Methods for treating igf-1r-related pediatric cancers with an insulin-like growth factor 1 receptor ligand conjugated to a cytotoxic agent

Abstract

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

Description

METHODS FOR TREATING IGF-1R-RELATED PEDIATRIC CANCERS WITH ANINSULIN-LIKE GROWTH FACTOR 1 RECEPTOR LIGAND CONJUGATED TO ACYTOTOXIC AGENTCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of 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 herein incorporated by reference in their entirety.FIELD

[0002] The presently disclosed subject matter relates generally to methods of treating pediatric cancers in particular by administering an IGF-1R ligand conjugated to a cytotoxic agent.REFERENCE TO A SEQUENCE LISTING

[0003] The Sequence Listing written in file name 614072SEQLIST.xml is 16.7 kilobytes, was created on May 23, 2024, and is hereby incorporated by reference.BACKGROUND

[0004] The insulin-like growth factor-1 receptor (IGF-1R) has been implicated broadly in the regulation of both normal immunity and autoimmune diseases. IGF-1 is a peptide of 70 amino acid residues having 40% identity with proinsulin. (Daughaday, W. H., et al., 1989, Endocrine Revs. 10:68). Insulin and IGF-1 have some cross-reactivity with each other's receptor. (Soos, M. A., et al., 1993, Biochem. J. 290:419.) IGF-1 is secreted by the liver into the circulatory system and stimulates growth of many cell types. IGF-1 is also produced by many cell types throughout the body, including many cancers, for autocrine and paracrine effects. IGF-1 production is stimulated by growth hormone. (Stewart, C. H., et al., 1996, Physiol. Revs. 76:1005; Yakar, S., et al., 2002, Endocrine 19:239).

[0005] IGF-1R is often found to be expressed at higher levels in cancer cells of adults 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 patients with cancer. Further, epidemiological studies have reported a positive association between circulating IGF-1 levels and various primary cancers, suchas breast, colorectal, 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, pre- and postmenopausal breast, lung, thyroid, and colorectal cancers (Ma et al., 1999; Renehan et al., 2004; Shi et al., 2001). These cancers occur in adults, not children.

[0006] Pediatric cancers are not the same as adult cancers. The type of cancer is not generally related to lifestyle. The cause of pediatric cancer is often unknown. How far cancers spread and how cancer is treated in children are usually different from adult cancers. Further, a child’s body and how the child will respond to treatments is unique as well.

[0007] There is currently a need for treatments against IGF-1R related pediatric cancers. The subject matter described herein addresses this need.BRIEF SUMMARY

[0008] In certain embodiments, the subject matter described herein is directed to methods for treating an insulin-like growth factor 1 receptor (IGF-lR)-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent.

[0009] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the IGF-lR-related pediatric cancer is selected from the group consisting of Ewing’s sarcoma, rhabdomyosarcoma, synovial sarcoma, neuroblastoma, osteosarcoma, Wilms’ tumor, Beckwith Wiedemann Syndrome associated tumors, desmoplastic small round cell tumor, and adrenocortical carcinoma.

[0010] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the IGF-lR-related pediatric cancer is selected from the group consisting of Ewing’s sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma and neuroblastoma.

[0011] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and acytotoxic agent, wherein the IGF-1R ligand, or portion or variant thereof comprises wildtype insulin-like growth factor 1 (IGF-1) (SEQ ID NO:3), wildtype insulin (SEQ ID NO: 10 and SEQ ID NO: 11), wildtype insulin-like growth factor 2 (IGF-2) (SEQ ID NO: 12), a variant of wildtype IGF-1 (SEQ ID NO:3), a variant of wildtype insulin (SEQ ID NO: 10 and SEQ ID NO: 11), or a variant of wildtype IGF-2 (SEQ ID NO: 12).

[0012] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the IGF-1R ligand, or portion or variant thereof comprises a variant of wildtype insulin-like growth factor 1 (IGF-1) (SEQ ID NO:3).

[0013] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the IGF-1R ligand, or portion or variant thereof comprises SEQ ID NO:2.

[0014] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the cytotoxic agent is a chemotherapeutic agent.

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

[0016] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the cytotoxic agent comprises a toxin.

[0017] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the cytotoxic agent comprises Clostridium perfringens enterotoxin, diphtheriatoxin, ricin chain A, Pseudomonas exotoxin, A chain toxins, a ribosome inactivating protein, oc-sarcin, aspergillin, or a ribonuclease.

[0018] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the IGF-1R ligand, or portion or variant thereof comprises SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein the methotrexate is covalently bound to a lysine of SEQ ID NO:2, and the IGF-lR-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.

[0019] These and other embodiments are described fully below.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figures 1A&C depict dose-response curves and ICsos for a conjugate described herein against Ewing’s sarcoma cell lines, A-673 (A) and CADO-ES1 (C). Figures 1B&D depict IGF- 1R expression levels in A-673 (B) and CADO-ES1 (D).

[0021] Figures 2A&C depict dose-response curves and ICsos for a conjugate described herein against Ewing’s sarcoma cell lines, RD-ES (A) and SK-ES-1 (C). Figures 2B&D depict IGF-1R expression levels in RD-ES (B) and SK-ES-1 (D).

[0022] Figure 3A depicts a dose-response curve and ICso for a conjugate described herein against a rhabdomyosarcoma cell line, SJCRH30. Figure 3B depicts IGF-1R expression levels in SJCRH30.

[0023] Figures 4A&C depict dose-response curves and ICsos for a conjugate described herein against osteosarcoma cell lines, 143B (A) and HOS (C). Figures 4B&D depict IGF-1R expression levels in 143B (B) and HOS (D).

[0024] Figures 5A&C depict dose-response curves and ICsos for a conjugate described herein against osteosarcoma cell lines, Saos-2 (A) and U-2OS (C). Figures 5B&D depict IGF-1R expression levels in Saos-2 (B) and U-2OS (D).

[0025] Figures 6A&C depict dose-response curves and ICsos for a conjugate described herein against neuroblastoma cell lines, IMR32 (A) and SK-N-AS (C). Figures 6B&D depict IGF-1R expression levels in IMR32 (B) and SK-N-AS (D).

[0026] Figure 7A depicts a dose-response curve and IC50 for a conjugate described herein against neuroblastoma cell line, SH-SY5Y. Figure 7B depicts IGF-1R expression levels in SH-SY5Y.

[0027] Figures 8 depicts a dose-response curve and IC50 for a conjugate described herein against adrenocortical carcinoma cell line SW-13.DETAILED DESCRIPTION

[0028] The subject matter described herein relates to methods of treating insulin-like growth factor 1 receptor (IGF-lR)-related pediatric cancers using a targeted therapy directed to IGF-1R, that comprises an IGF-1 or variant thereof in combination with a cytotoxic payload. Although past attempts at inhibiting IGF-1R with non-payload-bearing naked antibodies or small molecules demonstrated some clinical activity, to date, there has been no resulting approved therapy.

