Methods for treating IGF-1R-associated pediatric 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 pediatric 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 / 505,263, filed May 31, 2023, and U.S. Provisional Application No. 63 / 550,463, 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 pediatric cancers 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 614072SEQLIST.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 typically 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. Furthermore, epidemiological studies have reported a positive correlation between circulating IGF-1 levels and various primary cancers, such as breast cancer, colorectal cancer, and prostate cancer. A series of studies have shown that high levels of IGF-1 are associated with an 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). These cancers occur in adults, not children.

[0008] Childhood cancer differs from adult cancer. The type of cancer is often unrelated to lifestyle. The causes of childhood cancer are often unknown. The extent of cancer spread and the treatment of cancer in children typically differ from those in adults. Furthermore, children's bodies and their responses to treatment are unique.

[0009] There is a current need for treatments targeting IGF-1R-related pediatric cancers. The topic described in this article addresses this need. Summary of the Invention

[0010] In some embodiments, the subject matter described herein relates to a method for treating a subject with insulin-like growth factor 1 receptor (IGF-1R)-associated pediatric cancer, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion thereof or a variant thereof, and a cytotoxic agent.

[0011] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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 pediatric cancer is selected from the group consisting of: Ewing's sarcoma, rhabdomyosarcoma, synovial sarcoma, neuroblastoma, osteosarcoma, Wilms' tumor, Beckwith Wiedemann syndrome-related tumors, desmoplastic small round cell tumors, and adrenocortical carcinoma.

[0012] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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 pediatric cancer is selected from the group consisting of Ewing's sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma.

[0013] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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 wild-type insulin-like growth factor 1 (IGF-1) (SEQ ID NO: 3), wild-type insulin (SEQ ID NO: 10 and SEQ ID NO: 11), 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).

[0014] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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).

[0015] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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.

[0016] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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.

[0017] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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.

[0018] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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 comprises a toxin.

[0019] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion thereof or a variant thereof and a cytotoxic agent, wherein the cytotoxic agent comprises Clostridium perfringens enterotoxin, diphtheria toxin, ricin A, Pseudomonas exotoxin, A-streptotoxin, ribosome-inactivating protein, α-broom toxin, aspergillin, or ribonuclease.

[0020] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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, the cytotoxic agent is methotrexate, wherein the methotrexate is covalently bound to a lysine residue of SEQ ID NO: 2, and the IGF-1R-related pediatric cancer is selected from the group consisting of: Ewing's sarcoma, adrenocortical carcinoma, desmoplastic small round cell tumor, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma.

[0021] These and other embodiments are described in full below. Attached Figure Description

[0022] Figures 1A and 1C depict the dose-response curves and IC50 values ​​of the conjugates described herein against Ewing's sarcoma cell lines A-673 (A) and CADO-ES1 (C). 50 Figures 1B and 1D depict the IGF-1R expression levels in A-673 (B) and CADO-ES1 (D).

[0023] Figures 2A and 2C depict the dose-response curves and IC50 values ​​of the conjugates described herein against Ewing's sarcoma cell lines RD-ES (A) and SK-ES-1 (C). 50 Figures 2B and D depict the IGF-1R expression levels in RD-ES (B) and SK-ES-1 (D).

[0024] Figure 3A depicts the dose-response curves and IC50 of the conjugate described herein against the rhabdomyosarcoma cell line SJCRH30. 50 Figure 3B depicts the IGF-1R expression level in SJCRH30.

[0025] Figures 4A and 4C depict the dose-response curves and IC50 values ​​of the conjugates described herein against osteosarcoma cell lines 143B (A) and HOS (C). 50 Figures 4B and D depict the IGF-1R expression levels in 143B (B) and HOS (D).

[0026] Figures 5A and 5C depict the dose-response curves and IC50 values ​​of the conjugates described herein against the osteosarcoma cell lines Saos-2 (A) and U-2OS (C). 50 Figures 5B and D depict the IGF-1R expression levels in Saos-2 (B) and U-2OS (D).

[0027] Figures 6A and 6C depict the dose-response curves and IC50 values ​​of the conjugates described herein against the neuroblastoma cell lines IMR32 (A) and SK-N-AS (C). 50 Figures 6B and D depict the IGF-1R expression levels in IMR32 (B) and SK-N-AS (D).

[0028] Figure 7A depicts the dose-response curve and IC50 of the conjugate described herein against the neuroblastoma cell line SH-SY5Y. 50 Figure 7B depicts the IGF-1R expression level in SH-SY5Y.

[0029] Figure 8 The dose-response curves and IC50 values ​​of the conjugate described in this paper against the adrenocortical carcinoma cell line SW-13 were depicted. 50 . Detailed Implementation

[0030] The subject matter described herein relates to methods for treating insulin-like growth factor 1 receptor (IGF-1R)-related pediatric 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 a payload have shown some clinical activity, no approved therapies have been developed to date.

[0031] A number of aggressive cancers with unmet needs have established links to the IGF-1R pathway, where genetic alterations activate the IGF-1R pathway and / or increase IGF-1R expression, which unfortunately is often associated with poor outcomes. A specific conjugate, called LX-101, is well-tolerated and has shown single-agent activity in a previous phase 1 trial in adult patients with advanced, pre-treated cancer. However, further research is needed involving specific pediatric cancers that could lead to improved treatment for these vulnerable patients. This article discloses studies demonstrating potent antitumor activity of the conjugate against multiple IGF-1R-related pediatric cancer cell lines, further supporting the clinical development of the methods described herein to address the unmet needs in treating pediatric cancers.

