3-IGF-1 (SEQ ID NO: 6), des(1-3)-IGF-1 (SEQ ID NO: 7), long-IGF-1 (SEQ ID NO: 8), or long-G3-IGF-1 (SEQ ID NO: 9).

16. The method of claim 15, wherein the IGF-1R ligand comprises 765IGF (SEQ ID NO: 2).

17. The method according to any one of claims 1 to 16, wherein the IGF-1R ligand or a portion thereof or a variant thereof is covalently bound to the cytotoxic agent.

18. The method according to any one of claims 1 to 17, wherein the cytotoxic agent comprises a chemotherapeutic agent.

19. The method according to claim 18,The chemotherapeutic agents mentioned are amsacrine, azacytidine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, dacarbazine, docetaxel, and doxorubicin. Epirubicin, estramustine, etoposide, fluxuridine, fludarabine, fluorouracil, gemcitabine, hexamethylmelamine, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mecaptopurine, methotrexate, mitomycin C C) Mitotane, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin, plicamycin, Procarbazine, Raltitrexed, Semustine, Streptozocin, Temozolamide, Teniposide, Thioguanine, Thiotepa, Topotecan, Trimetrexate, Valrubicin, Vincristine, Vinblastine, Vindesine, or Vinorelbine.

20. The method of claim 19, wherein the chemotherapeutic agent is methotrexate.

21. The method according to any one of claims 1 to 17, wherein the cytotoxic agent comprises a toxin.

22. The method of claim 21, wherein the toxin comprises Clostridium perfringens enterotoxin, diphtheria toxin, ricin A, Pseudomonas exotoxin, A-chain toxin, ribosome inactivating protein, α-broom toxin, aspergillin, or ribonuclease.

23. The method of claim 22, wherein the toxin comprises Clostridium perfringens enterotoxin or a portion thereof or a variant thereof.

24. The method of claim 23, wherein the toxin comprises SEQ ID NO: 14 or SEQ ID NO:

15.

25. The method of claim 22, wherein the toxin comprises diphtheria toxin or a portion thereof or a variant thereof.

26. The method of claim 25, wherein the toxin comprises SEQ ID NO: 13 or SEQ ID NO:

16.

27. The method of any one of claims 1 to 26, wherein the subject (i) has not previously received treatment for the IGF-1R-related cancer; (ii) has previously received treatment for the IGF-1R-related cancer; (iii) has relapsed from previous treatment for the IGF-1R-related cancer; (iv) is refractory to previous treatment for the IGF-1R-related cancer; or (v) is susceptible to adverse reactions caused by other treatments for the IGF-1R-related cancer.

28. The method according to any one of claims 1 to 27, wherein the conjugate is administered in combination with one or more other therapies.

29. The method of claim 28, wherein the one or more other therapies comprise one or more of the following: surgery, systemic chemotherapy (preoperative or postoperative), and radiation therapy.