[0029] A variety of aggressive cancers of unmet need have well-established connections to the IGF-1R pathway with genetic alterations activating the IGF-1R pathway and / or high IGF-1R expression, which is unfortunately often correlated with poor outcomes. A particular conjugate known as LX-101 was well -tolerated and demonstrated single agent activity in previous Phase 1 trials of adult patients with advanced, pretreated cancers. However, there is a need for more studies involving specific pediatric cancers that lead to improved treatments for these vulnerable patients. Disclosed herein are studies that show a conjugate has potent anti-tumor activity against a variety of pediatric cancer cell lines related to IGF-1R, further supporting the clinical development of the methods described herein to address the unmet needs in treating pediatric cancers.

[0030] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented herein. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subj ect matter described herein covers 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 this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event that one or more of theincorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.I. Definitions

[0031] As used herein, a patient or subject and the like is any mammal suffering from an IGF-1R- related pediatric cancer. As used herein, the term “mammal” includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs, and sheep.

[0032] In certain embodiments, the patient or subject is a “pediatric patient” or “pediatric subject” (i.e., a human patient about or under the age of 21 years at the time of diagnosis or treatment). The term “pediatric” can be further divided into various subpopulations including: neonates (from birth through the first 28 days of life); infants (29 days of age to less than two years of age); children (two years of age to less than 12 years of age); and adolescents (12 years of age through 21 years of age (up to, but not including, the twenty-second birthday)).

[0033] As used herein, the term “conjugate” refers to a molecule comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent.

[0034] As used herein, the term “insulin-like growth factor 1 receptor (IGF-lR)-related pediatric cancer” refers to a cancer that is afflicting a subject, has an etiology that involves overexpression of IGF-1R or in which genetic alterations implicate the IGF-1R signaling pathway, such as IGF- 1R pathway activation, and is predominantly, although not always, found in pediatric subjects. Such cancers predominantly afflicting pediatric subjects are known to those of skill in the field.

[0035] As used herein, the term “cytotoxic agent” refers to any agent capable of preventing, delaying, reducing and / or reversing the activity, severity, and / or progression of the disease when treated in accordance with the methods described herein. Any suitable cytotoxic agent that results in cell killing can be used in the conjugate and in the method of treating an IGF-lR-related pediatric cancer.

[0036] As used herein, the term “residue” or “residue of’ a chemical moiety or compound refers to a chemical moiety or compound that is bound to a molecule, whereby through the binding, at least one covalent bond has replaced at least one atom of the original chemical moiety or compound, resulting in a residue of the chemical moiety or compound in the molecule.

[0037] As used herein, a subject is “refractory” to prior treatment if the subject has failed to achieve a response to a therapy such that the therapy is determined to not be therapeuticallyeffective, such as: failure to reach clinical endpoint, including any of response, extended duration of response, extended disease-free survival, relapse-free survival, and progression-free survival.

[0038] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0039] As used herein, the term “about,” when referring to a measurable value such as an amount of a compound or agent of the current subject matter, dose, time, temperature, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0040] As used herein, conditional language used herein, such as, among others, “can,” “could,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and / or steps. Thus, such conditional language is not generally intended to imply that features, elements and / or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and / or steps are included or are to be performed in any particular embodiment. The terms “comprising,” “including,” “having,” and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Also, the term “or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term “or” means one, some, or all of the elements in the list.

[0041] Definitions of additional terms may be set forth below.II. Methods of Treatment

[0042] In certain embodiments, the subject matter described herein is directed to a method for treating an insulin-like growth factor 1 receptor (IGF-lR)-related pediatric cancer in a subject, said method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent.

[0043] In certain embodiments, the subject is a pediatric subject under about 21 years of age. In certain embodiments, the patient is from birth through the first 28 days of life, from 29 days of age to less than two years of age, from two years of age to less than 12 years of age, or 12 years of ageto 21 years of age (up to, but not including, the twenty-first birthday). In certain embodiments, the patient is from birth through the first 28 days of life, from 29 days of age to less than 1 year of age, from one month of age to less than four months of age, from three months of age to less than seven months of age, from six months of age to less than 1 year of age, from 1 year of age to less than 2 years of age, from 2 years of age to less than 3 years of age, from 2 years of age to less than seven years of age, from 3 years of age to less than 5 years of age, from 5 years of age to less than 10 years of age, from 6 years of age to less than 13 years of age, from 10 years of age to less than 15 years of age, or from 15 years of age to less than 21 years of age.

[0044] In certain embodiments, the methods treat a subject in need thereof, which can further include performing a morphological diagnosis prior to administering the conjugate. The methods can further include performing molecular testing prior to administering the conjugate. In certain embodiments, the method includes performing morphological diagnosis and molecular testing prior to administering the conjugate.

[0045] In certain embodiments, IGF-1R is overexpressed in tumor cells of the IGF-lR-related pediatric cancer. In certain embodiments, the IGF-1R is overexpressed on tumor cells relative to non-tumor cells. In other embodiments, the overexpression of IGF-1R on tumor cells of said IGF- lR-related pediatric cancer confers poor prognosis. In certain embodiments, the overexpression of IGF-1R on tumor cells is measured by flow cytometry or immunohistochemistry.

[0046] In certain embodiments, the IGF-lR-related pediatric cancer has one or more genetic alterations that activate the IGF-1R signaling pathway. In certain embodiments, the genetic alteration is a mutation, gene fusion, gene amplification, or translocation.

[0047] In certain embodiments, the IGF-lR-related pediatric cancer is selected from the group consisting of Ewing’s sarcoma, rhabdomyosarcoma, synovial sarcoma, neuroblastoma, osteosarcoma, Wilms’ tumor, Beckwith Wiedemann Syndrome associated tumors, desmoplastic small round cell tumor and adrenocortical carcinoma. In certain aspects, the cancer is selected from the group consisting of Ewing’s sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma and neuroblastoma. In certain aspects, the cancer is selected from the group consisting of Ewing’s sarcoma, rhabdomyosarcoma, osteosarcoma, and neuroblastoma.

