A method for treating IGF-1R-related childhood cancer by conjugating an insulin-like growth factor 1 receptor ligand to a cytotoxic drug.

Abstract

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

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 505,263, filed on 31 May 2023, and U.S. Provisional Patent Application No. 63 / 550,463, filed on 6 February 2024, both of which are incorporated herein by reference.

[0002] The subject matter of this disclosure generally relates to methods for treating childhood cancer, particularly by administering IGF-1R ligands conjugated to cytotoxic agents.

[0003] (Reference to sequence listing)

[0004] The sequence listing, located in the filename 614072SEQLIST.xml, is 16.7 kilobytes in size, was created on May 23, 2024, and is incorporated herein by reference. [Background technology]

[0005] The insulin-like growth factor-1 receptor (IGF-1R) is widely involved in regulating both 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 into the circulatory system by the liver and stimulates the proliferation of many cell types. IGF-1 is also produced by many cell types throughout the body, including many cancers, due to its 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).

[0006] IGF-1R is often found to be expressed at higher levels in adult cancer cells than in normal cells of the same tissue type. Elevated IGF-1R activity promotes cancer cell proliferation, migration, and invasion, and is associated with tumor metastasis, treatment resistance, poor prognosis, and shortened survival in patients with cancer. Furthermore, epidemiological studies have reported a positive association between circulating IGF-1 levels and various primary cancers, including 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, breast cancer in the premenopausal and postmenopausal populations, 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.

[0007] Childhood cancers are not the same as adult cancers. The type of cancer is generally not related to lifestyle. The causes of childhood cancers are often unknown. The extent to which cancer spreads and how it is treated in children usually differ from those of adult cancers. Furthermore, children's bodies and their responses to treatment are unique.

[0008] Currently, there is a need for treatment for IGF-1R-related childhood cancers. The subject matter described herein addresses this need. [Overview of the Initiative]

[0009] In certain embodiments, the subject matter described herein relates to a method for treating insulin-like growth factor 1 receptor (IGF-1R)-related childhood 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.

[0010] In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood cancer in a subject, comprising administering to the subject a conjugate comprising an IGF-1R ligand, or a part or variant thereof, and a cytotoxic agent, wherein the IGF-1R-related childhood cancer is selected from the group consisting of Ewing's sarcoma, rhabdomyosarcoma, synovial sarcoma, neuroblastoma, osteosarcoma, Wilms' tumor, Beckwith-Wiedemann syndrome-related tumors, fibrous round cell tumors, and adrenocortical carcinoma.

[0011] In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood 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-related childhood cancer is selected from the group consisting of Ewing's sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma.

[0012] In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood 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 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).

[0013] In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood 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 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 IGF-1R-related childhood 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.

[0015] In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-associated childhood cancer in a subject, comprising administering 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 IGF-1R-associated childhood cancer in a subject, comprising administering 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 IGF-1R-related childhood cancer in a subject, comprising administering 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 IGF-1R-associated childhood 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 Clostridium perfringens enterotoxin, diphtheria toxin, lysine chain A, Pseudomonas exotoxin, A-chain toxin, ribosome inactivating protein, α-sarcin, aspergiline, or ribonuclease.

[0019] In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood cancer in a subject, comprising administering to the subject a conjugate comprising a conjugate containing 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, the methotrexate is covalently bound to the lysine of SEQ ID NO: 2, and the IGF-1R-related childhood cancer is selected from the group consisting of Ewing's sarcoma, adrenocortical carcinoma, fibroplastic round cell tumor, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma.

[0020] These and other embodiments are described in full below. [Brief explanation of the drawing]

[0021] [Figure 1] Figures 1A and 1C show the dose-response curves and IC50s of the conjugates described herein for the Ewing sarcoma cell lines, A-673 (A) and CADO-ES1 (C). Figures 1B and 1D show the IGF-1R expression levels in A-673 (B) and CADO-ES1 (D). [Figure 2]Figures 2A and 2C show the dose-response curves and IC50 of the conjugates described herein against the Ewing's sarcoma cell lines, RD-ES (A), and SK-ES-1 (C). Figures 2B and 2D show the IGF-1R expression levels in RD-ES (B) and SK-ES-1 (D). [Figure 3] Figure 3A shows the dose-response curve and IC50 of the conjugate described herein against the rhabdomyosarcoma cell line, SJCRH30. Figure 3B shows the IGF-1R expression level in SJCRH30. [Figure 4] Figures 4A and 4C show the dose-response curves and IC50 of the conjugates described herein against the osteosarcoma cell lines, 143B (A) and HOS (C). Figures 4B and 4D show the IGF-1R expression levels in 143B (B) and HOS (D). [Figure 5] Figures 5A and 5C show the dose-response curves and IC50 of the conjugates described herein against the osteosarcoma cell lines, Saos-2 (A) and U-2OS (C). Figures 5B and 5D show the IGF-1R expression levels in Saos-2 (B) and U-2OS (D). [Figure 6] Figures 6A and 6C show the dose-response curves and IC50 of the conjugates described herein against the neuroblastoma cell lines, IMR32 (A) and SK-N-AS (C). Figures 6B and 6D show the IGF-1R expression levels in IMR32 (B) and SK-N-AS (D). [Figure 7] Figure 7A shows the dose-response curve and IC50 of the conjugate described herein against the neuroblastoma cell line, SH-SY5Y. Figure 7B shows the IGF-1R expression level in SH-SY5Y. [Figure 8] Figure 8 shows the dose-response curve and IC50 of the conjugate described herein against the adrenocortical carcinoma cell line SW-13.

Mode for Carrying Out the Invention

[0022] The subject matter described herein relates to methods for treating insulin-like growth factor 1 receptor (IGF-1R)-associated childhood cancers using targeted therapies against IGF-1R, including IGF-1 or its variants combined with a cytotoxic payload. Attempts to inhibit IGF-1R using naked antibodies or small molecules without a payload have shown some clinical activity in the past, but to date, no therapies have been approved as a result.

[0023] A variety of invasive cancers with unaddressed needs have established associations with the IGF-1R pathway, exhibiting gene mutations and / or high expression of IGF-1R that activate the IGF-1R pathway. Unfortunately, these are often correlated with poor prognosis. A specific conjugate known as LX-101 demonstrated good tolerability and efficacy as a monotherapy in a previous Phase 1 trial in adult cancer patients with advanced, previously treated cancer. However, further research targeting specific pediatric cancers is needed to improve treatment options for these vulnerable patients. This specification discloses studies demonstrating that the conjugate possesses potent antitumor activity against various pediatric cancer cell lines associated with IGF-1R, further supporting the clinical development of the method described herein to address unaddressed needs in the treatment of pediatric cancers.

