WO2025222014A1

WO2025222014A1 - Compositions and methods for treating cancer

Info

Publication number: WO2025222014A1
Application number: PCT/US2025/025182
Authority: WO
Inventors: Haifeng Yang; Zhijiu ZHONG; John EISENBREY; Kevin Kayvan ZARRABI
Original assignee: Thomas Jefferson University
Current assignee: Thomas Jefferson University
Priority date: 2024-04-17
Filing date: 2025-04-17
Publication date: 2025-10-23
Anticipated expiration: 2026-10-17

Abstract

Provided are compositions comprising anti-melanoma cell adhesion molecule (MCAM) conjugated microbubbles and methods of use for the treatment of clear cell renal cell carcinoma (ccRCC).

Description

COMPOSITIONS AND METHODS FOR TREATING CANCER

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/635,195, filed April 17, 2024, the contents of which are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P30CA056036 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Anti-angiogenesis small receptor tyrosine kinase (RTK) inhibitors and tyrosine kinase inhibitors (TKI) target endothelial cells of the blood vessels of both tumorous and normal vasculature. However, anti-angiogenesis small RTK inhibitors and TKIs are reported to have strong side effects that may limit dosage and lead to therapy discontinuation.

There is a need in the art for reducing side effects and increasing the therapeutic index of these drugs by targeted delivery. The current invention satisfies this need in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of various embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. Figure 1 depicts representative immunohistochemistry (IHC) images demonstrating that melanoma cell adhesion molecule (MCAM) is expressed at a much higher level in human RCC tumor vasculature than in normal adjacent tissue (NAT). IHC images depict MCAM and CD31 expression in bordering RCC tumor vasculature and NAT (cancer vessels are marked by arrows; normal tissue vessels are marked by asterisks). Total intensity of strong positives in annotated NAT and Cancer images were quantified. TMCAM/TCDSI ratios were calculated.

Figure 2, comprising Figure 2A through Figure 2D, depicts representative IHC images demonstrating that MCAM is highly expressed in xenograft vasculature but not in normal mouse kidney vasculature. Figure 2A depicts a representative IHC analysis of MCAM and CD31 performed on consecutive cuts of tissues arrayed on the same slides. Figure 2B depicts representative IHC images of CD31 and MCAM stained samples from xenograft tumor made with RCC4 cells. Figures 2C and Figure 2D depict representative IHC images of CD31 and MCAM stained samples from normal mouse kidney.

Figure 3 depicts representative IHC images demonstrating that MCAM is highly expressed in clear cell renal cell carcinoma (ccRCC) tumor vasculature but much lower in normal vasculature in human organs and tissues. Consecutive cuts of human ccRCC and normal human organs and tissues were arrayed on the same slides, and analysis of MCAM and CD31 were performed as in Figure 2.

Figure 4 depicts representative IHC images demonstrating that MCAM is highly expressed in ccRCC tumor vasculature from stages I to IV. MCAM and CD31 expressions in human RCC tumor vasculature and NAT were analyzed. Representative images from Stage I and IV cancer and NAT are presented. Total intensity of positives in Normal and Cancer images were quantified. TMCAM/TCDSI ratios were calculated for RCC and Normal in stage I patients 6, 7, 8, 10, and 19.

Figure 5, comprising Figure 5 A through Figure 5F, depicts representative experimental results demonstrating that anti -MC AM-conjugated microbubbles had greater retention in xenograft tumors than in mouse kidneys. MCAM Ab-conjugated microbubbles retention in ccRCC tumors, but not in normal kidneys, is prolonged. Figure 5A depicts a representative image of 786-0 PBRMlsh94 subcutaneous xenograft RCC tumor with B-mode ultrasound (left side) and CEUS (right side) as baseline. Figure 5B, Figure 5C, and Figure 5D depict representative ultrasound images of tail-vein injected MCAM Ab-conjugated microbubbles in both modes after washing, pre-bursting, and after bursting, respectively. Images of non-immune IgG conjugated microbubbles before and after tail-vein injection were captured in the same way. Linear CEUS values were measured after imaging (Figure 5C and Figure 5D, right side). Figure 5E depicts a bar graph showing the differences in ultrasound signal intensity between MCAM Ab- conjugated and IgG-conjugated microbubbles. Figure 5F depicts a bar graph showing the results from the same experiment performed on mouse normal kidneys.