[0032] The subject matter currently disclosed will now be described more fully below. However, those skilled in the art, benefiting from the teachings presented herein, will conceive of numerous modifications and other embodiments of the subject matter to which it pertains. Therefore, it should be understood that the subject matter currently disclosed 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 encompasses all alternatives, modifications, and equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. If one or more of the incorporated documents, patents, and similar materials differ from or contradict this application, including but not limited to defined terminology, usage of terms, described techniques, etc., this application shall prevail.

[0033] I. Definition

[0034] As used herein, patients or subjects are any mammals with IGF-1R-related pediatric cancers. As used herein, the term "mammal" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cattle, pigs, and sheep.

[0035] In some embodiments, the patient or subject is a “pediatric patient” or “pediatric subject” (i.e., a human patient who is about or less than 21 years of age at the time of diagnosis or treatment). The term “pediatric” can be further subdivided into various subgroups, including: newborns (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 (until but not including their 22nd birthday)).

[0036] As used herein, the term "conjugate" refers to a molecule that contains an IGF-1R ligand or a portion or variant thereof, as well as a cytotoxic agent.

[0037] As used herein, the term "insulin-like growth factor 1 receptor (IGF-1R)-associated pediatric cancer" refers to cancers that affect subjects, have an etiology involving IGF-1R overexpression or in which genetic alterations involve IGF-1R signaling pathways, such as IGF-1R pathway activation, and are predominantly, but not always, found in pediatric subjects. It is known to those skilled in the art that this type of cancer predominantly affects pediatric subjects.

[0038] As used herein, the term "cytotoxic agent" means any agent that, when used to treat patients according to the methods described herein, is capable of preventing, delaying, reducing, and / or reversing the activity, severity, and / or progression of disease. Any suitable cytotoxic agent that causes cell killing may be used in conjugates and methods of treating IGF-1R-related pediatric cancers.

[0039] As used herein, the term “residue” or “residue of…” for a chemical part or compound means that, through binding with a molecule, at least one covalent bond has replaced at least one atom of the original chemical part or compound, thereby creating a chemical part or compound of residue in the molecule.

[0040] As used herein, if a subject fails to achieve a response to a therapy, such that the therapy is determined to be treatment-ineffective (e.g., failure to meet clinical endpoints, including any response; prolonged duration of response; prolonged disease-free survival, relapse-free survival, and progression-free survival), then the subject is “refractory” to prior treatment.

[0041] As used herein, “and / or” means and covers any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in an alternative (“or”) context.

[0042] As used herein, when referring to measurable values ​​such as amount, dosage, time, temperature, etc. of compounds or agents as in the present subject, the term “about” means covering a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0043] As used herein, conditional language such as “can,” “able,” “may,” “may,” “e.g.,” etc., unless otherwise specifically stated or otherwise understood in the context in which they are used, is generally intended to convey that certain embodiments include certain features, elements, and / or steps, while other embodiments do not include said certain features, elements, and / or steps. Therefore, such conditional language is not generally intended to imply in any way that a feature, element, and / or step is necessary for one or more embodiments, or that one or more embodiments must include logic for determining, with or without author input or prompting, whether such features, elements, and / or steps are included in any particular embodiment or will be performed. The terms “comprising,” “including,” “having,” etc., are synonymous and used in an inclusive manner, and do not exclude additional elements, features, actions, operations, etc. Similarly, the term “or” is used in its inclusive meaning (rather than in its exclusive meaning), and thus, for example, when used to connect a list of elements, the term “or” means one, some, or all of the elements in the list.

[0044] Definitions of other terms will be provided below.

[0045] II. Treatment Methods

[0046] In some embodiments, the subject matter described herein relates to a method for treating a subject with insulin-like growth factor 1 receptor (IGF-1R)-associated pediatric cancer, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand or a portion thereof or a variant thereof, and a cytotoxic agent.

[0047] In some embodiments, the subjects are pediatric subjects under approximately 21 years of age. In some embodiments, the patients are from birth to the first 28 days of life, from 29 days of age to under two years of age, from two years of age to under 12 years of age, or from 12 years of age to 21 years of age (up to but not including the 21st birthday). In some embodiments, the patients are from birth to the first 28 days of life, from 29 days of age to under 1 year of age, 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 one year of age to under 2 years of age, from two years of age to under 3 years of age, from two years of age to under 7 years of age, from three years of age to under 5 years of age, from five years of age to under 10 years of age, from six 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.

[0048] In some embodiments, the method treats a subject in need and may further include morphological diagnosis prior to administration of the conjugate. The method may further include molecular testing prior to administration of the conjugate. In some embodiments, the method includes both morphological diagnosis and molecular testing prior to administration of the conjugate.

[0049] In some embodiments, IGF-1R is overexpressed in tumor cells of IGF-1R-related pediatric cancers. In some embodiments, IGF-1R is overexpressed on tumor cells relative to non-tumor cells. In other embodiments, overexpression of IGF-1R on tumor cells of said IGF-1R-related pediatric cancers leads to a poor prognosis. In some embodiments, the overexpression of IGF-1R on tumor cells is detected by flow cytometry or immunohistochemistry.

[0050] In some embodiments, IGF-1R-related pediatric cancers have one or more genetic alterations that activate the IGF-1R signaling pathway. In some embodiments, the genetic alteration is a mutation, gene fusion, gene amplification, or translocation.

[0051] In some embodiments, IGF-1R-related pediatric cancers are selected from the group consisting of: Ewing's sarcoma, rhabdomyosarcoma, synovial sarcoma, neuroblastoma, osteosarcoma, Wilms' tumor, Beckwith-Wiedman syndrome-related tumors, desmoplastic small round cell tumors, and adrenocortical carcinoma. In some aspects, the cancers are selected from the group consisting of: Ewing's sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma. In some aspects, the cancers are selected from the group consisting of: Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, and neuroblastoma.