[0024] Herein, the subject matter of this disclosure will be described more fully. However, a person skilled in the art will, by benefiting from the teachings disclosed herein, come up with many variations and other embodiments of the disclosures herein. Therefore, it should be understood that the subject matter of this disclosure is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included in the appended claims. In other words, the subject matter described herein encompasses all substitutes, modifications, and equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. If one or more of the incorporated documents, patents, and similar materials differ from or contradict this application, including but not limited to defined terms, use of terms, and described techniques, this application shall control.

[0025] I. Definition As used herein, patient, or subject, is any mammal having IGF-1R-associated childhood cancer. 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.

[0026] In certain embodiments, the patient or subject is a “pediatric patient” or “pediatric subject” (i.e., a human patient aged 21 years or younger at the time of diagnosis or treatment). The term “pediatric” can be further divided into various subgroups, including neonates (from birth to the first 28 days of life), infants (29 days to under 2 years), children (2 years to under 12 years), and adolescents (12 years to 21 years (excluding their 22nd birthday)).

[0027] As used herein, the term “conjugate” refers to a molecule comprising an IGF-1R ligand, a part thereof, or a variant thereof, and a cytotoxic agent.

[0028] As used herein, the term “insulin-like growth factor 1 receptor (IGF-1R)-associated childhood cancer” refers to cancers affecting subjects that have an etiology involving overexpression of IGF-1R or in which genetic modification implies an IGF-1R signaling pathway, such as activation of the IGF-1R pathway, and are primarily, but not always, seen in pediatric subjects. Such cancers primarily affect pediatric subjects but are known to those skilled in the art.

[0029] As used herein, the term “cytotoxic agent” means any agent that, when administered according to the methods described herein, can prevent, delay, reduce, and / or reverse the activity, severity, and / or progression of a disease. Any suitable cytotoxic agent that results in cell killing may be used in methods for treating IGF-1R-associated childhood cancers.

[0030] As used herein, the terms “residue” or “residue of” a chemical moiety or compound means a chemical moiety or compound that is bonded to a molecule, thereby, through the bond, at least one covalent bond displaces at least one atom of the original chemical moiety or compound, resulting in a chemical moiety or compound residue within the molecule.

[0031] As used herein, a subject is “refractory” to prior treatment if it has failed to achieve a response to therapy to which the treatment is determined to be ineffective, for example, if it has failed to reach a clinical endpoint including any of the following: response, extension of the duration of response, extension of disease-free survival, extension of relapse-free survival, and progression-free survival.

[0032] As used herein, “and / or” means and encompasses one or more possible combinations of the related enumerated items, as well as the absence of any combination as interpreted as an alternative (or).

[0033] As used herein, the term “approximately” means that when referring to a measurable value such as the amount of a compound or drug, such as the current subject, dose, time, or temperature, it includes a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the specified amount.

[0034] When used herein, the conditional language used herein, such as in particular "can," "could," "might," "may," and "for example," is generally intended to convey that a particular embodiment includes certain features, elements, and / or steps, while other embodiments do not, unless otherwise stated or understood in the context in which they are used. Therefore, such conditional language is generally not intended to imply that features, elements, and / or steps are required in any way to one or more embodiments, or that one or more embodiments necessarily include logic for determining, with or without author input or instruction, whether these features, elements, and / or steps are included in or implemented in any particular embodiment. The terms "comprising," "including," and "having" are synonymous and used in a comprehensive, open manner, without excluding additional elements, features, actions, behaviors, etc. Furthermore, the term "or" is used in its inclusive sense (rather than its exclusive sense), for example, when used to connect a list of elements, meaning one, some, or all of the elements in the list.

[0035] Additional definitions of terms may be provided below.

[0036] II. Treatment Methods In certain embodiments, the subject matter described herein relates to a method for treating insulin-like growth factor 1 receptor (IGF-1R)-related childhood 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.

[0037] In certain embodiments, the subjects are children under approximately 21 years of age. In certain embodiments, patients are those born within the first 28 days, from 29 days to under 2 years of age, from 2 years to under 12 years of age, or from 12 years to 21 years of age (excluding their 21st birthday). In certain embodiments, patients are those born up to 28 days of age, from 29 days to under 1 year of age, from 1 month to under 4 months of age, from 3 months to under 7 months of age, from 6 months to under 1 year of age, from 1 year to under 2 years of age, from 2 years to under 3 years of age, from 2 years to under 7 years of age, from 3 years to under 5 years of age, from 5 years to under 10 years of age, from 6 years to under 13 years of age, from 10 years to under 15 years of age, or from 15 years to under 21 years of age.

[0038] In certain embodiments, the method may include treating the subject requiring treatment and performing a morphological diagnosis before administering the conjugate. The method may further include performing molecular testing before administering the conjugate. In certain embodiments, the method includes performing both morphological diagnosis and molecular testing before administering the conjugate.

[0039] In certain embodiments, IGF-1R is overexpressed in tumor cells of IGF-1R-associated childhood cancer. In certain embodiments, IGF-1R is overexpressed on tumor cells compared to non-tumor cells. In other embodiments, overexpression of IGF-1R in tumor cells of the IGF-1R-associated childhood cancer results in a poor prognosis. In certain embodiments, overexpression of IGF-1R on tumor cells is measured by flow cytometry or immunohistochemistry.

[0040] In certain embodiments, IGF-1R-associated childhood cancers have one or more genetic modifications that activate the IGF-1R signaling pathway. In certain embodiments, the genetic modification is a mutation, gene fusion, gene amplification, or translocation.

[0041] In certain embodiments, IGF-1R-related childhood cancers are selected from the group consisting of Ewing's sarcoma, rhabdomyosarcoma, synovial sarcoma, neuroblastoma, osteosarcoma, Wilms' tumor, Beckwith-Wiedemann syndrome-related tumors, fibrous round cell tumors, and adrenocortical carcinoma. In certain embodiments, cancer is selected from the group consisting of Ewing's sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma. In certain embodiments, cancer is selected from the group consisting of Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, and neuroblastoma.