Figure 6 depicts representative IHC images demonstrating that MCAM preferentially stains tumor vasculature on human ccRCC samples.

Figure 7 depicts representative IHC images of human kidney normal adjacent tissues.

Figure 8 depicts representative IHC images of human ccRCC.

Figure 9 depicts representative IHC images demonstrating that Anti-X strongly stains the tumor vasculature in xenografts formed by Ren-02 ccRCC cancer cells but weakly stained the blood vessels in normal mouse kidney.

Figure 10 depicts representative IHC images demonstrating that there is similar expression of MCAM to CD31 in xenograft made with 786-O-PBRMlsh94.

Figure 11 depicts representative IHC images demonstrating that there is lower expression of MCAM in the mouse colon.

Figure 12 depicts representative IHC images demonstrating that there is lower expression of MCAM in the mouse skin.

Figure 13 depicts representative IHC images demonstrating that there is lower expression of MCAM in the mouse lung.

Figure 14, comprising Figure 14A through Figure 141, depicts experimental results demonstrating that anti-MCAM antibody-conjugated microbubbles are not retained in the mouse kidneys longer than the non-targeting IgG-conjugated microbubbles. Figure 14A and Figure 14B depicts representative images of the kidney of a nude mouse imaged with B-mode ultrasound (Figure 14A, left side) and CEUS (Figure 14B, right side) as baseline. After tail-vein injection of anti-MCAM Ab-conjugated microbubbles, wash in (Figure 14B), pre burst (Figure 14C), and after burst (Figure 14D) ultrasound images of both modes were obtained. Before and after tail-vein injection of IgG-conjugated microbubbles images were captured the same way as in the first injection (Figure 14E through Figure 14H). Measurement of the linear CEUS values was performed afterwards (Figure 14C, Figure 14D, Figure 14G, and Figure 14H, right sides). Figure 141 depicts a representative quantification of the change in echo signal values for anti-MCAM Ab-conjugated microbubbles and IgG-conjugated microbubbles.

Figure 15 depicts representative IHC images demonstrating MCAM is expressed at high levels in human tumor vasculature of lung squamous cell carcinoma.

Figure 16 depicts representative IHC images demonstrating MCAM is expressed at high levels in human tumor vasculature of invasive breast cancer.

Figure 17 depicts representative IHC images demonstrating MCAM is expressed at high levels in human tumor vasculature of colon adenocarcinoma.

Figure 18 depicts representative IHC images demonstrating MCAM is expressed at high levels in human tumor vasculature of metastatic squamous cell carcinoma and pheochromocytoma.

Figure 19 depicts representative IHC images demonstrating MCAM is expressed at higher levels in human tumor vasculature of various tumors than in vasculatures of normal adjacent tissues in KD1502 TMA (kidney cancers).

Figure 20, comprising Figure 20A through Figure 20J, depicts representative experimental results demonstrating MCAM is highly expressed in xenograft vasculature but not in normal mouse kidney vasculature. Figure 20A depicts representative images of mouse xenograft tumors made with various human kidney cancer cell lines with different genotypes arrayed with a normal mouse kidney. Consecutive slides were cut from the array, and anti-CD31 and anti-MCAM immunohistochemistry (IHC) were performed on the respective slides. Figure 20B through Figure 20H depict representative magnified micrographs of anti-CD31 and anti- MCAM presented on RCC4 xenograft tumor (Figure 20B), mouse kidney collecting tubules (Figure 20C), mouse glomeruli (Figure 20D), 786-0 SCR (scramble shRNA) (Figure 20E), 786-0 sh-PBRMl (shRNA against PBRM1, a major tumor suppressor in ccRCC) (Figure 20F), Ren-02 SCR (scramble shRNA) (Figure 20G), and Ren-02 sh- BAP1 (shRNA against BAP1, another major tumor suppressor in ccRCC) (Figure 20H). Figure 201 depict representative images illustrating how the total intensity of positive signals for MCAM and CD31 IHC were quantified for each sample with Aperio ImageScope [vl2.4.6.5003]. Figure 20J depicts a graph presenting the relative ratios of TMCAM/TCD31.