[0052] In some embodiments, IGF-1R-related pediatric cancers belong to the Ewing's tumor family, which includes Ewing's sarcoma, extraosseous Ewing's tumor, and primitive neuroectodermal tumors. Ewing's sarcoma is an aggressive bone and soft tissue cancer typically characterized by a gene fusion between the Ewing's sarcoma breakpoint region 1 (EWSR1) and a gene encoding a member of the E26 transcription factor family. The most common fusion is EWSR1-FLI1 (Friend leukemia virus integration 1), which accounts for 85%-90% of ES cases. Ewing's sarcoma accounts for 2% of all childhood malignancies, with approximately 400-500 new patients annually in the United States, representing a pool of approximately 4,000 patients. Although about a quarter of these patients have metastatic disease at diagnosis, the high recurrence rate with local treatment leads researchers to believe that most patients have some form of micrometastasis at diagnosis. There are currently no approved therapies for Ewing's sarcoma. The most common treatment options are chemotherapy (most commonly [1] vincristine, doxorubicin, and cyclophosphamide, and [2] alternating regimens of ifosfamide and etoposide) and surgery, if possible. While outcomes can be positive for patients with localized disease, an estimated 35% of patients with localized disease eventually relapse after a treatment response. Recurrent and / or metastatic disease has a 5-year survival rate of 10%–15% and represents approximately 40%–50% of patients. These patients have very limited treatment options and represent a significant unmet medical need.

[0053] In some embodiments, IGF-1R-related pediatric cancer is rhabdomyosarcoma. Rhabdomyosarcoma is a pediatric soft tissue sarcoma with two main subtypes: embryonal rhabdomyosarcoma, which accounts for approximately 60% of rhabdomyosarcomas and is primarily malignant in early childhood; and alveolar rhabdomyosarcoma, which accounts for approximately 30% of rhabdomyosarcomas and is primarily malignant in patients aged 10–20 years. Approximately 700 new cases of rhabdomyosarcoma are diagnosed annually in the United States. No targeted therapy has been approved for rhabdomyosarcoma. Chemotherapy regimens (typically vincristine sulfate, actinomycin-D, and cyclophosphamide or VAC) are commonly used, with initial response rates between 20% and 40%. However, the relapse rate is high, exceeding 70%. For patients with localized disease, the standard of care is a combination of surgery, chemotherapy, and radiation therapy. Response rates for these modalities range from 70% to 85%, but one-third of patients relapse after initial response, and treatment outcomes are similar to those in the metastatic population. It is noteworthy that patients with the aforementioned genetic alterations / fusions generally perform worse with standard therapy compared to patients without fusion-positive disease. At least 15%–20% of patients are metastatic at diagnosis, a setting characterized by limited and largely ineffective treatment options. Metastatic and recurrent / refractory disease represents a major unmet medical need, with very limited treatment options, and accounts for approximately 25%–30% of the rhabdomyosarcoma population.

[0054] In some embodiments, IGF-1R-related pediatric cancer is synovial sarcoma. Synovial sarcoma is a soft tissue sarcoma originating from primitive mesenchymal cells and typically occurs in the lower extremities. Over 95% of synovial sarcoma cases are characterized by the gene fusion SS18-SSX. In the United States, approximately 1,000 patients are diagnosed with synovial sarcoma each year, with about 40% of these cases being pediatric patients. Although the 5-year survival rate ranges between 60% and 75%, response to treatment is rare, and advanced metastasis is common, as approximately 60% of patients eventually develop metastatic disease. There is no standard care for synovial sarcoma, and treatment options are limited, with surgery (sometimes amputation), chemotherapy, and radiation therapy being the primary options. While no treatment specifically for synovial sarcoma has been approved, many therapies have been approved under the category of soft tissue sarcomas. Most of these approvals are based on a response rate < 20% and a median progression-free survival (PFS) < 5 months.

[0055] In some embodiments, IGF-1R-related pediatric cancers are neuroblastomas. In the United States, approximately 60% of the approximately 750 patients diagnosed with neuroblastoma each year have high-risk disease, with a survival rate of less than 50% compared to the near 90% survival rate for low- and intermediate-risk disease. Current treatment for high-risk patients consists of chemotherapy, surgery (if possible), radiation therapy, stem cell transplantation, and combinations of dinutuximab (a GD-2 disialic acid ganglioside) with GM-CSF and isotretinoin. The response rate to these treatments is approximately 30%, with a 3-year event-free survival rate of approximately 45%. Recently, in November 2020, following two single-arm studies in 22 and 38 patients, another anti-GD-2 antibody, naxitamab, in combination with GM-CSF, received accelerated approval for relapsed / refractory high-risk neuroblastomas. Approval was based on response rates of 45% and 34%, respectively, with 30% and 23% of respondents having a response duration of at least 6 months.

[0056] In some embodiments, the IGF-1R-related pediatric cancer is osteosarcoma. Osteosarcoma is the most common malignant bone tumor in children and adolescents. Approximately 1,000 patients are diagnosed with osteosarcoma each year. Outcomes are particularly poor in metastatic patients, accounting for up to 30% of diagnoses. Only 20-30% of patients with metastatic disease achieve a durable response to first-line chemotherapy and surgery.

