Compositions and methods for image-guided brachytherapy

Abstract

Disclosed herein are methods, pharmaceutical compositions, and kits for image-guided brachytherapy. An example method includes navigating a hollow tube to a tumor in a subject using an imaging modality and administering a pharmaceutical composition through the hollow tube to the tumor. An example pharmaceutical composition includes a pharmaceutically acceptable excipient and a collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, the collection of self-assembling conjugates being included in the pharmaceutical composition at a concentration of no more than 300 µM.

Description

Attorney Docket No.028193-0060-WO01 COMPOSITIONS AND METHODS FOR IMAGE-GUIDED BRACHYTHERAPY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 702,195 filed on October 2, 2024, which is incorporated fully herein by reference. TECHNICAL FIELD

[0002] This disclosure relates to methods for image-guided brachytherapy and compositions that can be used in the methods. INTRODUCTION

[0003] Pancreatic cancer is a leading cause of cancer deaths in the United States, with a 5- year survival rate of only 11%. The development of effective therapies for pancreatic cancer, as well as other non-superficial cancers, is hence an area of unmet need. SUMMARY

[0004] In one aspect, described herein are methods for image-guided brachytherapy in a subject, the method including: navigating a hollow tube to a tumor in the subject using an imaging modality, the hollow tube having an internal diameter of no less than 0.4 mm; and administering a pharmaceutical composition through the hollow tube to the tumor, the pharmaceutical composition including a pharmaceutically acceptable excipient and a collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and wherein the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM.

[0005] In another aspect, described herein are kits including: a hollow tube having an internal diameter of no less than 0.4 mm; and a pharmaceutical composition, the pharmaceutical composition including a pharmaceutically acceptable excipient, and a collection of self- assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of noAttorney Docket No.028193-0060-WO01 greater than 37 °C, and the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM.

[0006] In another aspect, described herein are pharmaceutical compositions including: a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient includes a buffer and a contrast-enhancing agent selected from the group consisting of a CT contrast-enhancing agent, a PET contrast-enhancing agent, and a SPECT contrast-enhancing agent, the contrast-enhancing agent being included at no more than 50% by volume of the pharmaceutical composition; and a collection of self-assembling conjugates, each self- assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0008] FIG.1A is a plot showing whole-body mouse131I-labeled elastin-like polypeptide (ELP) activity normalized to the day one activity over the eight days following injection.

[0009] FIG.1B are bar graphs showing activity distribution in the tumor and thyroid of mice reported as a fraction of whole-body activity measured immediately prior to sacrifice. Error bars: one standard error of the mean; ns: p>0.05 (ANOVA).

[0010] FIG.2 are computed tomography (CT) images showing an example trajectory of a needle used in percutaneous injection of example124I-labeled ELP into pancreatic tissue. A 22 ga Chiba needle was advanced under CT guidance through the skin in a 40 kg male Yorkshire pig. Left-to-right: three CT scans demonstrating the plane of the needle tip as it was advanced stepwise towards the pancreatic tail (circled on far-right image).

[0011] FIG.3 are positron emission tomography (PET) / CT co-registered images showing an example124I-labeled ELP brachytherapy depot retained within the pancreas over six days. Left- to-right: t=0 hrs, 3 hrs, and 6 days (144 hrs) co-registered PET / CT images demonstrating retention of ELP within the pancreas. No peripheral activity was observed in tissues surrounding pancreas.Attorney Docket No.028193-0060-WO01

[0012] FIG.4 is a co-registered PET / CT image demonstrating small124I activity within thyroid of pig six days (144 hrs) following injection.

[0013] FIG.5 shows a plot of iodinated contrast fluid and its effect on conjugate phase behavior. ELP-Tyr7was resuspended in either 1X PBS, Isovue-300 iodinated contrast fluid, or a 1:1 volume / volume (v / v) mixture of each (50% Isovue) and optical turbidity was measured at 350 nm using a Cary 3500 UV-vis spectrophotometer on a temperature ramp from 10 °C to 40 °C (solid lines), which then returned to 10 °C (dashed lines).

[0014] FIG.6A are plots showing concentrations of lipase (left) and amylase (right) for three Yorkshire pigs injected with single 1 mL124I-labelled ELP depots in the pancreas using an Enzyme-Linked Immunosorbent Assay (ELISA).

[0015] FIG.6B are plots showing concentrations of lipase (left) and amylase (right) for a mini-pig with a single 1 mL124I-labeled ELP depots in the pancreas using ELISA. An increase in levels over 3x from baseline (t = 0 h) indicates potential acute pancreatitis. DETAILED DESCRIPTION 1. Definitions

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in practice or testing of the disclosed technology. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0017] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0018] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should alsoAttorney Docket No.028193-0060-WO01 be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0019] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.

[0020] “Amino acid” as used herein refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.

[0021] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide”, “protein,” and “peptide” are used interchangeably herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. “Domains” are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids in length. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units. A “motif” is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length. In some embodiments, a motifAttorney Docket No.028193-0060-WO01 includes 3, 4, 5, 6, or 7 sequential amino acids. A domain may be comprised of a series of motifs, which may be the same or different.

[0022] The terms "effective amount" or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results to treat a disease (e.g., cancer) or one or more of its symptoms and / or to prevent or reduce the risk of the occurrence or reoccurrence of the disease or disorder or symptom(s) thereof. In reference to cancer an effective or therapeutically effective amount can include an amount sufficient to, among other things, inhibit tumor growth and / or metastasis.

[0023] “Radionuclide,” “radioactive nuclide,” “radioisotope,” or “radioactive isotope” are used interchangeably herein to represent any atom that has excess nuclear energy and is, therefore, unstable. The excess energy may be emitted as gamma, alpha, beta, or a combination thereof. The radionuclide may provide irradiation through beta-particles, alpha-particles or Auger electrons.

[0024] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0025] The terms “treatment” or “treating” refer to the medical management of a subject with the intent to heal, cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the cancer. 2. Methods of Image-Guided Brachytherapy

[0026] Disclosed herein are methods for image-guided brachytherapy using ELPs. Brachytherapy is a version of radiotherapy where the radioactive source is placed inside or in close proximity to the tumor to maximize local dose absorption. Here, the disclosed methods deliver radiolabeled ELP conjugates to a tumor with image guidance. The methods use a largerAttorney Docket No.028193-0060-WO01 diameter hollow tube that can be beneficial for larger subjects, such as humans and large animals, in combination with administering an advantageous formulation of the radiolabeled ELP conjugate through the hollow tube to the tumor (e.g., in the tumor or adjacent to the tumor). Upon deposition of the radiolabeled ELP conjugate formulation at the tumor, it can phase transition into a viscous depot that retains the radionuclide at the injected site. The viscous depot can then be used to treat the subject having the tumor, image the tumor in the subject, or both.