Figure 21, comprising Figure 21Athrough Figure 21E, depicts representative experimental results demonstrating MCAM is highly expressed in xenograft vasculature but not in vasculatures of normal mouse organs. Figure 21 A through Figure 2 ID depict representative images of mouse xenograft tumors made with two human kidney cancer cell lines with different genotypes arrayed with normal mouse organs. Consecutive slides were cut from the array, and anti-CD31 and anti-MCAM IHC were performed on the respective slides. On each slide, the magnified micrographs of anti-CD31 and anti-MCAM IHC of 786-0 sh-PBRMl tumor and Ren-02 sh-BAPl tumor were presented as positive controls. Figure 2 IE depict representative magnified micrographs of anti-CD31 and anti-MCAM IHC of mouse stomach, intestine, colon, bladder, brain, heart, lung, muscle, pancreas and skin.

Figure 22, comprising Figure 22A through Figure 22C, depicts representative experimental results demonstrating MCAM is highly expressed in tumor vasculature but not in vasculatures of many normal human organs. Figure 22A and Figure 22B depict representative images of tissue microarrays holding normal human organs. Human kidney and ccRCC tissue slices added together with mouse xenograft tumor samples made with Ren-02 SCR and Ren-02 shB Pl cancers onto tissue microarrays holding normal human organs. The slides were stained with anti-CD31 (Figure 22A) or anti-MCAM (Figure 22B) for IHC respectively. Figure 22C depicts representative magnified micrographs of anti-CD31 and anti-MCAM IHC of human ccRCC tumor and human adrenal gland tumor were presented as positive controls. The magnified micrographs of anti-CD31 and anti-MCAM IHC of human larynx, adrenal gland, lung, liver, kidney, pancreas, esophagus, stomach, small intestine, colon, heart, pituitary gland, lymph node, skin, muscle, spleen, testis, salivary gland, diaphragm, prostate, cerebrum, cerebellum, cervix, ovary, uterus and breast. Figure 23, comprising Figure 23 A through Figure 231, depict representative experimental results demonstrating MCAM is expressed at higher levels in tumor vasculature of human ccRCC at various stages than in vasculatures of normal adjacent tissues. Figure 23 A depicts representative images of tissue microarrays demonstrating MCAM is expressed at higher levels in tumor vasculature of human Stage

1 ccRCC than in vasculatures of normal adjacent tissues. Figure 23B depicts representative IHC images demonstrating MCAM is expressed at higher levels in tumor vasculature of human Stage 1 ccRCC than in vasculatures of normal adjacent tissues. Figure 23C depicts representative quantification demonstrating MCAM is expressed at higher levels in tumor vasculature of human Stage 1 ccRCC than in vasculatures of normal adjacent tissues. Figure 23D depicts representative images of tissue microarrays demonstrating MCAM is expressed at higher levels in tumor vasculature of human Stage

2 ccRCC than in vasculatures of normal adjacent tissues. Figure 23E depicts representative IHC images demonstrating MCAM is expressed at higher levels in tumor vasculature of human Stage 2 ccRCC than in vasculatures of normal adjacent tissues. Figure 23G depicts representative tissue microarrays demonstrating MCAM is expressed at higher levels in tumor vasculature of human Stage 3 ccRCC than in vasculatures of normal adjacent tissues. Figure 23H depicts representative IHC images demonstrating MCAM is expressed at higher levels in tumor vasculature of human Stage 3 ccRCC than in vasculatures of normal adjacent tissues. Figure 231 depicts representative IHC images demonstrating MCAM is expressed at higher levels in tumor vasculature of human Stage 4 ccRCC than in vasculatures of normal adjacent tissues.

Figure 24 depicts representative IHC images demonstrating MCAM is expressed at higher levels in human tumor vasculature of various tumors than in vasculatures of normal adjacent tissues in KD1502 TMA (kidney cancers) and various urological cancers.