[0057] In some embodiments, IGF-1R-associated cancer is desmoplastic small round cell tumor (DRC). DRC is a highly aggressive sarcoma driven by a genetic translocation causing the gene fusion EWS-WT1. In the United States, approximately 100–150 patients are diagnosed with DRC each year. While most patients are adolescents and young adults, the disease can occur at any age. Treatment outcomes for these patients are poor, with reported 5-year survival rates ranging from 5% to 18% and reported 3-year event-free survival rates ranging from 7% to 10%. The most commonly used treatment is a combination of intensive chemotherapy and radiation, which produces a response in 50% of patients, but is ultimately not durable because almost all patients relapse. 50% of patients have metastatic disease, representing a group with an even worse prognosis.

[0058] In some embodiments, IGF-1R-related pediatric cancer is adrenocortical carcinoma. Adrenocortical carcinoma is a cancer that originates in the cortex or outer layer of the adrenal gland. Approximately 400–600 patients are diagnosed with adrenocortical carcinoma each year, of whom 50–100 are children or young adults. The only approved systemic therapy for adrenocortical carcinoma is mitotane, which was approved in 1970. However, mitotane has shown modest efficacy (reported response rates as low as 7%) and is associated with significant toxicities that limit its use. The only curative treatment is surgery, but this is not an option for most patients. Among patients eligible for surgery, up to 70% eventually relapse, even after successful surgery. In relapsed / refractory disease settings, combination chemotherapy consisting of gemcitabine and metronomic capecitabine is commonly used. In a study of 145 patients with adrenocortical carcinoma, this treatment resulted in a response rate of 4.9% and a median progression-free survival (PFS) of 3 months. Approximately 30%–50% of patients have metastatic disease, where treatment options are extremely limited, and the 5-year survival rate is less than 20%. Despite the availability of approved treatments, treatment options for adrenocortical carcinoma remain limited.

[0059] In the embodiments described herein, the conjugate may comprise a chemical conjugate in which the IGF-1R ligand and the cytotoxic agent are chemically linked together, either directly or via a chemical linker. In other embodiments, the conjugate is a recombinant gene 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 via a peptide bond. In some embodiments, the conjugate is a fusion protein described in U.S. Patent No. 9,675,671, which is hereby incorporated by reference in its entirety.

[0060] Methods for producing the conjugates described herein are known in the art. The nucleotide sequence encoding the IGF-1R ligand can be produced by standard recombinant DNA techniques or by protein synthesis techniques, cloned into a suitable expression vector using standard molecular biology techniques, expressed in bacterial, insect, or mammalian cells, and purified by any method known in the art for purifying proteins. The conjugates described herein comprising the IGF-1R ligand and a chemotherapeutic agent can be prepared by standard chemical and protein conjugation techniques and are described in U.S. Patent Nos. 7,811,982, 9,675,671, and 9,801,923, each of which is incorporated herein by reference in its entirety. The conjugates described herein comprising the IGF-1R ligand and a toxin 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 herein by reference in its entirety.

[0061] IGF-1R is a heterotetramer with kinase activity, consisting of two extracellular ligand-binding α subunits and two transmembrane β subunits, whose kinase activity 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, followed by IGF-2, while its affinity for insulin may be between 1 / 100 and 1 / 50. IGF-1R can also form mixed receptors by dimerizing with the insulin receptor. See Hakuno et al., *Journal of Molecular Endocrinology*, 61(1):T69-T86 (2018).

[0062] In some 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; mature insulin consists of two chains linked by disulfide bonds (corresponding to chain A of SEQ ID NO: 10 and chain B of SEQ ID NO: 11, thus two SEQ ID NOs are described), 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 a specific embodiment, 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).

[0063] In some embodiments, the IGF-1R ligand in the conjugate comprises a variant of IGF-1 with a reduced binding affinity for IGFBP compared to wild-type IGF-1 (SEQ ID NO: 3), or a variant of IGF-2 with a reduced binding affinity for IGFBP compared to wild-type IGF-2 (SEQ ID NO: 12). IGFBP belongs to a family of at least six proteins that bind to IGF-1 and IGF-2 with high affinity. IGFBP binds to most IGFs in circulation, thereby increasing their half-life, modulating their bioavailability, and generally inhibiting their ability to bind to IGF receptors. See Baxter, *American Journal of Physiology, Endocrinology & Metabolism*, 278(6):E967-76 (2000) and Allard et al., *Front Endocrinology* (Lausanne), 9;9:117 (2018). Therefore, variants of IGF-1 or IGF-2 with reduced binding to IGFBP exhibit greater biological activity in vivo.

[0064] IGF-1 variants with reduced binding affinity to 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) are replaced by the first 16 amino acids of human IGF-1 (SEQ ID NO: 3); R3-IGF-1 (SEQ ID NO: 6), in which glutamic acid at position 3 of native human IGF-1 (SEQ ID NO: 3) is replaced by arginine; and des(1-3)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(1-3)IGF-1 are described in Francis et al., Journal of Molecular Endocrinology 8(3):213-23 (1992). In some embodiments, the conjugate comprises IGF132 (SEQ ID NO: 4), R3-IGF-1 (SEQ ID NO: 6), or des(1-3)-IGF-1 (SEQ ID NO: 7).

[0065] In some embodiments, variants of IGF-1 have a higher affinity for IGF-1R than wild-type IGF-1 (SEQ ID NO: 3) for IGF-1R, or variants of IGF-2 have a higher affinity for IGF-1R than wild-type IGF-2 (SEQ ID NO: 12) for IGF-1R.

[0066] In some 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, as described above, facilitates protein purification and provides a site for conjugation with cytotoxic agents. 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 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 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 Val-Asn dipeptide, followed by a variant of human IGF-1 in which glutamic acid at position 3 of natural human IGF-1 (SEQ ID NO: 3) is replaced by glycine.