[0048] In certain embodiments, the IGF-lR-related pediatric cancer belongs to the Ewing’s Family of Tumors, which includes Ewing’s sarcoma, extraosseous Ewing tumor, and primitive neuroectodermal tumor. Ewing's sarcoma is an aggressive bone and soft tissue cancer, oftencharacterized by the presence of a genetic fusion between the Ewing’s sarcoma breakpoint region 1 (EWSR1) and a gene encoding a member of the E-twenty-six family of transcription factors. The most common fusion is EWSR1-FLI1 (Friend leukemia virus integration 1), which accounts for between 85-90% of ES. Ewing’s sarcoma accounts for 2% of all childhood malignancies, totaling around 400-500 new patients per year in the United States and a prevalent pool of about 4,000 patients. Although about a quarter of these patients present with metastatic disease at diagnosis, local treatments have a high rate of relapse, which has led researchers to believe that the majority of patients have some form of micrometastases at diagnosis. There are no approved therapies for Ewing’s sarcoma. The most common treatment options are chemotherapy (most often alternating regimens of [1] vincristine, doxorubicin, and cyclophosphamide and [2] ifosfamide and etoposide) and surgery, when possible. While outcomes can be positive in patients with local disease, an estimated 35% of patients with local disease end up relapsing after response on treatment. Recurrent and / or metastatic disease has a 5-year survival rate of 10-15% and represents around 40-50% of patients. These patients have very limited treatment options and represent a serious unmet medical need.

[0049] In certain embodiments, the IGF-lR-related pediatric cancer is rhabdomyosarcoma. Rhabdomyosarcoma is a pediatric soft tissue sarcoma with two major subtypes: embryonal rhabdomyosarcoma, which accounts for -60% of rhabdomyosarcoma and is primarily a malignancy of early childhood, and alveolar rhabdomyosarcoma, which accounts for -30% of rhabdomyosarcoma and is primarily a malignancy affecting patients aged 10-20 years. Approximately 700 new cases of rhabdomyosarcoma are diagnosed every year in the United States. There are no approved targeted therapies for rhabdomyosarcoma. Chemotherapy regimens (usually vincristine sulfate, actinomycin-D, and cyclophosphamide, or VAC) are often used, with initial response rates between 20 and 40%. However, relapse rates are high - greater than 70%. For patients presenting with local disease, standard of care is a combination of surgery, chemotherapy, and radiation therapy. These modalities see response rates between 70-85%, but a third of patients relapse after initial response, and treatment outcomes mirror that of the metastatic population. Notably, patients with the above-mentioned genetic alterations / fusions generally do worse with standard therapies than those without fusion positive disease. At least 15-20% of patients are metastatic at diagnosis, which is a setting where treatment options are lacking and largely ineffective. Metastatic and relapsed / refractory disease represents a major unmet medical need,with very limited treatment options, and makes up for about 25-30% of the rhabdomyosarcoma population.

[0050] In certain embodiments, the IGF-lR-related pediatric cancer is synovial sarcoma. Synovial sarcoma is a soft tissue sarcoma originating from primitive mesenchymal cells and generally arises in the lower extremities. Over 95% of synovial sarcoma cases are characterized by the gene fusion SS18-SSX. In the U.S., 1,000 patients are diagnosed with synovial sarcoma per year, with -40% of those cases being pediatric patients. Although 5-year survival rates are between 60 and 75%, responses on treatment are scarce, and late metastases are common, as -60% of patients end up with metastatic disease. There is no standard of care for synovial sarcoma, and treatment options are limited, with surgery (sometimes amputation), chemotherapy, and radiation being the leading options. While no treatments have been approved specifically for synovial sarcoma, many therapies have been approved under the umbrella of soft tissue sarcomas. Most of these approvals have been based on response rates <20% and median PFS <5 months.

[0051] In certain embodiments, the IGF-lR-related pediatric cancer is neuroblastoma. Approximately 60% of the -750 patients diagnosed with neuroblastoma every year in the United States present with high-risk disease, where survival is less than 50%, compared to low and intermediate risk disease, where survival is close to 90%. Current treatment for high-risk patients consists of chemotherapy, surgery (when possible), radiation therapy, stem cell transplant therapy, and dinutuximab (directed to the GD-2 disialoganglioside) combined with GM-CSF and isotretinoin. Response rates on these treatments are -30%, with 3-year event free survival rates of -45%. Most recently in November 2020, naxitamab, another anti-GD-2 antibody, received accelerated approval in combination with GM-CSF for relapsed / refractory high-risk neuroblastoma following two single-arm studies of 22 and 38 patients. The approval was based on response rates of 45% and 34%, with 30% and 23% of responders, respectively, having durations of response of at least 6 months.

[0052] In certain embodiments, the IGF-lR-related pediatric cancer is osteosarcoma. Osteosarcoma is the most common malignant bone tumor in children and adolescents. There are about 1,000 patients diagnosed with osteosarcoma per year. Metastatic patients have particularly poor outcomes and account for up to 30% of diagnoses. Only 20-30% of patients with metastatic disease have durable responses to first-line treatment with chemotherapy and surgery.

[0053] In some embodiments, the IGF-lR-involved cancer is desmoplastic small round cell tumor. Desmoplastic small round cell tumors are extremely aggressive sarcomas driven by a genetic translocation resulting in the gene fusion EWS-WT1. Approximately 100-150 patients are diagnosed with desmoplastic small round cell tumors in the US each year. While most patients are adolescents and young adults, the disease occurs at all ages. Treatment outcomes for these patients are dismal with 5-year survival rates reported between 5 and 18%, and 3-year event free survival reported between 7 and 10%. The most commonly used treatment is intense chemotherapy combined with radiation, which yields responses in 50% of patients that are ultimately not durable, as almost all patients relapse. 50% of patients present with metastatic disease, representing a population with an even worse prognosis.

[0054] In certain embodiments, the IGF-lR-related pediatric cancer is adrenocortical carcinoma. Adrenocortical carcinoma is a cancer that arises from the cortex, or the outer layer, of the adrenal gland. Approximately 400-600 patients are diagnosed with adrenocortical carcinoma every year, of which 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 (published response rates are as low as 7%) and is associated with significant toxi cities that limit its use. The only curative treatment is surgery, but for most patients this is not an option. In patients that are eligible for surgery, up to 70% of patients will eventually relapse even after successful surgery. In the relapsed / refractory disease setting, combination chemotherapy consisting of gemcitabine and metronomic capecitabine is often used. In a study of 145 adrenocortical carcinoma patients, this treatment yielded a 4.9% response rate and a median PFS of 3 months. Approximately 30-50% of patients present with metastatic disease, where treatment options are extremely limited and 5-year survival rates are less than 20%. Despite the availability of an approved treatment, treatment options for adrenocortical carcinoma remain limited.

[0055] In the embodiments described herein, the conjugate can comprise a chemical conjugate in which the IGF-1R ligand and the cytotoxic agent are chemically linked together, either directly or through a chemical linker. In other embodiments, the conjugate is a genetic recombinant in which the conjugate is expressed as a single polypeptide. When the conjugate is a recombinant conjugate, the translated conjugate preferably comprises a toxin, or portion or variant thereof, linked via apeptide bond to the TGF-1R ligand. In certain 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.