[0027] The method can include navigating a hollow tube to a tumor in the subject using an imaging modality. The hollow tube can be navigated to the tumor by imaging modalities used in the art, such as that for cardiovascular catheter placement and / or cancer treatment. Example image modalities include, but are not limited to, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and ultrasound. In some embodiments, the imaging modality is MRI, CT, PET, or ultrasound. In some embodiments, the imaging modality is CT or PET.

[0028] The hollow tube is one that can be used in the medical field to, e.g., administer therapies to a subject. Example hollow tubes that can be used in the disclosed methods include, but are not limited to, a needle, a catheter, a cannula, or a combination thereof. In some embodiments, the hollow tube is a needle or a catheter. In some embodiments, the hollow tube is a needle. In some embodiments, the hollow tube is a needle attached to a catheter.

[0029] The hollow tube has an inner or internal diameter (e.g., diameter of the lumen of the hollow tube) on the larger side. For example, the hollow tube can have an inner diameter of no less than 0.4 mm, no less than 0.5 mm, no less than 0.6 mm, no less than 0.7 mm, no less than 0.8 mm, no less than 0.9 mm, no less than 1 mm, no less than 1.3 mm, no less than 1.6 mm, no less than 2 mm, no less than 2.3 mm, no less than 2.7 mm, or no less than 3 mm. In some embodiments, the hollow tube has an inner diameter of no greater than 3.3 mm, no greater than 3 mm, or no greater than 2.7 mm. In some embodiments, the hollow tube has an inner diameter of no less than 0.4 mm to about 3.3 mm, such as about no less than 0.4 mm to about 3 mm, no less than 0.4 mm to about 2.7 mm, no less than 0.4 mm to about 2 mm, or no less than 0.4 mm to about 1.6 mm.

[0030] The hollow tube can be navigated along a distance in the subject that can extend from entering the subject to the tumor. This can include from the skin to the tumor or from the digestive tract (e.g., mouth) to the tumor. For example, the distance can be about 0.5 cm to about 152 cm (e.g., ~60 inches), 1 cm to about 152 cm, about 5 cm to about 150 cm, about 10 cm to about 125 cm, about 25 cm to about 110 cm, about 30 cm to about 100 cm, about 1 cm toAttorney Docket No.028193-0060-WO01 about 120 cm, about 20 cm to about 152 cm, or about 50 cm to about 152 cm. In some embodiments, the distance is no less than 0.5 cm, no less than 1 cm, no less than 5 cm, no less than 10 cm, no less than 20 cm, no less than 30 cm, no less than 40 cm, no less than 50 cm, or no less than 60 cm. In some embodiments, the distance is no greater than 152 cm (e.g., ~60 inches), no greater than 150 cm, no greater than 145 cm, no greater than 140 cm, or no greater than 125 cm. Because the hollow tube can be navigated along the above-listed distances, the hollow tube can also have a length that corresponds to the distance navigated. Accordingly, the distance the hollow tube is navigated can be applied to the length of the hollow tube. For the purposes of brevity, they will not be repeated here.

[0031] A number of different tumor types may be treated by the disclosed methods. For example, the tumor can be a pancreatic cancer, a liver cancer, a colorectal cancer, a cervical cancer, an ovarian cancer, a sarcoma, a breast cancer, a gastrointestinal cancer, or a melanoma. In some embodiments, the tumor is a pancreatic cancer, a liver cancer, a colorectal cancer, a cervical cancer, an ovarian cancer, a sarcoma, a breast cancer, or a gastrointestinal cancer. In some embodiments, the tumor is a pancreatic cancer, gastrointestinal cancer or a liver cancer. In some embodiments, the tumor is a pancreatic cancer. In some embodiments, the tumor is a solid tumor. The methods are particularly useful for tumors that are not superficial and require navigation of the administration through areas of the subject.

[0032] The subject (e.g., in need thereof) of the disclosed methods is generally not limited and can be any animal that is in need of a treatment for a tumor. Subject can mean a mammal that wants or is in need of the herein described pharmaceutical compositions or methods. The subject may be a human or a non-human animal. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a primate such as a human; a non- primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant. The subject may be male or female. In some embodiments, the subject is human.

[0033] The methods can provide advantageous benefits to the subject receiving the disclosed image-guided brachytherapy. For example, the collection of self-assembling conjugates or pharmaceutical composition thereof can be stably placed at the tumor after phase transitioning to a viscous depot. For example, the collection of self-assembling conjugates or pharmaceutical composition thereof can be retained at the tumor for at least 2 days (following administration), at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week,Attorney Docket No.028193-0060-WO01 or at least 2 weeks. Retention can also be measured by the remaining radionuclide activity of the collection of self-assembling conjugates at the tumor. For example, the collection of self- assembling conjugates can retain a radionuclide activity of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the administered radionuclide activity at the tumor at any of the days listed above for retention. In some embodiments, the collection of self-assembling conjugates retains a radionuclide activity of about 20% to about 95%, such as about 20% to about 80% or about 25% to about 75% of the administered radionuclide activity at the tumor at any of the days listed above for retention. Because the collection of self-assembling conjugates is stable at the tumor, it can effectively and consistently irradiate the tumor. This in turn can lead to better outcomes for the subject. A. Pharmaceutical Compositions

[0034] The disclosed pharmaceutical compositions can advantageously be used with larger diameter hollow tubes required for image-guided brachytherapy. The pharmaceutical composition includes a pharmaceutically acceptable excipient and a collection of self- assembling conjugates. The pharmaceutical compositions can include components at specific amounts or concentrations that allow the collection of self-assembling conjugates to be effectively delivered to the tumor. The components of the pharmaceutical composition can also be used to image the pharmaceutical composition. This can be useful for assessing where the pharmaceutical composition is located following administration, as well as identifying the location of the pharmaceutical composition or components thereof minutes, hours, or days after administration.

[0035] The pharmaceutical composition can also be imaged by an imaging modality. The pharmaceutical composition can be imaged by the radionuclide coupled to the elastin-like polypeptide, by a contrast-enhancing agent, or both. The pharmaceutical composition can be identified by the same or a different imaging modality that is used to navigate the hollow tube. Accordingly, the imaging modalities used to navigate the hollow tube can be applied to imaging the pharmaceutical composition. Because the pharmaceutical composition and the collection of self-assembly conjugates can be imaged, the method can also include imaging the tumor. i. Pharmaceutically Acceptable Excipients

[0036] The term “pharmaceutically acceptable excipient,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Examples of pharmaceutically acceptable excipients include, but are not limited to, buffering agents (e.g., phosphate buffered saline, artificial cerebrospinal fluid (aCSF), etc.), carbohydrates (e.g., glucose, trehalose, starch, etc.) solubilizers, solvents, antimicrobialAttorney Docket No.028193-0060-WO01 preservatives, antioxidants, suspension agents, penetration / absorption enhancers (e.g., DMSO, ethanol, pyrrolidones, and / or ionic liquids), contrast agents, or a combination thereof. In some embodiments, the pharmaceutically acceptable excipient includes a buffer, a contrast- enhancing agent, a radioactive biopolymer, a non-radioactive biopolymer, or a combination thereof. Example biopolymers include, but are not limited to, other elastin-like polypeptides or similarly intrinsically disordered polypeptides. In some embodiments, the pharmaceutically acceptable excipient includes a buffer, a contrast-enhancing agent, or a combination thereof. In some embodiments, the pharmaceutically acceptable excipient includes a buffer and a contrast- enhancing agent.