[0067] In some 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 some aspects, 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.

[0068] In some 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 some 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 some respects: (i) the binding affinity of a variant of wild-type IGF-1 to insulin-like growth factor binding protein (IGFBP) is lower than that of wild-type IGF-1 to say IGFBP, or the binding affinity of a variant of wild-type IGF-2 to say IGFBP is lower than that of wild-type IGF-2 to say IGFBP, and / or (ii) the binding affinity of a variant of wild-type IGF-1 to say IGF-1R is higher than that of wild-type IGF-1 to say IGF-1R, or the binding affinity of a variant of wild-type IGF-2 to say IGF-1R is higher than that of wild-type IGF-2 to say IGF-1R.

[0069] In some embodiments, the IGF-1R ligand or a portion thereof or a variant thereof comprises a leader sequence. In some aspects, the leader sequence comprises SEQ ID NO: 1.

[0070] In some 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).

[0071] In some embodiments, the IGF-1R ligand comprises 765IGF (SEQ ID NO: 2).

[0072] In some embodiments, the IGF-1R ligand or a portion thereof or a variant thereof is covalently bound to the cytotoxic agent.

[0073] In some embodiments, the cytotoxic agent comprises a chemotherapeutic agent. In some aspects, the chemotherapeutic agent is amsacrine, azacytidine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, dacarbazine, docetaxel, etc. Epirubicin, estramustine, etoposide, fluxuridine, fludarabine, fluorouracil, gemcitabine, hexamethylmelamine, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mecaptopurine, methotrexate, mitomycin C C) Mitotan, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, plicamycin, procarbazine, raltitrexed, semustine, streptozocin, temozolamide, teniposide, thioguanine, thiotepa, topotecan, trimetrexate, valrubicin, vincristine, vinblastine, vindesine, or vinorelbine. In some respects, the chemotherapeutic agent is methotrexate. In some respects, the chemotherapeutic agent is covalently bound to a lysine residue in the leader sequence. In some respects, a chemotherapeutic agent is one or more methotrexate residues that are covalently bound to any available lysine residue in the leader sequence.

[0074] In some embodiments, the conjugate comprises more than one cytotoxic agent that binds to an IGF-1R ligand. In some aspects, the conjugate may comprise one to twelve cytotoxic agents, or six to ten cytotoxic agents, or about eight cytotoxic agents. In some aspects, the conjugate may comprise one to twelve covalently bound cytotoxic agents, or six to ten covalently bound cytotoxic agents, or about eight covalently bound cytotoxic agents. In some aspects, the chemotherapy agent is covalently bound to any available position on the IGF-1R ligand. In some aspects, the chemotherapy agent is covalently bound to any available lysine residue. In some aspects, the chemotherapy agent is covalently bound to any available lysine residue (if present) in the leader sequence.

[0075] The leader sequence may incorporate a tag (such as a polyhistidine tag) that facilitates protein purification and provides a site for cytotoxic agent conjugation. In a specific embodiment, the leader sequence comprises SEQ ID NO: 1.

[0076] In some embodiments, the cytotoxic agent comprises a toxin. In some aspects, the toxin comprises Clostridium perfringens enterotoxin, diphtheria toxin, ricin A, Pseudomonas exotoxin, A-streptotoxin, ribosome-inactivating protein, α-fluocinolone, aspergillin, or ribonuclease. In some aspects, the toxin comprises Clostridium perfringens enterotoxin or a portion or variant thereof. In some aspects, the toxin comprises SEQ ID NO: 14 or SEQ ID NO: 15. In some aspects, the toxin comprises diphtheria toxin or a portion or variant thereof. In some aspects, the toxin comprises SEQ ID NO: 13 or SEQ ID NO: 16.

[0077] In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, 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, the cytotoxic agent is methotrexate, wherein the methotrexate is covalently bound to a lysine residue of SEQ ID NO: 2, and the IGF-1R-related pediatric cancer is selected from the group consisting of Ewing's sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma. In one aspect of these embodiments, the conjugate is LX-101 (a conjugate as described above, wherein the IGF-1R ligand is SEQ ID NO: 2, the cytotoxic agent is methotrexate, and wherein 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 methotrexates are present for each IGF-1R ligand). Methotrexate may covalently bind to the IGF-1R ligand, specifically to the lysine residues of SEQ ID NO: 2. The average number of methotrexate residues in each SEQ ID NO: 2 is 8. In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, the method comprising administering LX-101 to the subject, wherein the IGF-1R-related pediatric cancer is Ewing's sarcoma. In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, the method comprising administering LX-101 to the subject, wherein the IGF-1R-related pediatric cancer is adrenocortical carcinoma. In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, the method comprising administering LX-101 to the subject, wherein the IGF-1R-related pediatric cancer is rhabdomyosarcoma. In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, the method comprising administering LX-101 to the subject, wherein the IGF-1R-related pediatric cancer is osteosarcoma. In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, the method comprising administering LX-101 to the subject, wherein the IGF-1R-related pediatric cancer is synovial sarcoma. In some embodiments, the subject matter described herein relates to a method for treating a subject with IGF-1R-related pediatric cancer, the method comprising administering LX-101 to the subject, wherein the IGF-1R-related pediatric cancer is neuroblastoma. In all embodiments, each conjugate contains 6, 7, 8, 9, or 10 methotrexate residues.In all embodiments, the average number of methotrexate residues in each conjugate of the composition is 6, 7, 8, 9, or 10.