[0056] Methods for producing the conjugates described herein are known in the art. The nucleotide sequences encoding the IGF- 1R ligands can be produced by standard recombinant DNA techniques or by protein synthetic techniques, cloned into an appropriate expression vector using standard molecular biology techniques, expressed in bacterial, insect, or mammalian cells, and purified by any method known in the art for purification of a protein. Conjugates described herein comprising an IGF-1R ligand and a chemotherapeutic agent can be made by standard chemistry and protein conjugation techniques and are described in U.S. Patent No. 7,811,982; U.S. Patent No. 9,675,671; and U.S. Patent No. 9,801,923, each of which is incorporated by reference in its entirety. Conjugates described herein comprising an IGF-1R ligand and a toxin can be made as fusion proteins by standard recombinant DNA techniques and are described in U.S. Patent No. 8,017,102, which is hereby incorporated by reference in its entirety.

[0057] The IGF-1R is a heterotetramer consisting of two extracellular ligand-binding a subunits and two transmembrane b subunits with kinase activity that mediate signal transduction. The native ligands to the IGF-1R are IGF-1, IGF-2, and insulin. The IGF-1R has the highest affinity for IGF- 1, followed by IGF-2, and can bind to insulin with 50- to 100-fold lower affinity. IGF-1R can also form hybrid receptors by dimerization with the insulin receptor. See Hakuno et al. J Mol Endocrinol. 61(1):T69-T86 (2018).

[0058] In certain embodiments, the IGF-1R ligand in the conjugate comprises wildtype IGF-1 (SEQ ID NO:3), wildtype insulin (SEQ ID NOTO and SEQ ID NO:11; mature insulin consists of two chains connected by disulfide bonds, chain A, corresponding to SEQ ID NO: 10, and chain B, corresponding to SEQ ID NO: 11, hence the recitation of two SEQ ID NOs), or wildtype IGF-2 (SEQ ID NO: 12). In other embodiments, the IGF-1R ligand in the conjugate comprises a variant of wildtype IGF-1 (SEQ ID NOT), a variant of wildtype insulin (SEQ ID NO: 10 and SEQ ID NO:11), or a variant of wildtype IGF-2 (SEQ ID NO: 12). In a specific embodiment, the variant of wildtype IGF-1 is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to IGF-1 (SEQ ID NOT), said variant of wildtype insulin is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to insulin (SEQ ID NOTO and SEQ ID NO: 11), or said variant of wildtype IGF-2 is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to IGF-2 (SEQ ID NO: 12).

[0059] In certain embodiments, the IGF-1R ligand in the conjugate comprises a variant of IGF-1 that has reduced binding affinity for IGFBPs as compared to wildtype IGF-1 (SEQ ID NO:3) or a variant of IGF-2 that has reduced binding affinity for IGFBPs as compared to wildtype 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 IGFs in circulation, increasing their half-life, regulating their bioavailability, and generally inhibit their ability to bind to the IGF receptors. See Baxter, Am J Physiol Endocrinol Metab., 278(6):E967-76 (2000) and Allard et al. Front Endocrinol (Lausanne). 9;9: 117 (2018). Thus, variants of IGF-1 or IGF-2 that have reduced binding to IGFBPs have greater bioactivity in vivo.

[0060] IGF-1 variants with reduced binding affinity for IGFBPs are known in the art and include IGF132 (disclosed in U.S. Patent No. 4,876,242), in which the first 17 amino acids of the B chain of insulin (SEQ ID NO: 11) replace the first 16 amino acids of human IGF-1 (SEQ ID NO:3); R3- IGF-1 (SEQ ID NO:6), in which glutamic acid in position 3 of the native human IGF-1 (SEQ ID NO:3) is substituted by arginine; and des(l-3)IGF-l (SEQ ID NO:7), which lacks the first three amino acids of human IGF-1 (SEQ ID NO:3). R3-IGF-1 and des(l-3)IGF-l are described in Francis et al., J Mol Endocrinol. 8(3):213-23 (1992). In certain embodiments, the conjugate comprises IGF132 (SEQ ID NO:4), R3-IGF-1 (SEQ ID NO:6), or des(l-3)-IGF-l (SEQ ID NO:7).

[0061] In certain embodiments, the variant of IGF-1 has higher affinity for the IGF-1R than wildtype IGF-1 (SEQ ID NO:3), or the variant of IGF-2 has higher affinity for the IGF-1R than wildtype IGF-2 (SEQ ID NO: 12).

[0062] In certain embodiments, the IGF-1R ligand in the conjugate comprises 765IGF (SEQ ID NO:2), long-R3-IGF-l (SEQ ID NO:5), long-IGF-1 (SEQ ID NO:8), or long-G3-IGF-l (SEQ ID NO:9). 765IGF, long-R3 -IGF-1, long-IGF-1, and long-G3-IGF-l have N-terminal leader sequences that facilitate protein purification and provide sites for conjugation of cytotoxic agents, as described above. 765IGF (SEQ ID NO:2) comprises SEQ ID NO: 1 followed by R3-IGF-1 (SEQ ID NO:6); long-R3-IGF-l (SEQ ID NO:5) comprises the first 11 amino acids of methionyl porcine growth hormone, followed by a Val-Asn dipeptide, followed by R3-IGF-1 (SEQ ID NO:6); long- IGF-1 (SEQ ID NO:8) comprises the first 11 amino acids of methionyl porcine growth hormone, followed by a Val-Asn dipeptide, followed by human IGF-1 (SEQ ID NO:3); and long-G3-IGF-l comprises the first 11 amino acids of methionyl porcine growth hormone, followed by a Val-Asndipeptide, followed by a variant of human IGF-1 in which glutamic acid in position 3 of the native human IGF-1 (SEQ ID NO:3) is substituted by glycine.

[0063] In certain embodiments, the IGF-1R ligand comprises wildtype insulin-like growth factor 1, wildtype insulin, or wildtype insulin-like growth factor 2 (IGF-2). In certain aspects, the wildtype insulin-like growth factor 1 (IGF-1) comprises SEQ ID NO:3, wherein said wildtype insulin comprises SEQ ID NO: 10 or 11, and wherein said wildtype insulin-like growth factor 2 (IGF-2) comprises SEQ ID NO: 12.

[0064] In certain embodiments, the IGF- 1R ligand comprises a variant of wildtype IGF-1, a variant of wildtype insulin, or a variant of wildtype IGF-2. In certain aspects, the variant of wildtype IGF- 1 is at least 90% identical to SEQ ID NO:3, said variant of wildtype insulin is at least 90% identical to SEQ ID NO: 10 or 11, and said variant of wildtype IGF-2 is at least 90% identical to SEQ ID NO: 12.12. In certain aspects: (i) the variant of wildtype IGF-1 has reduced binding affinity for insulin-like growth factor binding proteins (IGFBPs) as compared to wildtype IGF-1, or the variant of wildtype IGF-2 has reduced binding affinity for IGFBPs as compared to wildtype IGF-2, and / or (ii) the variant of wildtype IGF-1 has increased affinity for the IGF-1R as compared to wildtype IGF-1, or the variant of wildtype IGF-2 has increased affinity for the IGF-1R as compared to wildtype IGF-2.