30. The method according to any one of claims 1 to 29, wherein the conjugate is administered at the following dosages: about 0.05 μEq / kg body weight, 0.10 μEq / kg body weight, 0.20 μEq / kg body weight, 0.40 μEq / kg body weight, 0.80 μEq / kg body weight, 1.0 μEq / kg body weight, 1.5 μEq / kg body weight, 1.6 μEq / kg body weight, 2.0 μEq / kg body weight, 2.5 μEq / kg body weight, 3.0 μEq / kg body weight, 3.5 μEq / kg body weight, 4.0 μEq / kg body weight, 4.5 μEq / kg body weight, 5.0 μEq / kg body weight, 5.5 μEq / kg body weight, 6.0 μEq / kg body weight, 6.5 μEq / kg body weight, 7.0 μEq / kg body weight, 7.5 μEq / kg body weight, 8.0 μEq / kg body weight, 8.5 μEq / kg body weight. 9.0 μEq / kg body weight, 9.5 μEq / kg body weight, or 10.0 μEq / kg body weight; or administered within the following dosage ranges: approximately 0.05–10.0 μEq / kg body weight, 0.1–8.0 μEq / kg body weight, 0.2–4.0 μEq / kg body weight, 0.3–3 μEq / kg body weight, 0.4–2.5 μEq / kg body weight, 0.05–0.5 μEq / kg body weight, 0.5–1.0 μEq / kg body weight, 1.0–1.5 μEq / kg body weight, 1.5–2.0 μEq / kg body weight, 2.0–2.5 μEq / kg body weight, 2.5–3.0 μEq / kg body weight, 3.0–3.5 μEq / kg body weight, 3.5–4.0 μEq / kg body weight, 4.0–4.5 μEq / kg body weight. μEq / kg body weight, 4.5-5.0 μEq / kg body weight, 5.0-5.5 μEq / kg body weight, 5.5-6.0 μEq / kg body weight, 6.0-6.5 μEq / kg body weight, 6.5-7.0 μEq / kg body weight, 7.0-7.5 μEq / kg body weight, 7.5-8.0 μEq / kg body weight, 8.0-8.5 μEq / kg body weight, 8.5-9.0 μEq / kg body weight, 9.0-9.5 μEq / kg body weight, or 9.5-10.0 μEq / kg body weight.

31. The method of claim 30, wherein the conjugate is administered at about 0.05 μEq / kg body weight, 0.10 μEq / kg body weight, 0.20 μEq / kg body weight, 0.40 μEq / kg body weight, 0.80 μEq / kg body weight, 1.6 μEq / kg body weight, or 2.5 μEq / kg body weight.

32. The method according to any one of claims 15 to 20 and 27 to 31, wherein the IGF-1R ligand is SEQ ID NO: 2, the cytotoxic agent is methotrexate, wherein each IGF-1R ligand of SEQ ID NO: 2 contains 6 to 10 methotrexate molecules, and the IGF-1R-related cancer is head and neck cancer.

33. The method according to any one of claims 15 to 20 and 27 to 31, wherein the IGF-1R ligand is SEQ ID NO: 2, the cytotoxic agent is methotrexate, wherein each IGF-1R ligand of SEQ ID NO: 2 contains 6 to 10 methotrexate molecules, and the IGF-1R-related cancer is triple-negative breast cancer.

34. The method according to any one of claims 15 to 20 and 27 to 31, wherein the IGF-1R ligand is SEQ ID NO: 2, the cytotoxic agent is methotrexate, wherein each IGF-1R ligand of SEQ ID NO: 2 contains 6 to 10 methotrexate molecules, and the IGF-1R-related cancer is bladder cancer.

35. The method according to any one of claims 15 to 20 and 27 to 31, wherein the IGF-1R ligand is SEQ ID NO: 2, the cytotoxic agent is methotrexate, wherein each IGF-1R ligand of SEQ ID NO: 2 contains 6 to 10 methotrexate molecules, and the IGF-1R-related cancer is gastrointestinal stromal tumor.

36. The method according to any one of claims 15 to 20 and 27 to 31, wherein the IGF-1R ligand is SEQ ID NO: 2, the cytotoxic agent is methotrexate, wherein each IGF-1R ligand of SEQ ID NO: 2 contains 6 to 10 methotrexate molecules, and the IGF-1R-associated cancer is adenoid cystic carcinoma.

37. The method according to any one of claims 1 to 32, wherein the head and neck cancer is HPV-.

38. The method according to any one of claims 1 to 31 and 35, wherein the gastrointestinal stromal tumor is wild-type.

39. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is head and neck cancer.

40. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is triple-negative breast cancer.

41. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is bladder cancer.

42. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is lung cancer.

43. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is colorectal cancer.

44. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is prostate cancer.

45. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is pancreatic cancer.

46. ​​The method according to claim 1 or 2, wherein the IGF-1R-related cancer is liver cancer.

47. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is esophageal cancer.

48. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is ovarian cancer.

49. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is kidney cancer.

50. The method according to claim 1 or 2, wherein the IGF-1R-related cancer is gastric cancer.

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