[0037] Contrast agents (also referred to herein as contrast-enhancing agents) can be part of the pharmaceutical composition to aid imaging of the pharmaceutical composition itself (during and after administration), to enhance imaging of the collection of self-assembling conjugates (during or after administration), improve imaging of the tumor, or a combination thereof. The contrast agent can be any type of contrast agent that improves the detectable signal for the imaging modality being used and that can be used with the collection of self-assembling conjugates. Example contrast agents include a CT contrast-enhancing agent, a PET contrast- enhancing agent, and a SPECT contrast-enhancing agent. In some embodiments, the contrast agent includes a CT contrast-enhancing agent or a PET contrast-enhancing agent. Example CT contrast enhancing agents include, but are not limited to, an iodine-based CT contrast- enhancing agent (e.g., iopamidol) and a barium-based CT contrast-enhancing agent (e.g., barium sulfate).

[0038] It has been found that there is a threshold amount of contrast-enhancing agent that can be used with the disclosed collection of self-assembling conjugates. In particular, it was found that using higher amounts of contrast-enhancing agents would affect phase transition properties of the self-assembling conjugate such that it phase transitions out of solution. To overcome this issue, it was found that a threshold concentration of no more than 50% of the contrast-enhancing agent by volume of the pharmaceutical composition allowed the elastin-like polypeptide and self-assembling conjugate thereof having a transition temperature of no greater than 37 °C to pass freely through the hollow tube and deposit at a tumor, where there it can phase transition to a stable depot. As such, the pharmaceutical composition can include the contrast-enhancing agent at no more than 50% by volume, such as no more than 49% by volume, no more than 48% by volume, no more 47% by volume, no more than 46% by volume, no more than 45% by volume, no more than 40% by volume, no more than 35% by volume, or no more than 30% by volume. In some embodiments, the pharmaceutical composition includesAttorney Docket No.028193-0060-WO01 the contrast-enhancing agent at no less than 0.1% by volume, no less than 0.5% by volume, no less than 1% by volume, or no less than 10% by volume. In some embodiments, the pharmaceutical composition includes the contrast-enhancing agent at about 0.1% to no more than 50% by volume, such as about 1% to no more than 50% by volume, about 5% to no more than 50% by volume, or about 10% to no more than 50% by volume. By volume in reference to the contrast enhancing agent refers to by volume of the pharmaceutical composition. In addition, the balance of the pharmaceutical composition can be the collection of self-assembling conjugates at a concentration as disclosed herein. ii. Collection of Self-Assembling Conjugates

[0039] The collection of self-assembling conjugates includes a plurality of self-assembling conjugates. Each self-assembling conjugate includes an elastin-like polypeptide coupled to a radionuclide. The collection can also include elastin-like polypeptides that are not coupled to a radionuclide. Elastin-like polypeptides (ELP) are thermally responsive polypeptides that exhibit phase transition behavior. “ELP” generally refers to a polypeptide comprising the pentapeptide repeat sequence (VPGX1G)p(SEQ ID NO:1), wherein X1is any amino acid and n is an integer greater than or equal to 1.

[0040] The elastin-like polypeptide can include an amino acid sequence of (VPGX1G)p(SEQ ID NO:2), wherein X1is any amino acid and p is 1 to 500. In some embodiments, p is an integer from 1 to 500, from 1 to 400, from 1 to 300, from 1 to 200, from 1 to 100, from 1 to 50, from 50 to 500, from 50 to 400, from 50 to 300, from 50 to 200, from 50 to 100, from 100 to 500, from 100 to 400, from 100 to 300, from 100 to 200, from 200 to 500, from 200 to 400, from 200 to 300, from 200 to 500, from 300 to 500, from 300 to 400, or from 400 to 500.

[0041] The elastin-like polypeptide can include an amino acid sequence of (VPGX2G)q(GrY)s(SEQ ID NO:3), wherein X2is any amino acid, q is 1 to 500, r is 0 to 10, and s is 1-250. In some embodiments, q is an integer from 1 to 500, from 1 to 400, from 1 to 300, from 1 to 200, from 1 to 100, from 1 to 50, from 50 to 500, from 50 to 400, from 50 to 300, from 50 to 200, from 50 to 100, from 100 to 500, from 100 to 400, from 100 to 300, from 100 to 200, from 200 to 500, from 200 to 400, from 200 to 300, from 200 to 500, from 300 to 500, from 300 to 400, or from 400 to 500. In some embodiments, r is an integer from 0 to 10, from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, from 0 to 1, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3, from 1 to 2, from 2 to 10, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, from 2 to 3, from 3 to 10, from 3 to 9, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 3 to 4, from 4 to 10, from 4 to 9, from 4 to 8, from 4 to 7, from 4 to 6, from 4 to 5, from 5 to 10, from 5 to 9, from 5 to 8, from 5 toAttorney Docket No.028193-0060-WO01 7, from 5 to 6, from 6 to 10, from 6 to 9, from 6 to 8, from 6 to 7, from 7 to 10, from 7 to 9, from 7 to 8, from 8 to 10, from 8 to 9, or from 9 to 10. In some embodiments, s is an integer from 1 to 250, from 1 to 200, from 1 to 150, from 1 to 100, from 1 to 50, from 50 to 250, from 50 to 200, from 50 to 150, from 50 to 100, from 100 to 250, from 100 to 200, from 100 to 150, from 150 to 250, from 150 to 200, or from 200 to 250.

[0042] In some embodiments, the elastin-like polypeptide includes an amino acid sequence of (VPGVG)m(GY)n(SEQ ID NO:4), wherein m is 50-250 and n is 1 to 50. In some embodiments, m is an integer from 50 to 200, from 50 to 150, from 50 to 100, from 100 to 250, from 100 to 200, from 100 to 150, from 150 to 200, from 150 to 200, or from 200 to 250. In some embodiments, m is 120. In some embodiments, n is an integer from 1 to 50, from 10 to 50, from 20 to 50, from 30 to 50, from 40 to 50, from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to 40. In some embodiments, n is 7. In some embodiments, m is 120 and n is 7.

[0043] In some embodiments, the elastin-like polypeptide includes an amino acid sequence of (VPGVG)120(GY) (SEQ ID NO:5), (VPGVG)120(GY)4(SEQ ID NO:6), or (VPGVG)120(GY)7(SEQ ID NO:7). In some embodiments, the elastin like polypeptide includes an amino acid sequence of (VPGVG)120(GY)7(SEQ ID NO:7).