DETAILED DESCRIPTION

The invention is based, in part, on the discovery that melanoma cell adhesion molecule (MCAM) is differentially expressed in cancerous tissues as compared with normal tissues and, thus, can be used as a biomarker and a target for the delivery of one or more therapeutic agents. Therefore, the invention relates to compositions and methods for treating and preventing cancer through targeted delivery of one or more therapeutic agents.

Definitions

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

As used herein, each of the following terms has the meaning associated with it in this section.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Natural antibodies are typically tetramers of immunoglobulin molecules. The term “antibody” as used herein encompasses antibody fragments, which refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Antibodies or antibody fragments as described herein may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fab, F(ab)2, Fab’, F(ab’)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antigen” or “Ag” as used herein is defined as a molecule that binds to an antibody or a T cell receptor. Any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. The present disclosure provides, but is not limited to, the use of partial nucleotide sequences. Moreover, an antigen need not be encoded by a “gene” at all. An antigen can be generated, synthesized, or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

“Cancer” or “tumor,” as used herein, refers to the abnormal growth or division of cells. Generally, the growth and/or life span of a cancer cell exceeds, and is not coordinated with, that of the normal cells and tissues around it. Cancers may be benign, pre-malignant or malignant. Cancer relevant to the invention occurs in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gall bladder, pancreas, etc.), respiratory system (e.g., larynx, lung, bronchus, etc ), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, genital system, (e.g., uterus, ovary, prostate, testis, etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eye, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

“Isolated” as used herein means (1) altered or removed from the natural state and/or (2) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting) and/or otherwise previously associated, and/or (3) designed, produced, prepared, and/or manufactured by the hand of man. In some embodiments, a nucleic acid or a peptide naturally present in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

As used herein, the term “sonoporation” refers to any ultrasound induced enhancement of cell membrane permeability, either with or without the presence of microbubbles.

As used herein, the term “microbubble” refers to any spherical arrangement of lipids creating an outer shell and an inner void space. The lipid layer may be modified to bind molecules in a stable manner.

As used herein, the “mechanical index” refers to any index of acoustic energy being delivered to a composition (i.e., for example, a tissue or a microbubble). Mathematically, a mechanical index is equal to the acoustic pressure (mPa) divided by the square root of the ultrasound frequency. UMTD microbubbles may have a mechanical index ranging between approximately 0.25-2.5, between 0.5-2.0, between 0.75-1.75, or between 1.0-1.5.

As used herein, the “bursting threshold” refers to any acoustic frequency that results in the lipid shell breakdown of a microbubble population, thereby releasing the stably bound nucleic acids. Such acoustic frequencies are usually generated by an ultrasound device operating at a frequency ranging between approximately 0.25-5 MHz, between approximately 0.5-2.5 MHz, between approximately 0.75-2.0 mHz, or between approximately 1.0-1.5 MHz. For example, a bursting threshold of UMTD microbubbles may be approximately between 1.3- 1.4 MHz.

The terms “subject,” “patient,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In some non-limiting embodiments, the patient, subject or individual is a mammal, bird, poultry, cattle, pig, horse, sheep, ferret, primate, dog, cat, guinea pig, rabbit, bat, or human.

A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.

In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, prevention, or eradication of at least one sign or symptom of a disease or disorder.

The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention is based, in part, on the discovery that delivery vehicles (e.g., a microbubble) directed to MCAM can deliver therapeutic agents to cancer cells and tissues. Accordingly, the present disclosure provides compositions comprising delivery vehicles that can be modified (e.g., with a targeting domain that binds a targeted molecule, such as MCAM) to deliver at least one therapeutic agent to treat cancer. In various embodiments, the composition comprises a delivery vehicle, a targeting domain, and at least one therapeutic agent.

Delivery Vehicle

In various embodiments, the delivery vehicle is a polymeric particle, inorganic particle, silica particle, liposome, micelle, multilamellar vesicle, or microbubble. In some embodiments, the delivery vehicle is a microbubble. In some embodiments, the delivery vehicle is an ultrasound enhancing microbubble.