[0078] In some embodiments, the subject (i) has not previously received treatment for IGF-1R-related pediatric cancer; (ii) has previously received treatment for IGF-1R-related pediatric cancer; (iii) has relapsed from previous treatment for IGF-1R-related pediatric cancer; (iv) is refractory to previous treatment for IGF-1R-related pediatric cancer; or (v) is susceptible to adverse reactions caused by other treatments for IGF-1R-related pediatric cancer. Therefore, in some embodiments, the IGF-1R-related pediatric cancer is recurrent, meaning the patient has relapsed after previous treatment. In some embodiments, the IGF-1R-related pediatric cancer is stage II, III, or IV. In some embodiments, the IGF-1R-related pediatric cancer has progressed to stage II, III, or IV during or after initial therapy.

[0079] 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.

[0080] Table 1 provides a list of the sequences cited in this paper.

[0081] Table 1.

[0082]

[0083]

[0084] 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 pediatric cancer. Typically, standard care options include surgery, systemic chemotherapy (pre- or post-operative), and / or radiation therapy.

[0085] In some embodiments, subjects with IGF-1R-related pediatric cancer treated according to the methods described herein have not previously received treatment for IGF-1R-related pediatric cancer.

[0086] In some embodiments, subjects with IGF-1R-related pediatric cancer treated according to the methods described herein have previously received treatment for IGF-1R-related pediatric cancer.

[0087] In some embodiments, the subjects had relapsed from previous treatment for IGF-1R-related pediatric cancer.

[0088] In some embodiments, the subjects were refractory to prior treatment for IGF-1R-related pediatric cancers.

[0089] In all embodiments, the conjugate was administered at a dose and frequency determined by the practitioner that was appropriate for the subject and the IGF-1R-related pediatric cancer being treated.

[0090] In some respects, the conjugates were administered at the following dosages: approximately 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. 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, 4.5–5.0 μEq / kg body weight. The concentrations are 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 some respects, the conjugate is applied 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 a chemotherapeutic agent group conjugated to an IGF-1R ligand. In some respects, the conjugate is administered at the following doses: approximately 0.05 mg / kg body weight, 0.10 mg / kg body weight, 0.15 mg / kg body weight, 0.20 mg / kg body weight, 0.25mg / 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, 0.50mg / 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, 0.75mg / 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 administered in the following dosage ranges: approximately 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. 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 The dosages are 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.

[0091] In some embodiments, the conjugate is applied daily, every other day, every three days, every four days, every five days, every six days, weekly, every two weeks, every three weeks, every four weeks, monthly, every two months, or every three months.

[0092] In some embodiments, the conjugate may be administered at a lower dose and / or frequency, at which the cytotoxic agent will be effective when administered as a single agent.

[0093] In some embodiments, the dosage and / or frequency may be specifically escalated in pediatric patients. In this regard, a dose of up to 0.80 μEq / kg body weight is administered more than once per week. In this regard, a dose of up to 0.80 μEq / kg body weight is administered at least once per week. In this regard, a dose greater than 1.6 μEq / kg body weight is administered at least once per week.

[0094] In some embodiments, the conjugate is administered at the maximum tolerated dose (MTD). As used herein, “MTD” refers to the highest dose of the agent that a single patient can tolerate, as determined by a practitioner. In other words, the MTD can be determined based on the side effects observed in a given patient.

[0095] In some embodiments, the method does not cause significant or 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 utilize insulin. Unacceptable hyperglycemia refers to a grade 3 or higher side effect as determined by the treating physician, and / or a side effect that cannot be controlled with diabetes medication and leads to discontinuation of treatment with the conjugate.

[0096] The conjugates described herein can be formulated into pharmaceutical compositions for use with the methods described herein. In some embodiments, the pharmaceutical composition comprises an effective amount of the conjugate and a pharmaceutically acceptable carrier or mediator. Such pharmaceutical compositions can be formulated to be administered to a subject and can be in any form that permits administration of the composition to a subject.

[0097] The materials used to prepare the pharmaceutical composition may be non-toxic in the amounts used. It will be apparent to those skilled in the art that the optimal dosage of the active ingredient in the pharmaceutical composition will depend on a variety of factors. These factors include, but are not limited to, the type of subject (e.g., human), the subject's overall health level, the type of condition the subject has, the purpose of the composition as part of a multi-drug regimen, the specific form of the composition, and the method of administration. The pharmaceutical composition contains an effective amount of the composition such that a suitable dosage will be obtained.

[0098] The term "carrier" refers to a diluent, adjuvant, or excipient administered with the composition containing the conjugate. Any adjuvant, stabilizer, thickener, lubricant, and colorant can be used. In one embodiment, the composition and pharmaceutically acceptable carrier are sterile when administered to a subject. When the composition is administered intravenously, water can be used as a carrier. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. If desired, the compositions of the present invention may also contain a small amount of pH buffer.

[0099] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may also 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 monoglycerides or diglycerides, which may be used as solvents or suspension media), polyethylene glycol, glycerol, cyclodextrin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; agents for pH adjustment such as hydrochloric acid; and agents for tonic adjustment such as sodium chloride or dextran. Parenteral compositions may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other materials. In some embodiments, physiological saline is an adjuvant. Injectable compositions may be sterile.

[0100] The compositions of this invention may be in the form of solutions, suspensions, tablets, pills, granules, capsules, liquid-containing capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other suitable form. Examples of suitable drug carriers are described in EW Martin's *Remington's Pharmaceutical Sciences*.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] In some embodiments, it may be necessary to apply the conjugate topically to the area requiring treatment. This can be achieved, for example, but not limited to, by: local infusion during surgery; topical application, such as in combination with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, which is a porous, non-porous, or gel-like material, including membranes or fibers such as silicone rubber membranes. In some embodiments, the conjugate may be injected intraperitoneally.