[0044] The elastin-like polypeptides can be expressed recombinantly in a host cell according to one of skill in the art. The elastin-like polypeptides can be purified by any means known to one of skill in the art. For example, the elastin-like polypeptides may be purified using chromatography, such as liquid chromatography, size exclusion chromatography, affinity chromatography, or a combination thereof. In some embodiments, the elastin-like polypeptides are purified without chromatography. In some embodiments, the elastin-like polypeptides are purified using inverse transition cycling.

[0045] The collection of self-assembling conjugates may include varying amounts of elastin- like polypeptide chains. For example, the collection of self-assembling conjugates may include about 10 to about 200 elastin-like polypeptide chains per collection, such as about 10 to about 100 or about 50 to about 200. Some or all of the elastin-like polypeptides can be coupled to a radionuclide as disclosed herein.

[0046] The radionuclide may be any of the radioisotopes known in the art capable of providing irradiation through beta-particles, alpha-particles, gamma rays, or Auger electrons. The radionuclide can also be an imaging radionuclide or a therapeutic radionuclide. Example radionuclides include, but are not limited to,131Cesium,137Cesium,60Cobalt,192Iridium,123Iodine,124Iodine,125Iodine,131Iodine,103Palladium,106Ruthenium,223Radium,226Radium,90Yttrium,177Lutetium,111Indium,186Rhenium,89Strontium,153Samarium,32Phosphorous,225Actinium,Attorney Docket No.028193-0060-WO01211Astatine,213Bismuth, and212Lead. In some embodiments, the radionuclide is an iodine radionuclide. In some embodiments, the radionuclide is123Iodine,124Iodine,125Iodine, or131Iodine.

[0047] Halogenic radionuclides (such as iodine or astatine) can be covalently conjugated to Tyr residues on the elastin-like polypeptide through electrophilic substitution or similar chemistries. Chelated radionuclides (including, but not limited to, lutetium, indium, rhenium, actinium, or lead) can be conjugated to a chelator (e.g., tetraxetan or DOTA) that is covalently conjugated to the elastin-like polypeptide through maleimide-cysteine or similar chemistries.

[0048] Each elastin-like polypeptide can be coupled with at least one radionuclide. In some embodiments, each elastin-like polypeptide is coupled with at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten radionuclides as described above. In some embodiments, the collection of self- assembling conjugates includes conjugates with different radionuclides. For example, the collection of self-assembling conjugates can include a first conjugate having an elastin-like polypeptide coupled to an iodine radionuclide and a second conjugate having an elastin-like polypeptide coupled to a cesium radionuclide.

[0049] The collection of self-assembling conjugates can have an effective amount or therapeutically effective amount of radionuclide activity. For example, the collection of self- assembling conjugates can have a radionuclide activity of about 1 μCi to about 100 μCi, such as about 5 μCi to about 95 μCi, about 10 μCi to about 90 μCi, about 15 μCi to about 85 μCi, about 20 μCi to about 80 μCi, about 1 μCi to about 60 μCi, or about 40 μCi to about 100 μCi. In some embodiments, the collection of self-assembling conjugates has a radionuclide activity of no greater than 100 μCi, no greater than 90 μCi, no greater than 80 μCi, no greater than 70 μCi, no greater than 60 μCi, or no greater than 50 μCi. In some embodiments, the collection of self- assembling conjugates has a radionuclide activity of no less than 1 μCi, no less than 5 μCi, no less than 10 μCi, no less than 15 μCi, no less than 20 μCi, or no less than 25 μCi.

[0050] The elastin-like polypeptide conjugated to the radionuclide has phase transition behavior. Phase transition refers to aggregation, which may occur sharply and, in some instances, reversibly at or above a transition temperature. A transition temperature (Tt) is a temperature at which the elastin-like polypeptide and conjugate thereof changes from one state to another, e.g., being soluble in solution to self-assembling with other ELPs and / or conjugates to form an aggregate (e.g. viscous depot). The Ttof the ELP and the conjugate thereof can be adjusted by varying the amino acid sequence of the elastin-like polypeptide, by varying the length of the polypeptide, or a combination thereof. The transition temperature may be noAttorney Docket No.028193-0060-WO01 greater than 45 °C, no greater than 42 °C, no greater than 40 °C, no greater than 39 °C, no greater than 38 °C, no greater than 37 °C, no greater than 36 °C, no greater than 35 °C, no greater than 30 °C, no greater than 25 °C, no greater than 20 °C, or no greater than 15 °C. In some embodiments, the transition temperature is no less than 10 °C, no less than 15 °C, no less than 20 °C, or no less than 25 °C. In some embodiments, the transition temperature is about 10 °C to about 45 °C, such as about 15 °C to about 40 °C, about 20 °C to about 37 °C, about 23 °C to about 37 °C, about 25 °C to about 35 °C, about 10 °C to about 37 °C, or about 15 °C to about 37 °C. Phase transition behavior is instilled in the conjugate by the ELP. Accordingly, both the ELP and the conjugate thereof can have the foregoing transition temperature properties.

[0051] The self-assembling conjugates of the collection may individually self-assemble into a variety of shapes and sizes. In some embodiments, the self-assembling conjugates may self- assemble into a micelle. In other words, the collection can be a micelle. The micelle may be rod- shaped or spherical, or the collection may include combinations of differently shaped nanoparticles. The micelle can have a varying average hydrodynamic radius. In some embodiments, the micelle has an average hydrodynamic radius of about 10 nm to about 100 nm, such as about 25 nm to about 75 nm or about 40 nm to about 60 nm. In some embodiments, the micelle has an average hydrodynamic radius of greater than 10 nm, greater than 20 nm, greater than 30 nm, greater than 40 nm, or greater than 50 nm. In some embodiments, the micelle has an average hydrodynamic radius of less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, or less than 50 nm. Particle size (e.g., hydrodynamic radius) can be measured via techniques used within the art such as, but not limited to, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) and dynamic light scattering.

[0052] The micelle may also be described by its average radius of gyration. For example, the micelle can have an average radius of gyration of about 10 nm to about 100 nm, such as about 25 nm to about 75 nm or about 40 nm to about 60 nm. In some embodiments, the micelle has an average radius of gyration of greater than 10 nm, greater than 20 nm, greater than 30 nm, greater than 40 nm, or greater than 50 nm. In some embodiments, the micelle has an average radius of gyration of less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, or less than 50 nm. Radius of gyration may also be characterized by dynamic light scattering.

[0053] Phase transition behavior may also enable purification of the self-assembling conjugate or elastin-like polypeptide using inverse transition cycling, thereby eliminating theAttorney Docket No.028193-0060-WO01 need for chromatography. “Inverse transition cycling” refers to a protein purification method for polypeptides having phase transition behavior, and the method may involve the use of the conjugate’s reversible phase transition behavior to cycle the solution through soluble and insoluble phases, thereby removing contaminants and eliminating the need for chromatography.