[0042] In certain embodiments, IGF-1R-associated childhood cancers belong to the Ewing tumor family, which includes Ewing sarcoma, extraskeletal Ewing tumors, and primitive neuroectodermal tumors. Ewing sarcoma is an invasive bone and soft tissue cancer often characterized by the presence of a gene fusion between the Ewing sarcoma breakpoint region 1 (EWSR1) and a gene encoding a member of the E-26 family of transcription factors. The most common fusion is EWSR1-FLI1 (Friend leukemia virus integration 1), accounting for 85–90% of ES cases. Ewing sarcoma accounts for 2% of all childhood malignancies, with approximately 400–500 new cases per year in the United States and a general pool of about 4,000 patients. About a quarter of these patients present with metastatic disease at diagnosis, but the high recurrence rate with local treatment leads researchers to believe that the majority of patients have some form of micrometastasis at diagnosis. There are no approved treatments for Ewing sarcoma. The most common treatment options are chemotherapy ([1] vincristine, doxorubicin, and cyclophosphamide, and [2] the most frequent alternating regimen of ifosfamide and etoposide) and surgery whenever possible. While patients with local lesions may have a good outcome, approximately 35% of patients with local lesions who respond to treatment eventually relapse. The 5-year survival rate for relapsed and / or metastatic disease is 10–15%, accounting for approximately 40–50% of patients. These patients have very limited treatment options and demonstrate a serious unmet medical need.

[0043] In certain embodiments, IGF-1R-associated childhood cancer is rhabdomyosarcoma. Rhabdomyosarcoma is a childhood soft tissue sarcoma with two main subtypes, primarily classified into two subtypes: embryonal rhabdomyosarcoma (accounting for approximately 60% of rhabdomyosarcomas and primarily occurring in infancy) and alveolar rhabdomyosarcoma (accounting for approximately 30% of rhabdomyosarcomas and primarily occurring in patients aged 10-20 years). In the United States, approximately 700 new cases of rhabdomyosarcoma are diagnosed annually. There are no approved targeted therapies for rhabdomyosarcoma. Chemotherapy regimens (usually vincristine sulfate, actinomycin-D, and cyclophosphamide, or VAC) are frequently used, with an initial response rate of 20-40%. However, the recurrence rate is high, exceeding 70%. For patients presenting with localized disease, the standard treatment is a combination of surgery, chemotherapy, and radiotherapy. These therapies show response rates of 70–85%, but one-third of patients relapse after the initial response, and treatment outcomes reflect those of the metastatic population. In particular, patients with the aforementioned genetic modifications / fusions generally have a poorer prognosis to standard therapy compared to patients without fusion-positive disease. At least 15–20% of patients are metastatic at diagnosis, which presents a situation with limited treatment options and little efficacy. Metastatic and recurrent / refractory disease represent a significant unmet medical need with very limited treatment options, accounting for approximately 25–30% of the rhabdomyosarcoma population.

[0044] In certain embodiments, IGF-1R-associated childhood cancer is synovial sarcoma. Synovial sarcoma is a soft tissue sarcoma originating from primitive mesenchymal cells and commonly occurs in the lower extremities. Over 95% of cases of synovial sarcoma are characterized by the gene fusion SS18-SSX. In the United States, 1,000 patients are diagnosed with synovial sarcoma annually, and approximately 40% of these cases are pediatric patients. The 5-year survival rate is 60–75%, but the response to treatment is poor, late metastasis is common, and approximately 60% of patients eventually develop metastatic disease. There is no standard treatment for synovial sarcoma, and treatment options are limited, with surgery (sometimes amputation), chemotherapy, and radiation being the primary options. There are no specifically approved treatments for synovial sarcoma, but many treatments are approved under the umbrella of soft tissue sarcomas. Most of these approvals are based on a response rate of less than 20% and a median PFS of less than 5 months.

[0045] In certain embodiments, IGF-1R-associated childhood cancer is neuroblastoma. In the United States, approximately 750 neuroblastomas are diagnosed annually, of which about 60% are high-risk, with a survival rate of less than 50%. In contrast, the survival rate for low-risk and intermediate-risk neuroblastoma is close to 90%. Current treatments for high-risk patients include chemotherapy, surgery (where possible), radiotherapy, stem cell transplantation, and dinutuximab (against GD-2 disiaroganglioside) in combination with GM-CSF and isotretinoin. The response rate to these treatments is approximately 30%, and the 3-year event-free survival rate is approximately 45%. More recently, in November 2020, another anti-GD-2 antibody, naxitamab, received accelerated approval in combination with GM-CSF for relapsed / refractory high-risk neuroblastoma following two single-arm trials involving 22 and 38 patients. Approval is based on response rates of 45% and 34%, respectively, with 30% and 23% of responding patients having a response duration of at least 6 months.

[0046] In certain embodiments, IGF-1R-associated childhood 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. Metastatic patients account for up to 30% of diagnoses, particularly those with a poor prognosis. Only 20–30% of patients with metastatic disease have a sustained response to first-line treatment with chemotherapy and surgery.

[0047] In some embodiments, IGF-1R-associated cancer is fibrous round cell tumor (ROC). RCC is a highly invasive sarcoma driven by a gene translocation, resulting in the gene fusion EWS-WT1. In the United States, approximately 100–150 patients are diagnosed with RCC 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 a reported 5-year survival rate of 5–18% and a 3-year recurrence-free survival rate of 7–10%. The most commonly used treatment is intensive chemotherapy combined with radiation, which ultimately yields a response in 50% of patients, but almost all patients relapse, and 50% of patients develop metastatic disease, representing a population with an even worse prognosis.

[0048] In certain embodiments, IGF-1R-related childhood cancer is adrenocortical carcinoma. Adrenocortical carcinoma is a cancer that originates from the cortex, the outer layer of the adrenal gland. Approximately 400–600 patients are diagnosed with adrenocortical carcinoma each year, of which 50–100 are children or young adults. The only approved systemic therapy for adrenocortical carcinoma is mitotane, approved in 1970. However, mitotane has shown moderate efficacy (low published response rate of 7%) and is associated with significant toxicity that limits its use. The only effective treatment is surgery, but this is not an option for most patients. In patients eligible for surgery, up to 70% of patients eventually recur even after successful surgery. In relapsed / refractory disease settings, combination chemotherapy with gemcitabine and regular capecitabine is often used. In a study of 145 patients with adrenocortical carcinoma, this treatment resulted in a 4.9% response rate and a median 3-month PFS. Approximately 30–50% of patients develop metastatic disease, 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.