[0065] In certain embodiments, the IGF-1R ligand, or portion or variant thereof, comprises a leader sequence. In certain aspects, the leader sequence comprises SEQ ID NO: 1.

[0066] In certain embodiments, the IGF-1R ligand comprises 765IGF (SEQ ID NO:2), IGF-132 (SEQ ID NO 4), long-R3-IGF-l (SEQ ID NO:5), R3-IGF-1 (SEQ ID NO:6), des(l-3)-IGF-l (SEQ ID NO:7), long-IGF-1 (SEQ ID NO:8), or long-G3-IGF-l (SEQ ID NO:9).

[0067] In certain embodiments, the IGF-1R ligand comprises 765IGF (SEQ ID NO:2).

[0068] In certain embodiments, the IGF-1R ligand, or portion or variant thereof, is covalently bound to the cytotoxic agent.

[0069] In certain embodiments, the cytotoxic agent comprises a chemotherapeutic agent. In certain aspects, the chemotherapeutic agent is amsacrine, azacytidine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine, hexamethylmelamine, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine,methotrexate, mitomycin C, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, plicamycin, procarbazine, ralitrexed, semustine, streptozocin, temozolamide, teniposide, thioguanine, thiotepa, topotecan, trimitrexate, valrubicin, vincristine, vinblastine, vindestine, or vinorelbine. In certain aspects, the chemotherapeutic agent is methotrexate. In certain aspects, the chemotherapeutic agent is covalently bound to a lysine residue in the leader sequence. In certain aspects, the chemotherapeutic agent is one or more methotrexate residues covalently bound to any available lysine residue in the leader sequence.

[0070] In certain embodiments, a conjugate comprises more than one cytotoxic agent bound to the IGF-1R ligand. In certain aspects, the conjugate can comprise one to 12 cytotoxic agents, or 6 to 10 cytotoxic agents, or about 8 cytotoxic agents. In certain aspects, the conjugate can comprise one to twelve covalently bound cytotoxic agents, or 6 to 10 covalently bound cytotoxic agents, or about 8 covalently bound cytotoxic agents. In certain aspects, the chemotherapeutic agent(s) are covalently bound to any available position on the IGF-1R ligand. In certain aspects, the chemotherapeutic agent(s) are covalently bound to any available lysine residue. In certain aspects, the chemotherapeutic agent(s) are covalently bound to any available lysine in the leader sequence when present.

[0071] Leader sequences can incorporate tags, such as polyhistidine tags, to facilitate protein purification, as well as provide sites for conjugation of cytotoxic agents. In a specific embodiment, the leader sequence comprises SEQ ID NO: 1.

[0072] In certain embodiments, the cytotoxic agent comprises a toxin. In certain aspects, the toxin comprises Clostridium perfringens enterotoxin, diphtheria toxin, ricin chain A, Pseudomonas exotoxin, A chain toxins, a ribosome inactivating protein, a-sarcin, aspergillin, or a ribonuclease. In certain aspects, the toxin comprises Clostridium perfringens enterotoxin, or a portion or variant thereof. In certain aspects, the toxin comprises SEQ ID NO: 14 or SEQ ID NO: 15. In certain aspects, the toxin comprises diphtheria toxin, or a portion or variant thereof. In certain aspects, the toxin comprises SEQ ID NO: 13 or SEQ ID NO: 16.

[0073] In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering to the subject a conjugate comprising an IGF-1R ligand, or portion or variant thereof, and a cytotoxic agent, wherein the IGF-1R ligand, or portion or variant thereof comprises SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein the methotrexate is covalently bound to a lysine of SEQID N0:2, and the IGF-lR-related pediatric cancer is selected from the group consisting of Ewing’s sarcoma, adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, synovial sarcoma and neuroblastoma. In a certain 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 up to 10, or at least 6 and up to 9, or at least 7 and up to 9, or at least 8 and up to 9 methotrexate are present for every IGF-1R ligand. A methotrexate can be covalently bound to the IGF-1R ligand, in particular, a lysine residue of SEQ ID NO:2. The average number of methotrexate residues per SEQ ID NO:2 is 8). In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering LX-101 to the subject, wherein the IGF-lR-related pediatric cancer is Ewing’ s sarcoma. In certain embodiments, the subj ect matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering LX-101 to the subject, wherein the IGF-lR-related pediatric cancer is adrenocortical carcinoma. In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering LX- 101 to the subject, wherein the IGF-lR-related pediatric cancer is rhabdomyosarcoma. In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR- related pediatric cancer in a subject, the method comprising administering LX-101 to the subject, wherein the IGF-lR-related pediatric cancer is osteosarcoma. In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering LX-101 to the subject, wherein the IGF-lR-related pediatric cancer is synovial sarcoma. In certain embodiments, the subject matter described herein is directed to methods for treating an IGF-lR-related pediatric cancer in a subject, the method comprising administering LX-101 to the subject, wherein the IGF-lR-related pediatric cancer is neuroblastoma. In all embodiments, the number of methotrexate residues per conjugate is 6, 7, 8, 9 or 10. In all embodiments, the average number of methotrexate residues per conjugate in a composition is 6, 7, 8, 9 or 10.

[0074] In certain embodiments, the subject (i) has not previously received treatment for the IGF- lR-related pediatric cancer; (ii) has previously received treatment for the IGF-lR-related pediatric cancer; (iii) has relapsed from previous treatment for the IGF-lR-related pediatric cancer; (iv) was refractory to previous treatment for the IGF-lR-related pediatric cancer; or (v) is susceptible toadverse reactions from other treatments for the IGF-lR-related pediatric cancer. Thus, in certain embodiments, the IGF-lR-related pediatric cancer is recurrent where the patient has relapsed after a previous treatment. In certain embodiments, the IGF-lR-related pediatric cancer is Stage II, III or IV. In certain embodiments, the IGF-lR-related pediatric cancer has progressed to Stage II, III or IV during or after an initial therapy.

[0075] In certain embodiments, the treatment results in a reduction in the growth of tumor cells in the subject. In certain embodiments, the reduction is caused by killing of IGF-lR-expressing tumor cells.

[0076] Table 1 provides a listing of sequences referenced herein.Table 1.

[0077] In certain embodiments, the methods described herein are part of a combination therapy. In particular, the methods described herein can be used either alone or in combination with standard of care treatment options for each type of pediatric cancer. In general, the standard of care options include surgery, systemic chemotherapy (either pre- or post-operatively) and / or radiation therapy.

[0078] In certain embodiments, the subject having an IGF-lR-related pediatric cancer treated in accordance with the methods described herein has not previously received treatment for the IGF- lR-related pediatric cancer.

[0079] In certain embodiments, the subject having an IGF-lR-related pediatric cancer treated in accordance with the methods described herein has previously received treatment for the IGF-lR- related pediatric cancer.