[0106] In some embodiments, the conjugate can be delivered in a controlled release system.

[0107] The following examples are provided in an illustrative rather than restrictive manner.

[0108] Example

[0109] Example 1—IGF-IR Expression Measurement

[0110] Table 2. Abbreviations used in this study

[0111]

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

[0113] Table 3. Cell lines

[0114]

[0115]

[0116] First, 1 million cells were resuspended in 100 μL PBS containing 2.5 μg of human Fc-blocker (BD Biosciences) and incubated in the dark at room temperature for 10 min to block. For staining, PECD221 (IGF-1R) antibody (clone 1H7, BD Biosciences) and eFluor780 live / dead dye (eBiosciences) were added to each sample and incubated in the dark at 4°C for 30 min. PE mouse IgG1,κ isotype controls (BD Biosciences) were used to set up isotype controls for each cell line. Then, 2 mL of PBS was added to each sample, the cells were gently resuspended, centrifuged at 500 ×g for 5 min, and the supernatant was discarded. The cells were washed twice more in PBS and then resuspended in 200 μL IC fixation buffer (eBiosciences) and incubated in the dark at room temperature for 30 min. Cells were washed with 2 mL PBS and resuspended in 250 μL PBS for collection. Data were acquired using a CytoFLEX S flow cytometer (Beckman Coulter) and analyzed using a Kaluza 2.0 (Beckman Coulter).

[0117] The obtained data are depicted in Figures 1-7. The greater fluorescence intensity of cells stained with PE CD221 antibody compared to cells stained with the isotype control indicates that cell lines A-673 (Figure 1B), CADO-ES1 (Figure 1D), RD-ES (Figure 2B), SK-ES-1 (Figure 2D), SJCRH30 (Figure 3B), 143B (Figure 4B), HOS (Figure 4D), Saos-2 (Figure 5B), U-2 OS (Figure 5D), IMR32 (Figure 6B), and SK-N-AS (Figure 6D) express IGF-1R on their surfaces. The SH-SY5Y cell line (Figure 7B) did not show a significant increase in IGF-1R fluorescence intensity compared to the isotype control. Therefore, no IGF-1R expression was found in SH-SY5Y cells at the levels of the other cell lines tested.

[0118] Example 2—Cytotoxicity assay in specific types of pediatric cancer cells

[0119] The cytotoxicity induced by LX-101 was evaluated on specific types of pediatric cancer cells. The cell lines in Table 6 were treated with nine concentrations of LX-101 at a 2.5-fold dilution, and cell viability was assessed.

[0120] Table 5. Abbreviations used in this scheme

[0121]

[0122] Approximately 7 days prior to treatment, thaw the frozen cell tubes in a water bath on dry ice. Slowly transfer the contents of each frozen tube into a 15 mL tube containing 10 mL of the culture medium shown in Table 6. Centrifuge the cells at 125 × g for 5 minutes at room temperature. Resuspend the cell clumps in preheated medium in a T-25 or T-75 flask and incubate at 37°C with 5% CO2 (except for cell lines SW-13 and SW-982 cultured at 37°C and 100% aeration). When the cell culture reaches approximately 80% confluence, passage the cells using TrypLE at a passage ratio of 1:3 to 1:6.

[0123] Table 6. Cell lines

[0124]

[0125]

[0126] One day prior to LX-101 treatment, cells were collected and counted during logarithmic growth using TrypLE. Cell numbers as shown in Table 7 were seeded into 100 μL per well of a 96-well plate according to the plate layout illustrated below. The plates were incubated overnight at 37°C and 5% CO2 (except for cell lines SW-13 and SW-982, which were cultured at 37°C and 100% aeration).

[0127] Table 7. Cell seeding

[0128]

[0129] Plate layout (one for each cell line)

[0130]

[0131] compound

[0132]

[0133] A 1 / 10 working stock solution of LX-101 was prepared in 1 mM HCl, resulting in a drug concentration of 0.4 μEq / ml in 1.9 mM HCl. Sterile Eppendorf tubes were prepared containing three times the final well concentration corresponding to the culture medium for each cell line (Table 6). First dilutions were prepared using the 1 / 10 working stock solution diluted in culture medium. Subsequent 2.5-fold dilutions were prepared using culture medium. Visual inspection for signs of precipitation was performed as the compound was diluted in culture medium.

[0134] For the media control, a 10 mM HCl stock solution was prepared by diluting HCl in cell culture-grade water, and the pH was confirmed to be between 1 and 3. Next, a working stock solution with a final concentration of 1.9 mM HCl was prepared. In Eppendorf, 75 μL of the working stock solution was added to 425 μL of culture medium to prepare 285 μM HCl at a final concentration three times the standard concentration.

[0135] As shown in Table 8, add 50 μL of preparative dilution of LX-101 or HCl to the appropriate wells to provide a total volume of 150 μL per well. Fill unused wells with 150 μL of PBS.

[0136] Table 8. LX-101 Titration

[0137]

[0138] After treatment, the plates were incubated at 37°C and 5% CO2 (except for cell lines SW-13 and SW-982 cultured at 37°C and 100% air). After four days, the plates were removed and equilibrated to room temperature for 30 minutes. A black sticker was placed at the bottom of the plate to block light. Then, 75 μL of CellTiter-Glo 2.0 reagent (Promega) was added to each well, and the contents were mixed on an orbital oscillator for 2 minutes. The plates were incubated at room temperature for another 10 minutes to stabilize the luminescence signal. The luminescence was recorded using an Envision 2104 multilabel microplate reader (PerkinElmer) with an integration time of 0.25–1 second per well.