[0049] In embodiments of the present invention, the conjugate may include a chemical conjugate in which the IGF-1R ligand and a cytotoxic agent are chemically linked together, either directly or via a chemical linker. In other embodiments, the conjugate is a recombinant organism in which the conjugate is expressed as a single polypeptide. When the conjugate is a recombinant conjugate, the translated conjugate preferably contains a toxin, or a portion thereof, or a variant linked to the IGF-1R ligand via a peptide bond. In certain embodiments, the conjugate is a fusion protein described in U.S. Patent No. 9,675,671, the whole of which is incorporated herein by reference.

[0050] Methods for preparing the conjugates described herein are known in the art. The nucleotide sequences encoding the IGF-1R ligand can be generated by standard recombinant DNA techniques or 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 protein purification. The conjugates of the present invention, comprising the IGF-1R ligand and chemotherapeutic agents, can be prepared by standard chemical and protein conjugate techniques, as described in U.S. Patents 7,811,982, 9,675,671, and 9,801,923, which are incorporated herein by reference. The conjugates described herein, comprising the IGF-1R ligand and toxins, can be prepared as fusion proteins by standard recombinant DNA techniques, as described in U.S. Patent 8,017,102, which is incorporated herein by reference.

[0051] IGF-1R is a heterotetramer consisting of two extracellular ligand-binding subunits and two transmembrane subunits that possess kinase activity to mediate signal transduction. The native ligands for IGF-1R are IGF-1, IGF-2, and insulin. IGF-1R has the highest affinity for IGF-1, followed by affinity for IGF-2, and can bind to insulin with an affinity 50–100 times lower. IGF-1R can also form a hybrid receptor by dimerization with the insulin receptor. See Hakuno et al. J Mol Endocrinol. 61(1):T69-T86(2018).

[0052] In certain embodiments, the IGF-1R ligand in the conjugate of the present invention comprises wild-type IGF-1 (SEQ ID NO: 3), wild-type insulin (SEQ ID NO: 10, and SEQ ID NO: 11), and mature insulin consists of two chains connected by a disulfide bond between chain A, corresponding to SEQ ID NO: 10, and chain B, corresponding to SEQ ID NO: 11, and thus comprises the enumeration of the two SEQ ID NOs, 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 certain embodiments, a 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), a 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 a 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).

[0053] In certain embodiments, the IGF-1R ligand in the conjugate includes a variant of IGF-1 with reduced binding affinity to IGFBP compared to wild-type IGF-1 (SEQ ID NO: 3), or a variant of IGF-2 with reduced binding affinity to IGFBP compared to wild-type IGF-2 (SEQ ID NO: 12). IGFBPs belong to a family of at least six proteins that bind to IGF-1 and IGF-2 with high affinity. IGFBPs bind to the majority of circulating IGF, increasing its half-life, regulating its bioavailability, and generally inhibiting its ability to bind to IGF receptors. See Am J Physiol Endocrinol Metab., 278(6):E967-76 (2000) and Allard et al. Front Endocrinol (Lausanne). 9;9:117(2018). Therefore, variants of IGF-1 or IGF-2 with reduced binding to IGFBP have higher bioactivity in vivo.

[0054] IGF-1 variants with reduced binding affinity to IGFBP are known in the art and include IGF132 (US Patent No. 4,876,242) (where the first 17 amino acids of the insulin B chain (SEQ ID NO: 11) are replaced with the first 16 amino acids of human IGF-1 (SEQ ID NO: 3)), R3-IGF-1 (SEQ ID NO: 6) (where the glutamic acid at position 3 of natural human IGF-1 (SEQ ID NO: 3) is replaced with arginine), and des(1-3)IGF-1 (SEQ ID NO: 7) (where the first three amino acids of human IGF-1 (SEQ ID NO: 3) are missing). R3-IGF-1 and des(1-3)IGF-1 are described in Francis et al., J Mol Endocrinol. 8(3):213-23 (1992). In some embodiments, the conjugate includes IGF132 (SEQ ID NO: 4), R3-IGF-1 (SEQ ID NO: 6), or des(1-3)-IGF-1 (SEQ ID NO: 7).

[0055] In certain embodiments, a variant of IGF-1 has a higher affinity for IGF-1R than wild-type IGF-1 (SEQ ID NO: 3), or a variant of IGF-2 has a higher affinity for IGF-1R than wild-type IGF-2 (SEQ ID NO: 12).

[0056] In some embodiments, the IGF-1R ligand in the conjugate includes 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 cytotoxic agents, as described above. 765IGF (SEQ ID NO: 2) contains SEQ ID NO: 1, followed by R3-IGF-1 (SEQ ID NO: 6); long-R3-IGF-1 (SEQ ID NO: 5) contains 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) contains 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 contains 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 the glutamic acid at position 3 of natural human IGF-1 (SEQ ID NO: 3) is substituted with glycine.

[0057] In certain embodiments, the IGF-1R ligand includes wild-type insulin-like growth factor 1, wild-type insulin, or wild-type insulin-like growth factor 2 (IGF-2). In certain embodiments, wild-type insulin-like growth factor 1 (IGF-1) includes SEQ ID NO: 3, wild-type insulin includes SEQ ID NO: 10 or SEQ ID NO: 11, and wild-type insulin-like growth factor 2 (IGF-2) includes SEQ ID NO: 12.

[0058] In certain embodiments, the IGF-1R ligand includes a variant of wild-type IGF-1, a variant of wild-type insulin, or a variant of wild-type IGF-2. In certain embodiments, the wild-type IGF-1 variant is at least 90% identical to SEQ ID NO: 3, the wild-type insulin variant is at least 90% identical to SEQ ID NO: 10 or 11, or the wild-type IGF-2 variant is at least 90% identical to SEQ ID NO: 12.12. In certain embodiments, (i) the wild-type IGF-1 variant has a reduced binding affinity to insulin-like growth factor-binding protein (IGFBP) compared to wild-type IGF-1, or the wild-type IGF-2 variant has a reduced binding affinity to IGFBP compared to wild-type IGF-2, and / or (ii) the wild-type IGF-1 variant has an increased affinity to IGF-1R compared to wild-type IGF-1, or the wild-type IGF-2 variant has an increased affinity to IGF-1R compared to wild-type IGF-2.

[0059] In certain embodiments, the IGF-1R ligand, or a portion thereof, or a variant thereof, includes a leader sequence. In certain embodiments, the leader sequence includes SEQ ID NO: 1.

[0060] In certain embodiments, the IGF-1R ligand includes 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).

[0061] In certain embodiments, the IGF-1R ligand includes 765IGF (SEQ ID NO: 2).