[0054] It was found that the self-assembling conjugate could not be included at higher concentrations in the pharmaceutical composition when used in conjunction with a larger diameter hollow tube (e.g., internal diameter of no less than 0.4 mm). In particular, it was found that using higher diameter hollow tubes would affect phase transition properties of the self- assembling conjugate such that it would phase transition out of solution within the hollow tube. To overcome this issue, it was found that a threshold concentration of no more than 300 μM would allow an elastin-like polypeptide and self-assembling conjugate thereof having a transition temperature of no greater than 37 °C to pass freely through the hollow tube and deposit at a tumor, where there it can phase transition to a depot. As such, the pharmaceutical composition can include the collection of self-assembling conjugates at a concentration of no more than 300 μM, no more than 290 μM, no more than 280 μM, no more than 270 μM, no more than 260 μM, no more than 250 μM, no more than 225 μM, no more than 200 μM, no more than 175 μM, no more than 150 μM, no more than 125 μM, no more than 100 μM, no more than 75 μM, no more than 50 μM, no more than 25 μM, no more than 10 μM, no more than 1 μM, or no more than 0.1 μM. In some embodiments, the pharmaceutical composition includes the collection of self- assembling conjugates at a concentration of no less than 0.01 μM, no less than 0.1 μM, no less than 1 μM, or no less than 10 μM. In some embodiments, the pharmaceutical composition includes the collection of self-assembling conjugates at a concentration of about 0.01 μM to no more than 300 μM, such as no less than 0.01 μM to no more than 300 μM, about 1 μM to no more than 300 μM, about 10 μM to no more than 300 μM, or about 50 μM to no more than 300 μM. iii. Example Pharmaceutical Compositions

[0055] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient includes a buffer and a contrast-enhancing agent selected from the group consisting of a CT contrast-enhancing agent, a PET contrast-enhancing agent, and a SPECT contrast-enhancing agent, the contrast- enhancing agent being included at no more than 50% by volume of the pharmaceutical composition; and a collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and the collection of self-Attorney Docket No.028193-0060-WO01 assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM. B. Administration

[0056] The pharmaceutical composition can be administered therapeutically. In therapeutic applications, the pharmaceutical composition can be administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this can be referred to as a “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular pharmaceutical composition regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0057] The pharmaceutical composition can be delivered via a variety of routes to the subject and guided to the tumor. Example delivery routes include, but are not limited to, percutaneously, endoscopically, intravenously, intracerebroventricularly, intrathecally, and subcutaneously. In some embodiments, the pharmaceutical composition is administered percutaneously or endoscopically. In some embodiments, the pharmaceutical composition is administered percutaneously and is guided by CT. In some embodiments, the pharmaceutical composition is administered endoscopically and is guided by ultrasound.

[0058] The pharmaceutical composition can be administered at varying times and frequency. For example, the pharmaceutical composition can be administered at least once (e.g., as a single dose) over 100 days, such as 90 days, 80 days, 70 days, 60 days, 50 days, 40 days, 30 days, 25 days, 20 days, 15 days, 10 days, 5 days, or 1 day. In addition, the pharmaceutical composition can be administered such that there is more than one collection of self-assembling conjugates deposited at the tumor. For example, the pharmaceutical composition can be administered 1x, 2x, 3x, 4x, 5x, or more over 100 days – or a period of time listed above.

[0059] Suitable in vivo dosage to be administered and the particular mode of administration can vary depending upon the age, weight, the severity of the affliction, and subjects treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies and in vitro studies.

[0060] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose inAttorney Docket No.028193-0060-WO01 the management of the disorder of interest can vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, can also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0061] The pharmaceutical compositions described herein may be administered with additional compositions to prolong stability, delivery, and / or activity of the pharmaceutical composition, or combined with additional therapeutic agents, or provided before or after the administration of additional therapeutic agents. 3. Kits

[0062] Also disclosed herein are kits that can be used in the disclosed methods. The kit can include a hollow tube as disclosed herein having an internal diameter of no less than 0.4 mm. The kit can also include a pharmaceutical composition as disclosed herein. The pharmaceutical composition can include a disclosed pharmaceutically acceptable excipient, and the disclosed collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C. The collection of self-assembling conjugates can be included in the pharmaceutical composition at a concentration of no more than 300 μM.

[0063] The kit may include other components. Such components may be provided individually or in combinations and may provide in any suitable container such as a vial, a bottle, or a tube. Examples of such components include, but are not limited to, one or more additional reagents, such as one or more dilution buffers; one or more reconstitution solutions; one or more storage buffers, and the like. Components (e.g., reagents) may also be provided in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). Suitable buffers include, but are not limited to, phosphate buffered saline, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof.

[0064] In addition to the above-mentioned components, the kit can include instructions for using the components of the kit to, e.g., practice the disclosed methods. The instructions are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In yet other embodiments,Attorney Docket No.028193-0060-WO01 the actual instructions are not present in the kit but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0065] The description of the methods, hollow tubes, pharmaceutical compositions, pharmaceutically acceptable excipients, and collection of self-assembling conjugates may be applied to the disclosed kits.

[0066] The disclosed technology has multiple aspects, illustrated by the following non-limiting examples. 4. Examples Example 1 Injectable Liquid Brachytherapy for Treating Pancreatic Cancer

[0067] This example describes a local radiotherapy that destroys tumors from within using elastin-like polypeptide (ELP) technology. ELPs are recombinant biopolymers that exhibit lower critical solution temperature phase behavior. Above a sequence-defined transition temperature, an ELP will undergo liquid-liquid phase separation and transition into a viscous, gel-like coacervate. The approach provided herein is designed to treat a cancer, such as pancreatic cancer, by using an ELP that is covalently conjugated to a therapeutic radionuclide, such as iodine-131 (131I). When injected intratumorally, it immediately transitions into a depot upon heating to body temperature, undergoes radiation-induced crosslinking, and retains nearly all injected radioactivity within the tumor. With numerous in vivo studies demonstrating efficacy in mice, here it is reported the first successful administration of this class of biopolymer within a porcine model that recapitulates the delivery challenges posed in human pancreatic cancer. Using interventional radiology, a CT image-guided injection of ELP brachytherapy labeled with iodine-124 (124I)—a positron-emitting radionuclide— was performed to visualize deposition of the ELP depot within pancreatic space and confirm retention of the depot, and nearly all radioactivity, over the following six days. This study demonstrates the translatability and promise of localized “liquid brachytherapy” platform, laying the groundwork for future use in the clinic. Materials & MethodsAttorney Docket No.028193-0060-WO01

[0068] As concentration increases, the phase transition temperature of an ELP will decrease, and vice versa. To enable injection of ELP into the pancreas of a large animal, it is essential that the ELP does not coacervate inside the long needle and clog it. ELP was first injected at various concentrations through a catheter in a 37 °C incubator to simulate body temperature. Once ELP concentrations that could be injected without clogging the catheter ex vivo were identified, they were radiolabeled with131I and injected at 1000, 400, and 300 μM ELP into BxPC3-luc2 tumors in mice. Deposition of activity was confirmed by measuring whole-body radioactivity daily. After 1 week, tumors and thyroids were excised and radioactivity compared in each with the whole- body value. The porcine experiment was then performed using the lower concentration, improved ELP formulation.