[0080] In certain embodiments, the subject has relapsed from previous treatment for the IGF-lR- related pediatric cancer.

[0081] In certain embodiments, the subject was refractory to previous treatment for the IGF-lR- related pediatric cancer.

[0082] In all embodiments, the conjugate is administered at a dose and frequency that is appropriate for the subject and the IGF-lR-related pediatric cancer being treated at the discretion of the practitioner.

[0083] In certain aspects, the conjugate is administered at a dose of about 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 pEq / kg of body weight or at a dose range of about 0.05-10.0, 0.1-8.0, 0.2-4.0, 0.3-3, 0.4-2.5, 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 pEq / kg of body weight. In certain aspects, the conjugate is administered at about 0.05, 0.10, 0.20, 0.40, 0.80, 1.6, or 2.5 pEq / kg of body weight. A pEq is equivalent to a pmol of chemotherapeutic agent groups conjugated to the IGF-1R ligand. In certain aspects, the conjugate is administered at a dose of about 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, 1.3, 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, 4.5, 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, 7.7, 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, 1 1.1 , 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, 14 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 nig / kg of body weight or at a dose range of about 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, 10.5-11.0, 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 of body weight, where a mg refers to the amount of IGF-1R ligand present in the conjugate.

[0084] In certain embodiments, the conjugate is administered daily, every other day, every three days, every four days, every five days, every six days, once per week, once every two weeks, once every three weeks, once every four weeks, once per month, every two months, or every three months.

[0085] In certain embodiments, the conjugate can be administered at a lower dose and / or frequency at which the cytotoxic agent would have an effect when dosed as a single agent.

[0086] In certain embodiments, the dose and / or frequency can be escalated in a pediatric patient in particular. In this aspect, doses of up to 0.80 pEq / kg of body weight are given more than once per week. In this aspect, doses of up to 0.80 pEq / kg of body weight are given at least once per week. In this aspect, doses of greater than 1.6 pEq / kg of body weight are given at least once per week.

[0087] In some embodiments, the conjugate is dosed at the maximum tolerated dose (MTD). “MTD,” as used herein, refers to the highest dose of an agent that an individual patient can tolerate as determined by the practitioner. In other words, side effects in a given patient can determine the MTD.

[0088] In certain embodiments, the methods do not cause appreciable or unacceptable hyperglycemia in the subject. In certain aspects, the methods do not cause unacceptable hyperglycemia in the subject. Hyperglycemia is another term for high blood glucose and can occur when there is insufficient insulin in the body or when the body cannot utilize insulin properly. Unacceptable hyperglycemia refers to an adverse effect of grade 3 or higher, as determined by a treating physician, and / or one that cannot be controlled with diabetic medications and leads to discontinuation of treatment with the conjugate.

[0089] 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 a conjugate and a pharmaceutically acceptable carrier or vehicle. Such pharmaceutical compositions can be formulated to be suitable for administration to a subject and can be in any form that allows for the composition to be administered to a subject.

[0090] Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the overall health of the subject, the type of condition the subject has, the use of the composition as part of a multi-drug regimen, the particular form of the composition, and the manner of administration. The pharmaceutical compositions comprise an effective amount of a composition such that a suitable dosage will be obtained.

[0091] The term “carrier” refers to a diluent, adjuvant or excipient, with which a composition comprising a conjugate is administered. Any auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a subject, the compositions and pharmaceutically acceptable carriers are sterile. Water may be a carrier when the composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The present compositions, if desired, can also contain minor amounts of pH buffering agents.

[0092] The liquid pharmaceutical compositions, whether they are solutions, suspensions, or other like form, can 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 mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; agents for the adjustment of pH such as hydrochloric acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in an ampoule, a disposable syringe, or a multiple-dose vial made of glass, plastic or othermaterial. In some embodiments, physiological saline is an adjuvant. An injectable composition may be sterile.

[0093] The present compositions can take the form of solutions, suspensions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Examples of suitable pharmaceutical carriers are described in Remington ’s Pharmaceutical Sciences by E.W. Martin.

[0094] In some embodiments, the compositions are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human subjects. Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where a composition is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0095] The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a composition with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are complexes that can non-covalently interact with a composition so as to facilitate dissolution or homogeneous suspension of the composition in the aqueous delivery system.

[0096] The conjugates described herein can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, e.g., microparticles, microcapsules, capsules, etc., and may be useful for administering a composition that comprises the conjugate. Methods of administration may include, but are not limited to, oral administration and parenteral administration; parenteral administration including, but not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin. The mode of administration is left to the discretion of the practitioner, and will depend, in-part, upon the site of the medical condition. In certain embodiments, the conjugate is administered intravenously, subcutaneously, or intramuscularly.

[0097] In certain embodiments, the conjugates are administered parenterally. In certain embodiments, the conjugates are administered intravenously. In certain embodiments, the conjugates are administered by continuous infusion. In certain embodiments, the conjugates are administered by an infusion that lasts for 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours.

[0098] In certain embodiments, it can be desirable to administer conjugates locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application, e.g., in conjunction with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In certain embodiments, conjugates can be injected intraperitoneally.

[0099] In certain embodiments, the conjugates can be delivered in a controlled release system.

[0100] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1 - IGF-IR Expression AssaysTable 2. Abbreviations used in this study

[0101] The expression of IGF-1R was determined for the cell lines shown in Table 3 via FACS.The MCF7 cell line was used as positive control.Table 3. Cell lines

[0102] First, 1 million cells were resuspended in 100 pL PBS with 2.5pg of human Fc-Block (BD Biosciences) and incubated at RT for 10 minutes in the dark to block. For staining, a PE CD221 (IGF-1R) antibody (clone 1H7, BD Biosciences) and an eFluor780 live / dead dye (eBiosciences) were added to each sample and incubated for 30 minutes at 4 °C in the dark. A PE Mouse IgGl, K isotype control (BD Bioscience) was used to set the isotype control for each cell line. Then, 2 mb of PBS was added to each sample, the cells were gently resuspended and centrifuged at 500 x for 5 minutes and the supernatant was discarded. The cells were washed two more times in PBS, then resuspended in 200 pL of IC Fixation Buffer (eBioscience) and incubated at RT for 30 minutes in the dark. The cells were washed with 2mL of PBS and resuspended in 250 pL PBS for acquisition. Data was acquired with a CytoFLEX S flow cytometer (Beckman Coulter) and analyzed using Kaluza 2.0 (Beckman Coulter).