[0062] In certain embodiments, the IGF-1R ligand, or a portion or variant thereof, is covalently bound to a cytotoxic agent.

[0063] In certain embodiments, the cytotoxic agent includes a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent is amsacrin, azacitidine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, fulocuridine, fludarabine, fluorouracil, gemcitabine, hexamethylmelamine, idarubicin, ifosfamide, These are irinotecan, lomustine, mechloretamine, melphalan, mercaptopurine, methotrexate, mitomycin C, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, plicamycin, procarbazine, laritrexed, semustine, streptozocin, temozolamide, teniposide, thioguanine, thiotepa, topotecan, trimitrexate, barrubicin, vincristine, vinblastine, vindestine, or vinorelbine. In certain embodiments, the chemotherapeutic agent is methotrexate. In certain embodiments, the chemotherapeutic agent is covalently bonded to a lysine residue in the leader sequence. In certain embodiments, the chemotherapeutic agent is one or more methotrexate residues covalently bonded to any available lysine residue in the leader sequence.

[0064] In certain embodiments, the conjugate comprises two or more cytotoxic agents bound to the IGF-1R ligand. In certain embodiments, the conjugate may comprise 1 to 12 cytotoxic agents, or 6 to 10 cytotoxic agents, or about 8 cytotoxic agents. In certain embodiments, the conjugate may comprise 1 to 12 covalently bound cytotoxic agents, or 6 to 10 covalently bound cytotoxic agents, or about 8 covalently bound cytotoxic agents. In certain embodiments, the chemotherapeutic agent is covalently bound to any available position on the IGF-1R ligand. In certain embodiments, the chemotherapeutic agent is covalently bound to any available lysine residue. In certain embodiments, the chemotherapeutic agent, if present, is covalently bound to any available lysine in the leader sequence.

[0065] The leader sequence can incorporate tags such as polyhistidine tags, facilitate protein purification, and provide sites for the conjugate of cytotoxic agents. In certain embodiments, the leader sequence includes Sequence ID No. 1.

[0066] In some embodiments, the cytotoxic agent includes a toxin. In certain embodiments, the toxin includes Clostridium perfringens enterotoxin, diphtheria toxin, lysine chain A, Pseudomonas exotoxin, A-chain toxin, ribosome inactivating protein, α-sarcin, aspergiline, or ribonuclease. In certain embodiments, the toxin includes Clostridium perfringens enterotoxin, or a part thereof, or a variant thereof. In certain embodiments, the toxin includes SEQ ID NO: 14 or SEQ ID NO: 15. In certain embodiments, the toxin includes diphtheria toxin, or a part thereof, or a variant thereof. In certain embodiments, the toxin includes SEQ ID NO: 13 or SEQ ID NO: 16.

[0067] In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood cancer in elephants, comprising administering 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, the methotrexate is covalently bound to the lysine of SEQ ID NO: 2, and the IGF-1R-related childhood cancer is selected from the group consisting of Ewing's sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and neuroblastoma. In certain embodiments of these embodiments, the conjugate is LX-101 as described above (the conjugate described above, wherein the IGF-1R ligand is SEQ ID NO: 2, the cytotoxic agent is methotrexate, and at least 6 to a maximum of 10, or at least 6 to a maximum of 9, or at least 7 to a maximum of 9, or at least 8 to a maximum of 9 methotrexate residues are each bound to the IGF-1R ligand. Methotrexate can be covalently bound to the IGF-1R ligand, particularly to the 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 described herein is a method for treating IGF-1R-related childhood cancer in a subject, comprising administering LX-101 to the subject, wherein the IGF-1R-related childhood cancer is Ewing's sarcoma. In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood cancer in a subject, comprising administering LX-101 to the subject, wherein the IGF-1R-related childhood cancer is adrenocortical carcinoma. In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood cancer in a subject, comprising administering LX-101 to the subject, wherein the IGF-1R-related childhood cancer is rhabdomyosarcoma. In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood cancer in a subject, comprising administering LX-101 to the subject, wherein the IGF-1R-related childhood cancer is osteosarcoma.In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood cancer in a subject, comprising administering LX-101 to the subject, wherein the IGF-1R-related childhood cancer is synovial sarcoma. In certain embodiments, the subject matter described herein relates to a method for treating IGF-1R-related childhood cancer in a subject, comprising administering LX-101 to the subject, wherein the IGF-1R-related childhood cancer is neuroblastoma. In certain embodiments, the number of methotrexate residues per conjugate is 6, 7, 8, 9, or 10. In certain embodiments, the average number of methotrexate residues per conjugate in the composition is 6, 7, 8, 9, or 10.

[0068] In certain embodiments, the subjects are (i) not previously treated for IGF-1R-related childhood cancer, (ii) previously treated for IGF-1R-related childhood cancer, (iii) relapsed from previous treatment for IGF-1R-related childhood cancer, (iv) refractory to previous treatment for IGF-1R-related childhood cancer, or (v) susceptible to adverse reactions from other treatments for IGF-1R-related childhood cancer. Therefore, in certain embodiments, IGF-1R-related childhood cancer relapses if the patient has relapsed after previous treatment. In certain embodiments, IGF-1R-related childhood cancer is stage II, III, or IV. In certain embodiments, IGF-1R-related childhood cancer progresses to stage II, III, or IV during or after initial therapy.

[0069] In certain embodiments, the treatment results in a reduction of tumor cell growth in the subject. In certain embodiments, the reduction is caused by the killing of IGF-1R expressing cells.

[0070] Table 1 provides a list of specific sequences referenced herein. [Table 1-1] [Table 1-2]

[0071] In certain embodiments, the methods described herein are part of a combination therapy. In particular, the methods described herein can be used alone or in combination with standard treatment options for each type of childhood cancer. Generally, standard treatment options include surgery, systemic chemotherapy (either pre- or post-operative), and / or radiotherapy.

[0072] In certain embodiments, subjects having IGF-1R-associated childhood cancer treated according to the method described herein have not previously received treatment for IGF-1R-associated childhood cancer.

[0073] In certain embodiments, subjects having IGF-1R-associated childhood cancer treated according to the method described herein have previously received treatment for IGF-1R-associated childhood cancer.

[0074] In certain embodiments, the subjects are those who have experienced recurrence after prior treatment for IGF-1R-associated childhood cancer.

[0075] In certain embodiments, the subjects were refractory to previous treatments for IGF-1R-related childhood cancer.

[0076] In all embodiments, the conjugate is administered at a dose and frequency appropriate to the subject, and IGF-1R-related childhood cancers are treated at the physician's discretion.