[0069] Porcine Experiment: A 40 kg male Yorkshire pig was positioned prone on a CT scanner. From a scout CT scan, a lateral route from the skin into the splenic lobe of the pancreas was planned. A 22g Chiba needle was advanced into the target portion of the pancreas under intermittent CT guidance. Intervening bowel, liver, and spleen was displaced using hydrodissection. When the treatment needle was advanced into the pancreas, 1 cc of ELP containing 300 Ci of124I activity was injected. The needles were then removed, and high- resolution PET / CT was performed to assess for successful124I-ELP deposition at t=0, 3, and 144 hrs (six days) following administration. Results & Discussion

[0070] In previous mouse studies, where the conjugate was directly injected though a needle into tumors, an ELP concentration of 1000 μM was chosen. When testing this concentration ex vivo, the ELP immediately clogged the catheter and needle upon heating (Table 1). Lower- concentration ELP formulations exhibit no differences in131I retention within the tumor. This is an interesting and surprising finding given that previous studies showed that lower ELP concentrations in vivo exhibited lower retention at the site of administration (see Liu et al., Cancer Research, 2012, November 15; 72(22) 5956-5965).

[0071] Mice (Nu / nu, n=5) were subcutaneously inoculated with BxPc3-luc2 pancreatic tumors in the hind flank and treated with131I-ELP brachytherapy at the 1000, 400, and 300 μM concentrations described in Table 1. As concentration was decreased, it was observed that concentrations below 400 μM could be injected with no perceptible resistance, suggesting that testing formulations at 1000, 400, and 300 μM in the in vivo mouse study could be used. Compared to the control, both new formulations exhibited near-identical depot-forming behavior as well as retention of injected activity for one week following injection (FIG.1A). At that time,Attorney Docket No.028193-0060-WO01 the tumor and thyroid were harvested from the mice and found no significant differences in measured activities in any of the formulations (FIG.1B). Based on this, it was decided to inject an ELP concentration of 300 μM in our pig experiment. Table 1: ELP concentration determines injectability through a catheter ex vivo. ELP was formulated at 1000, 500, 400, 300, 250, and 100 μM. A 5.2 fr catheter containing a 22 ga needle was pre-heated to 37oC in a circulating air incubator, and 1 mL of ELP at each concentration was injected through the catheter. Physical resistance to injection was observed, and the ELP was collected in a tared 1.5 mL centrifuge tube, frozen, and lyophilized to measure mass of deposited ELP as a fraction of original ELP mass used.

[0072] Percutaneous injection (FIG.2) of the124I-labeled ELP into the pig pancreas was successful, with the PET / CT scan at t=0 hrs demonstrating deposition of the injected ELP within the local site (FIG.3). At t=3 hrs, a follow-up scan confirmed retention of the ELP depot at this site. Six days following this, a final scan was performed and the124I-labeled ELP depot were observed in the same position within the pancreas tissue, demonstrating successful depot formation and retention. No other detectable activity was observed under PET, besides a small amount of124I activity within the thyroid of the animal (FIG.4).

[0073] The results of this study demonstrated that when pancreatic tumor xenografts were injected with ELP brachytherapy depots of 1000, 400, or 300 M, there was no change in the fraction of the injected dose retained within the mice at all three concentrations (measured beginning 24-hrs after treatment to account for initial depot formation at the injection site. Furthermore, evaluating the distribution of131I activity in the local tumor versus the thyroid revealed no significant uptake of the radionuclide in all three concentration groups. The results of this study validate that the radiolabeled ELP exhibits near-identical depot forming behavior and therapeutic efficacy at the lower concentration (300 M), which is essential for injection through long, clinically relevant needle configurations in large animals.Attorney Docket No.028193-0060-WO01

[0074] The results of this experiment represent a first-in-pig approach to injecting biopolymer brachytherapy. It is believed that this is the first example of an attempt to deliver injectable, persistent radiotherapy within an internal organ that cannot be reached with conventional rod- or seed-brachytherapy. Example 2 Modifications of Injection Procedure

[0075] Given leakage of124I-ELP that was observed from pancreatic and liver depot injections in previous pigs, it was sought to develop an alternate strategy which could reliably deposit ELP into target tissue without incurring significant losses due to escape into circulation. It was determined that a promising strategy would be to perform a “test injection” of non- radioactive, but highly iodinated contrast fluid that could be visualized rapidly on a CT scan. Retention of the contrast bolus at the needle tip would indicate that the ELP would be safe to follow with, while immediate leakage of contrast fluid would indicate that the needle position must be adjusted prior to injecting124I-ELP.

[0076] A different pig strain than the Yorkshire pigs was also employed. While the Yorkshire breed is ubiquitous due to its history as a breed for meat production, its deposition of subcutaneous fat can be limited, being up to 70% lean as a consequence of breeding strategies over decades. While deposition of adipose tissue in healthy human pancreas is relatively limited, there is normally noticeable fat surrounding the pancreas which can be imaged via CT scans. For the purposes of studying intra-pancreatic injections of ELP brachytherapy, additional retroperitoneal fat surrounding the pancreas would significantly enhance injectability by enlarging the volume of target tissue.

[0077] The Minnesota mini-pig (colloquially known as a “mini-pig”) is a breed which, despite its smaller stature, is known for containing greater subcutaneous and visceral fat deposits than the Yorkshire breed. This breed can better recapitulate the visceral fat which would be visible in human patients upon injection and thus was elected to use this breed for this example.

[0078] A male ~35 kg Minnesota Mini-pig (National Swine Research and Resource Center, University of Missouri, Colombia, MO) was prepared for the injection procedure, being transported, anesthetized, and placed on the bed of a PET / CT scanner. The anesthetized pig was imaged using a scout CT scan to determine a path for a trans-splenic approach to the tail of the pancreas. A 22 Ga Chiba needle was advanced under intermittent CT guidance to the target location and a 1 mL test injection of iodinated contrast fluid (Isovue-300, Bracco Diagnostics, Princeton, NJ) was hand-injected through a 3 mL syringe attached to the sample inlet port onAttorney Docket No.028193-0060-WO01 the needle. Following injection, a CT scan was immediately commenced to visualize distribution of the contrast bolus.

[0079] The initial contrast fluid injection exhibited immediate leakage along the path of the needle and into the surrounding tissue, which was similar behavior to the124I-ELP in previous experiments. The needle was advanced <1 cm further and felt a clear indication of puncture through a facial plane of—what was presumed—pancreatic tissue. The second injection of contrast fluid, followed by immediate CT scan, confirmed retention of the contrast bolus around the needle tip with no visible leakage. This confirmed that injection of the124I-ELP at the same location could be pursued.