[0103] The resulting data are depicted in Figures 1-7. The greater fluorescence intensity of cells stained with the PE CD221 antibody as compared to cells stained with the isotype control indicate the cell lines A-673 (Fig. IB), CADO-ES1 (Fig. ID) RD-ES (Fig. 2B), SK-ES-1 (Fig. 2D), SJCRH30 (Fig. 3B), 143B (Fig. 4B), HOS (Fig. 4D), Saos-2 (Fig. 5B), U-2 OS (Fig. 5D), IMR32 (Fig. 6B), and SK-N-AS (Fig. 6D) express IGF-1R on their surface. The cell line SH-SY5Y (Fig. 7B) did not show a significant elevation in fluorescent intensity for IGF-1R staining as compared to the isotype control. Thus, SH-SY5Y cells were not found to express IGF-1R at the level of the other cell lines tested.Example 2 - Cytotoxicity Assays in Specific Types of Pediatric Cancer Cells

[0104] LX-101-induced cytotoxicity was evaluated on specific types of pediatric cancer cells. The cell lines in Table 6 were treated with LX-101 at 9 concentrations using 2.5-fold dilutions and cell viability was assessed.Table 5. Abbreviations used in this protocol

[0105] Approximately 7 days before treatment, cryovials of the frozen cells were transferred to a water bath on dry ice to thaw. The contents of each cryovial were slowly transferred to a 15 mL tube containing 10 mL of the culture medium indicated in Table 6. The cells were centrifuged at 125 *g for 5 minutes at RT. The cell pellet was resuspended in pre-warmed culture media in a T- 25 or T-75 flask and incubated at 37°C with 5% CO2 (except cell lines SW-13 and SW-982 which were cultured at 37°C and 100% air). The cells were subcultured using TrypLE at a subcultivation ratio of 1 :3 to 1 :6 when the cell culture reached approximately 80% confluence.Table 6. Cell lines

[0106] One day prior to LX-101 treatment, the cells were collected during logarithmic growth using TrypLE and counted. The number of cells indicated in Table 7 was seeded in lOOpL per well of a 96-well plate according to the plate layout shown below. The plates were incubated overnight at 37°C, 5% CO2 (except cell lines SW-13 and SW-982 which were cultured at 37°C and 100% air).Table 7. Cell SeedingPlate layout (one per cell line):E: Empty well containing or PBSV: Vehicle control (95 pM HCl)U: Untreated cells (cells + medium)C1-C9: Nine concentration levels of test article LX-101 drug substance lot LIR0023Compounds* 1 uEq =1 umole of methotrexate groups in the drug LX-101.

[0107] A 1 / 10 working stock solution of LX-101 was prepared in 1 mM HC1, such that the concentration of the working stock solution was 0.4 pEq / ml of drug in 1.9 mM HC1. Sterile Eppendorf tubes were prepared containing 3x the final well concentrations in the medium corresponding to each cell line (Table 6). The first dilution was prepared using the 1 / 10 working stock diluted in media. Subsequent 2.5-fold dilutions were prepared using media. A visual check was performed for signs of precipitation when the compound was diluted in media.

[0108] For the vehicle control, a 10 mM HC1 stock solution was prepared by diluting HC1 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 HC1. In an Eppendorf, 75 pL of the working stock solution was added to 425 pl media to make 3x final concentration of 285 pM HC1.

[0109] As indicated in Table 8, 50 pL of the prepared dilutions of LX-101 or HC1 was added to the appropriate wells, providing a total volume of 150 pL per well. Unused wells were filled with 150 pL of PBS.Table 8. LX-101 Titrations

[0110] Following treatment, the plates were incubated at 37°C, 5% CO2 (except cell lines SW-13 and SW-982 which were cultured at 37°C and 100% air). After four days, the plates were removed and equilibrated to RT for 30 minutes. A black sticker was placed on the bottom of the plates to block light. Then, 75 pL CellTiter-Glo 2.0 Reagent (Promega) was added to each well and the contents were mixed on an orbital shaker for 2 minutes. The plates were incubated for an additional10 minutes at RT to stabilize the luminescence signal. The luminescence was recorded with an Envision 2104 multi-label microplate reader (PerkinElmer) using an integration time of 0.25-1 second per well.

[0111] LX-101 concentrations based on IGF-1 variant protein content were derived by dividing drug concentrations based on methotrexate content by 8, the average number of methotrexate groups per IGF-1 protein as determined by MALDI-TOF. ICso values were calculated using GraphPad PRISM software.Viability (% of control) = (LumTest article-Lumblank control) / (Lumvehicle-Lumblank eontrol)xl00%.Results

[0112] The cytotoxicity of lot LIR00223 of LX-101 was tested on MCF7 breast cancer cells as a reference. As shown in Table 10, the average ICso of 3 independent experiments with lot LIR0023 was found to be 31 nM.Table 10. Cytotoxicity of LX-101 (lot LIR0023) on MCF7 breast cancer2Absolute IC50s based on LX101 derived by dividing the IC50s based on MTX content by the average number MTX groups conjugated per IGF-1 variant protein (i.e., 8), as determined by MALFLTOF

[0113] Following the procedure described above, cytotoxicity data were collected for LX-101 (lot LIR0023) with the following cell lines, as shown in Table 9. The resulting data are depicted in Figures 1-8. LX-101 had the greatest effect on the osteosarcoma cell lines 143B and HOS and the adrenocortical carcinoma cell line SW-13, with maximum inhibition values greater than 94% and absolute ICsos less than 10 nM (based on the concentration of IGF-1R ligand which includes approximately 8 covalently bound methotrexate molecules). Significant inhibition of four different Ewing’s sarcoma cell lines and three different neuroblastoma lines was observed following treatment with LX-101 resulting in absolute ICsos of less than 30 nM. Alveolar rhabdomyosarcomaline SJCRH30 and osteosarcoma line U2OS were also inhibited by LX-101 with absolute IC50 values of 23 nM and 32 nM, respectively.

[0114] One of the four osteosarcoma lines tested, Saos-2, was not significantly inhibited by LX- 101, despite detectable IGF-1R expression. Interestingly, one of the neuroblastoma cell lines, SH- SY5Y did not show high IGF-1R expression, but still was inhibited by LX-101 with an absolute ICso of 30 nM. These data suggest that the activity of LX-101 against cancer cells is not necessarily correlated to the level of IGF-1R expression on the cancer cell.Table 9. Cytotoxicity of LX-101 (lot LIR0023) on cancer cell lines’Absolute IC50s based on MTX content in LX-101, as measured by absorbance at 305 nm2Absolute IC50s based on LX101 derived by dividing the IC50s based on MTX content by the average number MTX groups conjugated per IGF-1 variant protein (i.e., 8), as determined by MALFI-TOF

[0115] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0116] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practicing the subj ect matter described herein. The present disclosure is in no way limited to just the methods and materials described.

[0117] Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs.

[0118] Throughout this specification and the claims, the words “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. It is understood that embodiments described herein include “consisting of’ and / or “consisting essentially of’ embodiments.