[0077] In certain embodiments, the conjugate is administered in doses of approximately 0.05, 0.10, 0.20, 0.40, 0.80, 1.0, 1.5, 1.6, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 equivalents / kg body weight, or approximately 0.05-10.0, 0.1-8.0, 0.2-4.0, 0.3-3, 0.4-2.5 The drug is administered in dose ranges of 0.05-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, 4.0-4.5, 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, or 9.5-10.0 equivalents / kg body weight. In certain embodiments, the conjugate is administered at approximately 0.05, 0.10, 0.20, 0.40, 0.80, 1.6, or 2.5 Eq / kg body weight per kg of body weight. The equivalent amount is equivalent to the moles of the chemotherapeutic group conjugated to the IGF-1R ligand. In certain embodiments, the conjugate is approximately 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1, 1.2, 1 0.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4 0.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7 0.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 1 In doses of 4.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, or 16.5 mg / kg body weight, or approximately 0.05-0.5, 0.5-1.0 1.0~1.5, 1.5~2.0, 2.0~2.5, 2.5~3.0, 3.0~3.5, 3.5~4.0, 4.0~4.5, 4.5~5.0, 5.0~5.5, 5.5~6.0, 6.0~6.5, 6.5~7.0, 7.0~7.5, 7.5~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, 9.5~10.0, 10.0~10.5, 10.5~11. It is administered in dose ranges of 0, 11.0-11.5, 11.5-12.0, 12.0-12.5, 12.5-13.0, 13.0-13.5, 13.5-14.0, 14.0-14.5, 14.5-15.0, 15.0-15.5, 15.5-16.0, or 16.0-16.5 mg / kg body weight, where mg refers to the amount of IGF-1R ligand present in the conjugate.

[0078] In certain embodiments, the conjugate is administered daily, every other day, every three days, every four days, every five days, every six days, 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.

[0079] In certain embodiments, the conjugate may be administered at a lower dose and / or frequency than the cytotoxic agent would be effective when administered as a monotherapy.

[0080] In certain embodiments, the dose and / or frequency may be gradually increased, particularly in pediatric patients. In this embodiment, a dose of up to 0.80 equivalents / kg body weight is administered at least twice a week. In this embodiment, a dose of up to 0.80 equivalents / kg body weight is administered at least once a week. In this embodiment, a dose greater than 1.6 equivalents / kg body weight is administered at least once a week.

[0081] In some embodiments, the conjugate is administered at the maximum tolerated dose (MTD). As used herein, "MTD" refers to the maximum dose of the drug that an individual patient can tolerate, as determined by a physician. In other words, the side effects in a given patient can determine the MTD.

[0082] In certain embodiments, the method does not cause significant or unacceptable hyperglycemia in the subject. Hyperglycemia is another term for hyperglycemia, which can occur when there is insufficient insulin in the body or when the body is unable to adequately utilize insulin. Unacceptable hyperglycemia refers to adverse effects of grade 3 or higher as determined by the attending physician, and / or adverse effects that cannot be controlled with diabetes medications and lead to discontinuation of treatment with the conjugate of the present invention.

[0083] The conjugates described herein can be formulated in pharmaceutical compositions 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 a pharmaceutical composition can be formulated to be suitable for administration to a subject and may be in any form that allows the composition to be administered to a subject.

[0084] The materials used in the preparation of the pharmaceutical composition may be nontoxic in the amount used. It will be apparent to those skilled in the art that the optimal dose of the active ingredient in the pharmaceutical composition depends on various factors. These factors include, but are not limited to, the type of subject (e.g., human), the overall health status of the subject, the type of condition the subject has, the use of the composition as part of a multi-drug regimen, the specific form of the composition of the present invention, and the method of administration. The pharmaceutical composition contains an effective amount of the composition so as to yield a suitable dose.

[0085] The term "carrier" refers to a diluent, adjuvant, or excipient on which the composition of the present invention is administered. Optional auxiliaries, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, when administered to a subject, the composition of the present invention and the pharmaceutically acceptable carrier are sterilized. Water may be the carrier when the composition of the present invention is administered intravenously. Saline solution, as well as aqueous solutions of dextrose and glycerol, may also be used as liquid carriers, particularly in injectable solutions. The composition may also contain a small amount of pH buffering agent if desired.

[0086] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline solution, physiological saline, Ringer's solution, or isotonic sodium chloride; fixing oils such as synthetic monoglycerides or diglycerides that can function as a solvent or suspension medium; polyethylene glycol, glycerin, cyclodextrin, propylene glycol, or other solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates, or phosphates; pH adjusters such as hydrochloric acid; and osmotic regulators such as sodium chloride or glucose. Parenteral compositions may be enclosed 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.

[0087] This composition may take the form of a solution, suspension, tablet, pill, pellet, capsule, liquid-containing capsule, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, or any other form suitable for use. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin.

[0088] In some embodiments, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to human subjects. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer solution. Optionally, the composition may also include a solubilizer. The composition for intravenous administration may optionally include a local anesthetic, such as a lignocaine, to reduce pain at the injection site. Generally, the components are supplied in unit dosage forms, either separately or mixed together, as dry lyophilized powder or anhydrous concentrate, in sealed containers such as ampoules or sachets indicating the amount of the active agent. When the composition is administered by infusion, it can be dispensed using, for example, an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline may be provided so that the components can be mixed before administration.

[0089] Pharmaceutical compositions can be prepared using methodologies well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining the composition with water to form a solution. Surfactants can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are conjugates that can interact non-covalently with the composition of the present invention to facilitate its dissolution or homogeneous suspension in an aqueous delivery system.

[0090] The conjugates described herein may be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through the epithelium or mucosal lining (e.g., oral mucosa, rectum, and intestinal mucosa). Administration may be systemic or topical. Various delivery systems, such as microparticles, microcapsules, and capsules, are known and may be useful for administering the compositions of the present invention. Methods of administration include, but are not limited to, oral and parenteral administration, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intracerebral, intraventricular, subarachnoid, vaginal, transdermal, rectal, inhalation, or topical administration to the ear, nose, eye, or skin. The mode of administration is at the discretion of the physician and in part depends on the site of the condition. In certain embodiments, the conjugate is administered intravenously, subcutaneously, or intramuscularly.

[0091] In certain embodiments, the conjugate is administered parenterally. In certain embodiments, the conjugate is administered intravenously. In certain embodiments, the conjugate is administered by continuous infusion. In certain embodiments, the conjugate is administered by infusions lasting 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours.