Microbubbles are important contrast agents for diagnostic, theranostic, or therapeutic purposes in that they can provide simultaneous and co-localized contrast for imaging and drug carrying and delivering capacity for targeted therapy. The imaging modality and therapeutic trigger is ultrasound, which is focused to microscale events distributed throughout the insonified vasculature. A gas core, e.g. air, perfluorobutane, etc. provides the mechanism for ultrasound backscatter. Gas bubbles of this size in aqueous media are unstable owing to surface tension effects and require a stabilizing shell. The shell may be composed of surfactants, lipids, proteins, polymers, or a combination of these materials.

Microbubbles have useful effects when they are insonified by ultrasound. At low acoustic pressures, an insonified microbubble produces a backscattered echo that can be used to detect and locate the microbubble. The microbubble can therefore be used as a contrast agent in ultrasound imaging. The echogenicity, or relative strength of the backscattered signal, is strongest near the microbubble resonance frequency. Microbubbles of a few micrometers in diameter resonate at frequencies in the 1-10 MHz range which is the range of typical ultrasound clinical imaging scanners. Thus, microbubbles are highly echogenic to conventional ultrasound. Additionally, microbubbles scatter ultrasound nonlinearly. Imaging pulse sequences with modulated phase, frequency and amplitude can be used to separate the microbubble and tissue signals with high fidelity.

At higher acoustic pressures, the microbubble may become unstable during oscillation and fragment into daughter microbubbles. Fragmentation is a useful means of eliminating the contrast agent signal within the transducer focus. Microbubble fragmentation is being employed to measure reperfusion in tumor and cardiac tissue and in ultrasound molecular imaging protocols.

At acoustic pressures just below the fragmentation threshold, a microbubble will undergo dissolution, e.g. for drug delivery. At high acoustic pressures and lower frequencies inertial cavitation occurs and can be exploited for drug delivery.

Various microbubble products are available commercially, including microbubbles marketed under the trade names ALBUNEX®, DEFINITY®, TRUST Biosonics, and OPTISON®. In some embodiments, the microbubbles used in the procedures described herein are selected from such commercially available materials and are further modified to include targeting moieties as described herein.

In some embodiments, microbubbles (including multi-layered microbubbles) are prepared using methods known in the art; for example, according to the process reported in Liu et al., J. Controlled Release, 114 (2006) 89-99, and references cited therein. In some embodiments, microbubbles are prepared according to the process reported in Hu et al., J. Controlled Release, 147 (2010) 154-162, and references cited therein. In some embodiments, microbubbles are prepared according to the process reported in Hernot et al., Adv. Drug Delivery Rev. 60 (2008) 1153-1166, and references cited therein. In some embodiments, microbubbles are prepared according to the process reported in Geers et al., J. Controlled Release 148 (2010) e57-e73 (abstracts), and references cited therein. In some embodiments, microbubbles are prepared according to the process reported in Tinkov et al., J. Controlled Release 143 (2010) 143-150, and reference cited therein. Additional synthetic details for preparing (untagged) microbubbles can be found in Mayer et al., Adv. Drug Delivery Rev. 60 (2008) 1177- 1192. The procedures from any of the above-cited references can be modified so as to prepare the targeting microbubbles of interest.

The microbubbles may include a shell surrounding a hollow core. In some embodiments, the shell is composed of bio-lipids, proteins (e.g., albumin), surfactants, biocompatible polymers, or any combination thereof. Specific examples of such materials are provided herein below. In some embodiments, the shell is pegylated. In some embodiments, the hollow core is filled with a gas or low boiling fluid, and examples of such gases and fluids are also provided herein below. The microbubbles are designed with a shape and size to nucleate cavitation, which refers to the formation and collapse of gaseous microbubbles. The violent collapse of cavitation bubbles releases energy that can cause the fragmentation of an adjacent mass.

In some embodiments, the microbubbles described herein are modified to carry a chemical tag (referred to herein as “targeting moieties,” “targeting domain,” or “functional moieties”) on or near their surface. Such tags are selected to target a specific location, mass, molecule or structure in vivo. Because of the targeting, microbubbles concentrate at the targeted location, mass, molecule or structure and can be used in therapeutic treatments as described herein.