[0139] LX-101 concentration based on IGF-1 variant protein content was obtained by dividing the drug concentration based on methotrexate content by 8, as determined by the average number of methotrexate groups per IGF-1 protein using MALDI-TOF. IC50 was calculated using GraphPad PRISM software. 50 value.

[0140] Vitality (% relative to control) = (Lum 测试制品 -Lum 空白对照 ) / (Lum 媒剂 -Lum 空白对照 ) × 100%.

[0141] result

[0142] The cytotoxicity of LX-101 (lot number LIR00223) was tested on MCF7 breast cancer cells as a reference. Table 10 shows the mean IC50 of three independent assays for lot number LIR0023. 50 It was found to be 31 nM.

[0143] Table 10. Cytotoxicity of LX-101 (batch number LIR0023) against MCF7 breast cancer

[0144]

[0145] Following the above procedure, cytotoxicity data of LX-101 (batch number LIR0023) in the following cell lines were collected, as shown in Table 9. The obtained data are depicted in Figures 1-8. LX-101 had the greatest effect on osteosarcoma cell lines 143B and HOS, as well as adrenocortical carcinoma cell line SW-13, with a maximum inhibition value greater than 94% and an absolute IC50 value greater than 94%. 50 Less than 10 nM (based on the concentration of IGF-1R ligands, which comprises approximately 8 covalently bound methotrexate molecules). Significant inhibition was observed in four different Ewing's sarcoma cell lines and three different neuroblastoma cell lines after treatment with LX-101, resulting in absolute IC50. 50 Less than 30 nM. The alveolar rhabdomyosarcoma line SJCRH30 and the osteosarcoma line U2OS were also inhibited by LX-101, with absolute IC50 values ​​of 23 nM and 32 nM, respectively.

[0146] Although IGF-1R expression was detectable, one of the four osteosarcoma cell lines tested, Saos-2, was not significantly inhibited by LX-101. Interestingly, one of the neuroblastoma cell lines, SH-SY5Y, did not show high IGF-1R expression but was still inhibited by LX-101, with an absolute IC50 value of [missing information]. 50 The value was 30 nM. These data suggest that the activity of LX-101 against cancer cells is not necessarily related to the expression level of IGF-1R on cancer cells.

[0147] Table 9. Cytotoxicity of LX-101 (batch number LIR0023) against cancer cell lines

[0148]

[0149]

[0150] Although efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), some experimental errors and biases should be taken into account.

[0151] Those skilled in the art will recognize many methods and materials similar to or equivalent to those described herein, which can be used to practice the subjects described herein. This disclosure is by no means limited to the methods and materials described.

[0152] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject pertains.

[0153] Throughout this specification and claims, unless the context otherwise requires, the word "comprise / comprises / comprising" is used in a non-exclusive sense. It should be understood that the embodiments described herein include embodiments that are "composed of" and / or "substantially composed of".

[0154] Where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (to one-tenth of the lower limit unit, unless the context clearly indicates otherwise), as well as any other stated value or intermediate value within the stated range, is covered within the invention. Also covered are the upper and lower limits of these smaller ranges that may be independently included within smaller ranges, subject to any specific excluded limits within the stated range. Where the stated range includes one or both limits, ranges excluding any one or both of those included limits are also included.

[0155] Benefiting from the teachings presented in the foregoing description and associated drawings, those skilled in the art to which this subject matter pertains will conceive of numerous modifications and other embodiments set forth herein. Therefore, it should be understood that the 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. While specific terminology is used herein, it is used only in a general and descriptive sense and is not intended for limiting purposes.

Claims

1. A method for treating a subject with insulin-like growth factor 1 receptor (IGF-1R)-related pediatric 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 of claim 1, wherein the subject is a pediatric subject aged from about 1 day to about 21 years.

3. The method according to claim 1, wherein the IGF-1R-related pediatric cancer is selected from the group consisting of: Ewing's sarcoma, rhabdomyosarcoma, synovial sarcoma, neuroblastoma, osteosarcoma, Wilms' tumor, Beckwith Wiedemann syndrome-related tumors, desmoplastic small round cell tumors, and adrenocortical carcinoma.

4. The method according to claim 3, wherein the IGF-1R-related pediatric cancer is selected from the group consisting of Ewing's sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma.

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 pediatric cancer.

6. The method according to any one of claims 1 to 5, wherein the IGF-1R-related pediatric 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 pediatric cancer; (ii) has previously received treatment for the IGF-1R-related pediatric cancer; (iii) has relapsed from previous treatment for the IGF-1R-related pediatric cancer; (iv) is refractory to previous treatment for the IGF-1R-related pediatric cancer; or (v) is susceptible to adverse reactions caused by other treatments for the IGF-1R-related pediatric 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-associated pediatric cancer is Ewing's sarcoma.

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 pediatric cancer is adrenocortical carcinoma.

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 pediatric cancer is rhabdomyosarcoma.

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 pediatric cancer is osteosarcoma.

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-related pediatric cancer is synovial sarcoma.

37. 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 pediatric cancer is neuroblastoma.

38. 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 pediatric cancer is a desmoplastic small round cell tumor.

39. The method according to claim 1 or 2, wherein the IGF-1R-related pediatric cancer is Ewing's sarcoma.

40. The method according to claim 1 or 2, wherein the IGF-1R-related pediatric cancer is adrenocortical carcinoma.

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

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

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

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

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