[0092] In certain embodiments, it may be desirable to administer the conjugate topically to the area requiring treatment. This can be achieved, for example, by injection, by catheter, by suppository, or by implant, for example, by local injection during surgery or by topical application in conjunction with postoperative wound dressings, where the implant is made of porous, non-porous, or gelatinous material, including a membrane such as a cialastic membrane or fiber. In certain embodiments, the conjugate may be injected intraperitoneally.

[0093] In certain embodiments, the conjugate may be delivered via a controlled emission system.

[0094] The following examples are provided for illustrative purposes only and not for limiting purposes. [Examples]

[0095] Example 1 - IGF-1R Expression Assay [Table 2]

[0096] IGF-1R expression was determined via FACS in the cell lines shown in Table 3. The MCF7 cell line was used as a positive control. [Table 3]

[0097] First, 1,000,000 cells were resuspended in 100 μL of PBS containing 2.5 μg of human Fc block (BD Biosciences) and incubated in the dark at room temperature for 10 minutes to block. For staining, PE CD221 (IGF-1R) antibody (clone 1H7, BD Biosciences) and eFluor780 bio / dead stain (eBiosciences) were added to each sample and incubated in the dark at 4°C for 30 minutes. Isotype controls for each cell line were established using PE mouse IgG1, κ isotype control (BD Biosciences). Next, 2 mL of PBS was added to each sample, the cells were gently resuspended, and the cells were centrifuged at 500 × g for 5 minutes, with the supernatant discarded. The cells were washed twice more in PBS and then resuspended in 200 μL of IC fixation buffer (eBioscience) and incubated in the dark at room temperature for 30 minutes. Cells were washed with 2 mL of PBS and resuspended in 250 μL of PBS for acquisition. Data were acquired using a CytoFLEX S flow cytometer (Beckman Coulter) and analyzed using a Kaluza 2.0 (Beckman Coulter).

[0098] The obtained data are shown in Figures 1 to 7. The greater fluorescence intensity of cells stained with PE CD221 antibody compared to cells stained with isotype controls 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 surface. Cell line SH-SY5Y (Figure 7B) did not show a significant increase in fluorescence intensity for IGF-1R staining compared to isotype controls. Therefore, SH-SY5Y cells were found not to express IGF-1R at the same levels as the other cell lines tested.

[0099] Example 2 - Cytotoxicity assay in specific types of pediatric cancer cells

[0100] LX-101-induced cytotoxicity was evaluated in specific types of pediatric cancer cells. The cell lines listed in Table 6 were treated with LX-101 at nine different concentrations using 2.5-fold dilutions, and cell viability was assessed. [Table 4]

[0101] Approximately 7 days prior to treatment, the cryovials of frozen cells were thawed by transferring them to a water bath on dry ice. The contents of each cryovial were slowly transferred to a 15 mL tube containing 10 mL of the culture medium shown in Table 6. The cells were centrifuged at 125 × g for 5 minutes at room temperature. The cell pellet was resuspended in pre-warmed culture medium in a T-25 or T-75 flask and incubated at 37°C with 5% CO2 (except for cell lines SW-13 and SW-982, which were cultured at 37°C and 100% air). When the cell culture reached approximately 80% confluence, the cells were subcultured using TrypLE at a subculturing ratio of 1:3 to 1:6. [Table 5]

[0102] One day prior to LX-101 treatment, cells were collected and counted during logarithmic growth using TrypLE. The number of cells 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 and 5% CO2 (except for cell lines SW-13 and SW-982, which were cultured at 37°C and 100% air). [Table 6] [Table 7] [Table 8]

[0103] A 1 / 10 working stock solution of LX-101 was prepared in 1 mM HCl, with a concentration of 0.4 μEq / ml of the drug in 1.9 mM HCl. Sterile Eppendorf tubes were prepared to contain three times the final well concentration in the culture medium corresponding to each cell line (Table 6). The first dilution was prepared using a 1 / 10 working stock diluted in culture medium. Subsequent 2.5-fold dilutions were prepared using culture medium. When the compound was diluted in culture medium, visual inspection was performed for signs of precipitation.

[0104] For the vehicle 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 was prepared with a final concentration of 1.9 mM HCl. In an Eppendorf filter, 75 μL of the working stock solution was added to 425 μL of culture medium to triple the final concentration of 285 μM HCl.

[0105] As shown in Table 8, 50 μL of a prepared dilution of LX-101 or HCl was added to the appropriate wells to obtain a total volume of 150 μL per well. Unused wells were filled with 150 μL of PBS. [Table 9]

[0106] After treatment, the plates were incubated at 37°C in 5% CO2 (except for cell lines SW-13 and SW-982, which were cultured at 37°C and 100% air). After 4 days, the plates were removed and equilibrated at room temperature for 30 minutes. A black sticker was placed at the bottom of the plate to block light. 75 μL of CellTiter-Glo 2.0 reagent (Promega) was then added to each well, and the contents were mixed on an orbital shaker for 2 minutes. The plates were incubated at room temperature for a further 10 minutes to stabilize the luminescence signal. Luminescence was recorded using an Envision 2104 multi-label microplate reader (PerkinElmer) with an integration time of 0.25–1 second per well.

[0107] The LX-101 concentration based on IGF-1 variant protein content was derived by dividing the drug concentration based on methotrexate content by 8, which is the average number of methotrexate groups per IGF-1 protein determined by MALDI-TOF. 50 The values ​​were calculated using GraphPad PRISM software. Survival rate (percentage of control) = (Lum 被験物質 -Lum ブランク対照 ) / (Lum ビヒクル -Lum ブランク対照 ) × 100%.

[0108] result

[0109] The cytotoxicity of lot LIR00223 of LX-101 was tested as a reference on MCF7 breast cancer cells. As shown in Table 10, the mean IC of three independent experiments using lot LIR0023. 50 It was found to be 31 nM. [Table 10]

[0110] Following the above procedure, cytotoxicity data were collected for LX-101 (lot LIR0023) using the following cell lines as shown in Table 9. The data obtained are shown 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 exceeding 94% and an absolute IC 50 less than 10 nM (based on the concentration of the IGF-1R ligand containing approximately eight covalently bound methotrexate molecules). After treatment with LX-101, significant inhibition of four different Ewing's sarcoma cell lines and three different neuroblastoma cell lines was observed, with an absolute IC 50 less than 30 nM. The alveolar rhabdomyosarcoma cell line SJCRH30 and the osteosarcoma cell line U2OS were also inhibited by LX-101 with absolute IC50 values of 23 nM and 32 nM, respectively.