[0119] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of the range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these small ranges which may independently be included in the smaller rangers is also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0120] Many modifications and other embodiments set forth herein will come to mind to one skilled in the art to which this subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

WHAT IS CLAIMED IS:

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

2. The method of claim 1, wherein said subject is a pediatric subject from about 1 day to about 21 years of age.

3. The method of claim 1, wherein said IGF-lR-related pediatric cancer is selected from the group consisting of Ewing’s sarcoma, rhabdomyosarcoma, synovial sarcoma, neuroblastoma, osteosarcoma, Wilms’ tumor, Beckwith Wiedemann Syndrome associated tumors, desmoplastic small round cell tumor, and adrenocortical carcinoma.

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

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

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

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

8. The method of any one of claims 1-7, wherein said IGF-1R ligand comprises wildtype insulin-like growth factor 1, wildtype insulin, or wildtype insulin-like growth factor 2 (IGF-2).

9. The method of claim 8, wherein said wildtype insulin-like growth factor 1 (IGF-1) comprises SEQ ID NO:3, wherein said wildtype insulin comprises SEQ ID NO: 10 or 11, and wherein said wildtype insulin-like growth factor 2 (IGF-2) comprises SEQ ID NO: 12.

10. The method of any one of claims 1-7, wherein said IGF-1R ligand comprises a variant of wildtype IGF-1, a variant of wildtype insulin, or a variant of wildtype IGF-2.

11. The method of claim 10, wherein said variant of wildtype IGF-1 is at least 90% identical to SEQ ID NO:3, said variant of wildtype insulin is at least 90% identical to SEQ ID NO: 10 or11. and said variant of wildtype IGF-2 is at least 90% identical to SEQ ID NO: 12.

12. The method of claims 10 or 11, wherein: (i) said variant of wildtype IGF-1 has reduced binding affinity for insulin-like growth factor binding proteins (IGFBPs) as compared to wildtype IGF-1 , or said variant of wildtype IGF-2 has reduced binding affinity for IGFBPs as compared to wildtype IGF-2 , and / or (ii) said variant of wildtype IGF-1 has increased affinity for the IGF-1R as compared to wildtype IGF-1 , or said variant of wildtype IGF-2 has increased affinity for the IGF-1R as compared to wildtype IGF-2.

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

14. The method of claim 13, wherein said leader sequence comprises SEQ ID NO:1.

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

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

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

18. The method of any one of claims 1-17, wherein said cytotoxic agent comprises a chemotherapeutic agent.

19. The method of claim 18, wherein said chemotherapeutic agent is amsacrine, azacytidine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decarbazine, docetaxel,doxorubicin, epirubicin, estramustine, etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine, hexamethylmelamine, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin C, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, plicamycin, procarbazine, ralitrexed, semustine, streptozocin, temozolamide, teniposide, thioguanine, thiotepa, topotecan, trimitrexate, valrubicin, vincristine, vinblastine, vindestine, or vinorelbine.

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

21. The method of any one of claims 1-17, wherein said cytotoxic agent comprises a toxin.

22. The method of claim 21, wherein said toxin comprises Clostridium perfringens enterotoxin, diphtheria toxin, ricin chain A, Pseudomonas exotoxin, A chain toxins, a ribosome inactivating protein, a-sarcin, aspergillin, or a ribonuclease.

23. The method of claim 22, wherein said toxin comprises Clostridium perfringens enterotoxin, or a portion or 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 said toxin comprises diphtheria toxin, or a portion or 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 1-26, wherein said subject (i) has not previously received treatment for said IGF-lR-related pediatric cancer; (ii) has previously received treatment for said IGF-lR-related pediatric cancer; (iii) has relapsed from previous treatment for said IGF-lR-related pediatric cancer; (iv) was refractory to previous treatment for said IGF-lR- related pediatric cancer; or (v) is susceptible to adverse reactions from other treatments for said IGF-lR-related pediatric cancer.

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

29. The method of claim 28, wherein said one or more other therapies comprises one or more of the following: surgery, systemic chemotherapy (either pre- or post-operatively) and radiation therapy30. The method of any one of claims 1-29, wherein said conjugate is administered at a dose of about 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 pEq / kg of body weight or at a dose range of about 0.05-10.0, 0.1-8.0, 0.2-4.0, 0.3-3, 0.4-2.5, 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 pEq / kg of body weight.

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

32. The method of any one of claims 15-20 and 27-31, wherein the IGF-1R ligand is SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein there are 6 to 10 methotrexate molecules for every IGF-1R ligand of SEQ ID NO:2, and the IGF-lR-related pediatric cancer is Ewing’s sarcoma.

33. The method of any one of claims 15-20 and 27-31, wherein the IGF-1R ligand is SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein there are 6 to 10 methotrexate molecules for every IGF-1R ligand of SEQ ID NO:2, and the IGF-lR-related pediatric cancer is adrenocortical carcinoma.

34. The method of any one of claims 15-20 and 27-31, wherein the IGF-1R ligand is SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein there are 6 to 10 methotrexate molecules for every IGF-1R ligand of SEQ ID NO:2, and the IGF-lR-related pediatric cancer is rhabdomyosarcoma.

35. The method of any one of claims 15-20 and 27-31, wherein the IGF-1R ligand is SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein there are 6 to 10 methotrexate molecules for every IGF-1R ligand of SEQ ID NO:2, and the IGF-lR-related pediatric cancer is osteosarcoma.

36. The method of any one of claims 15-20 and 27-31, wherein the IGF-1R ligand is SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein there are 6 to 10 methotrexate molecules for every IGF-1R ligand of SEQ ID NO:2, and the IGF-lR-related pediatric cancer is synovial sarcoma.

37. The method of any one of claims 15-20 and 27-31, wherein the IGF-1R ligand is SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein there are 6 to 10 methotrexate molecules for every IGF-1R ligand of SEQ ID NO:2, and the IGF-lR-related pediatric cancer is neuroblastoma.

38. The method of any one of claims 15-20 and 27-31, wherein the IGF-1R ligand is SEQ ID NO:2, the cytotoxic agent is methotrexate, wherein there are 6 to 10 methotrexate molecules for every IGF-1R ligand of SEQ ID NO:2, and the IGF-lR-related pediatric cancer is desmoplastic small round cell tumor.

39. The method of claim 1 or 2, wherein the IGF-lR-related pediatric cancer is Ewing’s sarcoma.

40. The method of claim 1 or 2, wherein the IGF-lR-related pediatric cancer is adrenocortical carcinoma.

41. The method of claim 1 or 2, wherein the IGF-lR-related pediatric cancer is rhabdomyosarcoma.

42. The method of claim 1 or 2, wherein the IGF-lR-related pediatric cancer is osteosarcoma.

43. The method of claim 1 or 2, wherein the IGF-lR-related pediatric cancer is synovial sarcoma.

44. The method of claim 1 or 2, wherein the IGF-lR-related pediatric cancer is neuroblastoma.

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