[0080] Upon injecting 1 mL of124I-ELP (intended to deposit ~300 Ci of124I activity) into the tail of the pancreas, the target area was imaged with co-registered PET / CT. Initial deposition of ELP into the pancreatic tissue was successful, with an immediate PET / CT scan of the area demonstrating relatively uniform depot formation that nearly enveloped the remaining contrast fluid visible in the site. At 0.5 h, the follow-up scan demonstrated a reduction in activity of 30 Ci, which can be attributed to either free iodine loss or loss of ELP during depot formation. At 7 d, the depot was well-defined, with a calculated activity of 46 Ci. Based on the activity at 0.5 h, the calculated expected activity at 168 h was 64.93 Ci. The discrepancy of activity at this time could not be attributed to124I in any other tissues, as the only other detectable activity was 2.5 Ci in the thyroid, meaning the activity was lost early-on after depot formation. Example 3 Contrast Agent Can Affect Administration

[0081] Because iodinated contrast fluid contains high concentrations of dissolved salts (e.g., 300 mg iopamidol / mL solution), solutions of ELP dissolved in significant amounts of this contrast fluid were hypothesized, without being bound by a particular theory, to exhibit changes in the Tt. This could explain potential increases in ELP loss from the depot if the Ttis elevated by the contrast fluid. To test this, ELP-Tyr7was dissolved at a 300 M concentration in either 1X PBS (control), 100% Isovue-300 contrast fluid, or a 1:1 v / v mixture of the two. The thermally- responsive phase behavior of the various mixtures was measured using optical turbidimetry at temperatures from 10 °C to 40 °C (FIG.5). It was found that the Ttof ELP-Tyr7 is significantly elevated (from ~23 °C to ~35 °C) when mixed 1:1 with Isovue. It was also observed that as the proportion of contrast fluid increased, the ELP became less likely to fully solubilize. This was determined based on the presence of either a haze in the solution (observed in the 1:1 Isovue:PBS sample) or visible aggregates that sank to the bottom of the sample tube (in theAttorney Docket No.028193-0060-WO01 100% Isovue sample). These observations were confirmed with Cary scans that showed baseline increases in absorbance as the percentage of Isovue in the sample increased, with the 100% Isovue sample exhibiting clear signs of aggregation with minimal changes in absorbance as the temperature changed.

[0082] These findings indicate that further investigation is required to fully understand the effects of pre-injecting iodinated contrast fluid prior to I-ELP. While the importance of confirming retention of the injection was demonstrated prior to administering the radioactive ELP, it is possible that this contrast fluid slightly elevated the ELP Ttin vivo and either slowed depot- formation or caused some of the ELP to diffuse more quickly from the injection site. One could design a study in which the concentration of contrast fluid is titrated to a minimum level to still visualize the bolus with CT, which may minimize effects on ELP Tt. An alternative method could involve waiting a certain amount of time for the contrast bolus to dissipate with normal interstitial fluid flow, or to inject normal saline in the local site to accelerate this process. Example 4 Safety of124I-ELP in the Pancreas and Liver

[0083] In cases of acute pancreatic injury, acute pancreatitis (inflammation of the pancreas) can manifest in the form of elevated enzymes such as pancreatic lipase or amylase, where a greater than 3x increase in baseline levels is generally considered an indicator of possible pancreatitis. Given that this definition has been used by other researchers conducting porcine studies, these criteria were adopted to evaluate whether acute pancreatitis occurred in response to124I-ELP injection.

[0084] To measure pancreatic lipase and amylase levels, blood samples were collected at various time points from each pig and harvested serum. Briefly, blood samples were allowed to rest at room temperature for 1 h following collection, centrifuged for 10 mins at 1500 x g, the supernatant was transferred to a fresh tube, and centrifuged again for 10 mins at 1500 x g. The final supernatant (purified serum) was collected, and levels of lipase and amylase were measured using a porcine-specific ELISA kits for the corresponding enzymes (MyBioSource, San Diego, CA). In the first pig, no increase in either lipase or amylase levels was observed at any time point (FIG.6A). For pigs 2 and 3, which each exhibited significant leakage of the ELP depot from the pancreatic injection site, elevated levels of lipase and amylase were seen at 168 h that were near or above 3x the baseline values (FIG.6A). These levels can be indications of acute pancreatic injury because of the ELP leakage in the surrounding vasculature. It is possible that this response is also due to vascular injury because of transecting an artery or vein (whichAttorney Docket No.028193-0060-WO01 facilitated entry of the ELP into circulation) by the needle itself. The mini-pig that was test- injected with contrast fluid prior to the ELP, did not exhibit elevated lipase or amylase levels (FIG.6B), suggesting that locally-retained124I-ELP (which does not leak from the injected site) does not itself cause acute pancreatic injury.

[0085] It was confirmed that for all four pigs (three Yorkshire and one mini-pig), the procedure itself was well-tolerated, with all pigs fully recovering, ambulating, eating normally, and not exhibiting signs of pain or distress which were not managed within the 48h following the procedure with prescribed analgesics. There were also no alarming indications of ill-health (such as body weight drop). Based on these observations, an injection of124I-ELP in the pancreas and / or liver was found not to cause adverse reactions (beyond those caused by the trajectory of the needle itself) and is well-tolerated.

[0086] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure.

[0087] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.

[0088] For reasons of completeness, the following Embodiments are provided.

[0089] Clause 1. A method for image-guided brachytherapy in a subject, the method comprising: navigating a hollow tube to a tumor in the subject using an imaging modality, the hollow tube having an internal diameter of no less than 0.4 mm; and administering a pharmaceutical composition through the hollow tube to the tumor, the pharmaceutical composition including a pharmaceutically acceptable excipient and a collection of self- assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and wherein the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM.

[0090] Clause 2. The method of clause 1, wherein the hollow tube is navigated along a path of no less than 0.5 cm.

[0091] Clause 3. The method of clause 1 or 2, wherein the hollow tube has a length of no less than 1 cm.

[0092] Clause 4. The method of any one of clauses 1-3, wherein the hollow tube is a needle, a catheter, a cannula, or a combination thereof.Attorney Docket No.028193-0060-WO01

[0093] Clause 5. The method of any one of clauses 1-4, wherein the image modality is magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), or single-photon emission computed tomography (SPECT).

[0094] Clause 6. The method of any one of clauses 1-5, wherein the pharmaceutically acceptable excipient comprises a buffer, a contrast-enhancing agent, a radioactive biopolymer, a non-radioactive biopolymer, or a combination thereof.

[0095] Clause 7. The method of clause 6, wherein the contrast-enhancing agent comprises a CT contrast-enhancing agent, a PET contrast-enhancing agent, or a SPECT contrast-enhancing agent.

[0096] Clause 8. The method of any one of clauses 1-7, wherein the pharmaceutical composition comprises the collection of self-assembling conjugates at a concentration of no less than 0.01 to no more than 300 μM.

[0097] Clause 9. The method of any one of clauses 1-8, wherein the pharmaceutical composition is identified in the subject following administration using the image modality.

[0098] Clause 10. The method of any one of clauses 1-9, wherein the elastin-like polypeptide comprises an amino acid sequence of (VPGX1G)p, wherein X1is any amino acid and p is 1 to 500.