[0111] One of the four osteosarcoma lines tested, Saos-2, was not significantly inhibited by LX-101 despite detectable IGF-1R expression. Interestingly, one neuroblastoma cell line, SH-SY5Y, which did not show high levels of IGF-1R expression, was still inhibited by LX-101 with an absolute IC 50 of 30 nM. These data suggest that the activity of LX-101 against cancer cells does not necessarily correlate with the level of IGF-1R expression on the cancer cells. [Table 11]

[0112] Efforts have been made to ensure accuracy with respect to the numbers (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be taken into account.

[0113] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in practicing the subject matter described herein. The present disclosure is not limited to the methods and materials described.

[0114] Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this subject belongs.

[0115] Throughout this specification and the claims, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense. Embodiments described herein are understood to include embodiments consisting of and / or essentially consisting of embodiments.

[0116] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intervening value up to one-tenth of the lower limit unit is included between the upper and lower limits of the range, and any other listed or intervening values ​​within that listed range. The upper and lower limits of any smaller ranges that may independently be included in these smaller ranges are also included and are subject to any specifically excluded limits within the specified range. If the listed range includes one or both limits, it also includes the range that excludes one or both of those limits.

[0117] Many modifications and other embodiments of the invention described herein will be apparent to those skilled in the art who benefit from the teachings presented in the foregoing description and the accompanying drawings. It should be understood that the subject matter of this disclosure is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included in the appended claims. Certain terms are used herein, but they are used only in a general and descriptive sense, and not for limiting purposes.

Claims

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

2. The method according to claim 1, wherein the subject is a child from about 1 day old to about 21 years old.

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

4. The method according to claim 3, wherein the IGF-1R-related childhood 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 tumor cells of IGF-1R-related childhood cancer.

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

7. The method according to claim 6, wherein the gene modification 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 according to claim 8, wherein the wild-type insulin-like growth factor 1 (IGF-1) comprises SEQ ID NO: 3, the wild-type insulin comprises SEQ ID NO: 10 or 11, and 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 according to claim 10, wherein the wild-type IGF-1 variant is at least 90% identical to SEQ ID NO: 3, the wild-type insulin variant is at least 90% identical to SEQ ID NO: 10 or 11, and the wild-type IGF-2 variant 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 reduced binding affinity to insulin-like growth factor-binding protein (IGFBP) compared to wild-type IGF-1, or the wild-type IGF-2 variant has a reduced binding affinity to IGFBP compared to wild-type IGF-2, and / or (ii) the wild-type IGF-1 variant has an increased affinity to IGF-1R compared to wild-type IGF-1, or the wild-type IGF-2 variant has an increased affinity to IGF-1R compared to wild-type IGF-2.

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

14. The method according to claim 13, wherein the leader array includes sequence number 1.

15. The method according to any one of claims 1 to 7 and 10 to 14, wherein the IGF-1R ligand includes 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 according to 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 part 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 aforementioned chemotherapeutic agents include amsacrin, azacitidine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, phloxuridine, fludarabine, fluorouracil, gemcitabine, hexamethylmelamine, idarubicin, ifosfamide, irinotecan, and romus. The method according to claim 18, wherein the agent is tin, mechloretamine, melphalan, mercaptopurine, methotrexate, mitomycin C, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, plicamycin, procarbazine, laritrexed, semustine, streptozocin, temozolamide, teniposide, thioguanine, thiotepa, topotecan, trimitrexate, barrubicin, vincristine, vinblastine, vindestine, or vinorelbine.

20. The method according to 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 according to claim 21, wherein the toxin comprises Clostridium perfringens enterotoxin, diphtheria toxin, lysine chain A, Pseudomonas exotoxin, A-chain toxin, ribosome inactivating protein, α-sarcin, aspergiline, or ribonuclease.

23. The method according to claim 22, wherein the toxin comprises Clostridium perfringens enterotoxin, a part thereof, or a variant thereof.

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

15.

25. The method according to claim 22, wherein the toxin comprises diphtheria toxin, a part thereof, or a variant thereof.

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

16.

27. The method according to any one of claims 1 to 26, wherein the subject is (i) not previously treated for IGF-1R-related childhood cancer, (ii) previously treated for IGF-1R-related childhood cancer, (iii) relapsed from previous treatment for IGF-1R-related childhood cancer, (iv) refractory to previous treatment for IGF-1R-related childhood cancer, or (v) susceptible to side effects from other treatments for IGF-1R-related childhood 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 according to claim 28, wherein the one or more other therapies include one or more of surgery, systemic chemotherapy (either preoperative or postoperative), and radiotherapy.

30. The aforementioned conjugate is administered in doses of approximately 0.05, 0.10, 0.20, 0.40, 0.80, 1.0, 1.5, 1.6, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 equivalents / kg body weight, or approximately 0.05 to 10.0, 0.1 to 8.0, 0.2 to 4.0, 0.3 to 3, 0.4 to 2.5, 0.05 to 0.5, 0.5 to The method according to any one of claims 1 to 29, administered in a dose range of 1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, 4.0-4.5, 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, or 9.5-10.0 equivalents / kg body weight.

31. The method according to claim 30, wherein the conjugate is administered at a dose of approximately 0.05, 0.10, 0.20, 0.40, 0.80, 1.6, or 2.5 equivalents / 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, there are 6 to 10 methotrexate molecules for each IGF-1R ligand of SEQ ID NO: 2, and the IGF-1R-related childhood 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, there are 6 to 10 methotrexate molecules for each IGF-1R ligand of SEQ ID NO: 2, and the IGF-1R-related childhood 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, there are 6 to 10 methotrexate molecules for each IGF-1R ligand of SEQ ID NO: 2, and the IGF-1R-related childhood 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, there are 6 to 10 methotrexate molecules for each IGF-1R ligand of SEQ ID NO: 2, and the IGF-1R-related childhood 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, there are 6 to 10 methotrexate molecules for each IGF-1R ligand of SEQ ID NO: 2, and the IGF-1R-related childhood 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, there are 6 to 10 methotrexate molecules for each IGF-1R ligand of SEQ ID NO: 2, and the IGF-1R-related childhood 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, there are 6 to 10 methotrexate molecules for each IGF-1R ligand of SEQ ID NO: 2, and the IGF-1R-related childhood cancer is a fibrinogenic round cell tumor.

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

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

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

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

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

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

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