[0099] Clause 11. The method of any one of clauses 1-10, wherein the elastin-like polypeptide comprises an amino acid sequence of (VPGX2G)q(GrY)s, wherein X2is any amino acid, q is 1 to 500, r is 0-10, and s is 1-250.

[0100] Clause 12. The method of any one of clauses 1-11, wherein the elastin-like polypeptide comprises an amino acid sequence of (VPGVG)m(GY)n, wherein m is 50-250 and n is 1 to 50.

[0101] Clause 13. The method of clause 12, wherein m is 120 and n is 7.

[0102] Clause 14. The method of any one of clauses 1-13, wherein the collection of self- assembling conjugates has a radionuclide activity of about 1 μCi to about 100 μCi.

[0103] Clause 15. The method of any one of clauses 1-14, wherein the radionuclide is123I,124I,125I, or131I.

[0104] Clause 16. The method of any one of clauses 1-15, wherein the collection of self- assembling conjugates is a micelle.

[0105] Clause 17. The method of any one of clauses 1-16, wherein administration is done percutaneously or endoscopically.

[0106] Clause 18. The method of any one of clauses 1-17, wherein the tumor is a solid tumor.Attorney Docket No.028193-0060-WO01

[0107] Clause 19. The method of any one of clauses 1-18, wherein the tumor is a pancreatic cancer, a liver cancer, a colorectal cancer, a cervical cancer, an ovarian cancer, a sarcoma, a breast cancer, a gastrointestinal cancer, or a melanoma.

[0108] Clause 20. The method of any one of clauses 1-19, wherein the collection of self- assembling conjugates is retained at the tumor for at least 2 days.

[0109] Clause 21. The method of any one of clauses 1-20, wherein the subject is a human.

[0110] Clause 22. A kit comprising: a hollow tube having an internal diameter of no less than 0.4 mm; and a pharmaceutical composition, the pharmaceutical composition including a pharmaceutically acceptable excipient, and a collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM.

[0111] Clause 23. A pharmaceutical composition comprising: a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient includes a buffer and a contrast- enhancing agent selected from the group consisting of a CT contrast-enhancing agent, a PET contrast-enhancing agent, and a SPECT contrast-enhancing agent, the contrast-enhancing agent being included at no more than 50% by volume of the pharmaceutical composition; and a collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM.

[0112] Clause 24. The pharmaceutical composition of clause 23, wherein the contrast- enhancing agent is an iodine-based CT contrast-enhancing agent or a barium-based CT contrast-enhancing agent.Attorney Docket No.028193-0060-WO01 SEQUENCES (VPGX1G)p(SEQ ID NO:1), wherein X1is any amino acid and n is an integer greater than or equal to 1 (VPGX1G)p(SEQ ID NO:2), wherein X1is any amino acid (VPGX2G)q(GrY)s(SEQ ID NO:3) (VPGVG)m(GY)n(SEQ ID NO:4) (VPGVG)120(GY) (SEQ ID NO:5) (VPGVG)120(GY)4(SEQ ID NO:6) (VPGVG)120(GY)7(SEQ ID NO:7)

Claims

Attorney Docket No.028193-0060-WO01 CLAIMS What is claimed is:

1. A method for image-guided brachytherapy in a subject, the method comprising: navigating a hollow tube to a tumor in the subject using an imaging modality, the hollow tube having an internal diameter of no less than 0.4 mm; and administering a pharmaceutical composition through the hollow tube to the tumor, the pharmaceutical composition including a pharmaceutically acceptable excipient and a collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and wherein the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM.

2. The method of claim 1, wherein the hollow tube is navigated along a path of no less than 0.5 cm.

3. The method of claim 1, wherein the hollow tube has a length of no less than 1 cm.

4. The method of claim 1, wherein the hollow tube is a needle, a catheter, a cannula, or a combination thereof.

5. The method of claim 1, wherein the image modality is magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), or single-photon emission computed tomography (SPECT).

6. The method of claim 1, wherein the pharmaceutically acceptable excipient comprises a buffer, a contrast-enhancing agent, a radioactive biopolymer, a non-radioactive biopolymer, or a combination thereof.

7. The method of claim 6, wherein the contrast-enhancing agent comprises a CT contrast- enhancing agent, a PET contrast-enhancing agent, or a SPECT contrast-enhancing agent.

8. The method of claim 1, wherein the pharmaceutical composition comprises the collection of self-assembling conjugates at a concentration of no less than 0.01 to no more than 300 μM.Attorney Docket No.028193-0060-WO01 9. The method of claim 1, wherein the pharmaceutical composition is identified in the subject following administration using the image modality.

10. The method of claim 1, wherein the elastin-like polypeptide comprises an amino acid sequence of (VPGX1G)p, wherein X1is any amino acid and p is 1 to 500.

11. The method of claim 1, wherein the elastin-like polypeptide comprises an amino acid sequence of (VPGX2G)q(GrY)s, wherein X2is any amino acid, q is 1 to 500, r is 0-10, and s is 1- 250.

12. The method of claim 1, wherein the elastin-like polypeptide comprises an amino acid sequence of (VPGVG)m(GY)n, wherein m is 50-250 and n is 1 to 50.

13. The method of claim 12, wherein m is 120 and n is 7.

14. The method of claim 1, wherein the collection of self-assembling conjugates has a radionuclide activity of about 1 μCi to about 100 μCi.

15. The method of claim 1, wherein the radionuclide is123I,124I,125I, or131I.

16. The method of claim 1, wherein the collection of self-assembling conjugates is a micelle.

17. The method of claim 1, wherein administration is done percutaneously or endoscopically.

18. The method of claim 1, wherein the tumor is a solid tumor.

19. The method of claim 1, wherein the tumor is a pancreatic cancer, a liver cancer, a colorectal cancer, a cervical cancer, an ovarian cancer, a sarcoma, a breast cancer, a gastrointestinal cancer, or a melanoma.

20. The method of claim 1, wherein the collection of self-assembling conjugates is retained at the tumor for at least 2 days.Attorney Docket No.028193-0060-WO01 21. The method of claim 1, wherein the subject is a human.

22. A kit comprising: a hollow tube having an internal diameter of no less than 0.4 mm; and a pharmaceutical composition, the pharmaceutical composition including a pharmaceutically acceptable excipient, and a collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM.

23. A pharmaceutical composition comprising: a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient includes a buffer and a contrast-enhancing agent selected from the group consisting of a CT contrast-enhancing agent, a PET contrast-enhancing agent, and a SPECT contrast-enhancing agent, the contrast-enhancing agent being included at no more than 50% by volume of the pharmaceutical composition; and a collection of self-assembling conjugates, each self-assembling conjugate including a radionuclide coupled to an elastin-like polypeptide, wherein the elastin-like polypeptide has a transition temperature of no greater than 37 °C, and the collection of self-assembling conjugates is included in the pharmaceutical composition at a concentration of no more than 300 μM.

24. The pharmaceutical composition of claim 23, wherein the contrast-enhancing agent is an iodine-based CT contrast-enhancing agent or a barium-based CT contrast-enhancing agent.

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