Alternatively or in addition, the microbubbles can be used to transport a load of material (e.g., a therapeutic agent) within the core to a specific mass, location, or structure in vivo. For example, gas-filled microbubbles are synthesized with one or more tags for targeting a specific location, tissue, tumor, or mass. The microbubbles are delivered to the target as part of a pharmaceutically acceptable formulation. Upon attachment to or association with the target, cavitation is induced with consequent disruption or fragmentation of the target.

The contents of the microbubble can vary with application. In some embodiments, the microbubble contains air, CO2, a fluorinated or perfluorinated gas (e.g. a perfluorinated alkane such as perfluoropropane), another gas, or mixtures thereof. In other embodiments, the microbubble may contain a low boiling (e.g., normal boiling point less than about 30° or 35° C ). This allows that a deflated microbubble may be injected into the patient, said microbubble inflating as it heats to physiological temperatures (ca. 37° C ). In other embodiments, the microbubbles can be fdled partially or completely with a payload other than a gas, such as a pharmaceutically active agent, a therapeutic agent, a cytotoxic agent, an imaging agent, or the like.

The microbubbles are intended for delivery to the site of a targeted mass or tissue that is to be reduced in size or eliminated. The microbubbles are tagged with a targeting domain so that they selectively bind or associate with the target.

Various sizes and shapes of microbubbles are suitable based on the specific intended applications. In some embodiments, the microbubbles are selected from spherical, ellipsoidal, disk-shaped, and asymmetric shapes. In some embodiments, the shape of the microbubbles is not static. For example, in some embodiments, the unperturbed microbubbles may be spherical, but the microbubbles may adopt a different shape such as ellipsoidal or disk-shaped when an external force (e.g., a flowing fluid such as blood) is present.

In some embodiments, the microbubbles have an average diameter (wherein “average diameter” refers to the largest dimension for non-spheroidal shapes) between 0.1 pm and 10 pm, or between 0.5 pm and 10 pm, or between 1 pm and 10 pm. In some embodiments, the average diameter is between 0.5 pm and 3 pm, or between 1 pm and 2 pm. In some embodiments, the microbubbles have an average diameter less than 10 pm, or less than 5 pm, or less than 1 pm, or less than 0.5 pm, or less than 0.1 pm. In some embodiments, the microbubbles have an average diameter greater than 0.1 pm, or greater than 0.5 gm, or greater than 1 gm, or greater than 5 gm, or greater than 10 gm. In some embodiments, the microbubbles have an average diameter less than 1.6 gm. In some embodiments, the microbubbles have an average diameter that is smaller than blood vessels. In some embodiments, the microbubbles have an average diameter that is smaller than red blood cells. The synthetic processes described herein allow the production of microbubbles of various sizes and materials. It will be appreciated that use of the term “microbubbles” is not intended to limit the size of the microbubbles to any particular range (e.g., micron diameters).

In some embodiments, the microbubbles are targeted to the mass of interest by the attachment of a targeting agent, targeting domain, or tag, for example to the surface of the microbubble. For example, microbubbles can be chemically functionalized using a variety of techniques, the details of such techniques being dependent on the exact chemical moiety to be attached.

By way of non-limiting examples, the targeting domain can be attached to the delivery vehicle using avidin-biotinylation, peggylation, a linker, a peptide, a peptide linker, a hydrophobic peptide, a hydrophobic peptide linker, covalent bond, ionic bond, etc. The targeting domain is chosen based on properties of the target tissue, target cell, target molecule, or target mass as well as the structure and chemical properties of the microbubbles. A variety of targeting domains may be used, some of which are described in more detail herein.

Targeting domains and other functional groups can be attached asymmetrically or in patterns as needed for a particular application. In some embodiments there is directional modification of the surface of the microbubbles. For some applications, only one part of the surface of the microbubble is functionalized with a tagging domain in order to direct energy toward or away from the intended target.

Targeting Domain

In some embodiments, the invention encompasses a delivery vehicle (e.g., microbubble) comprising a targeting domain that directs the delivery vehicle to specific target molecule, target cell, or target tissue as mediated by binding of the targeting domain to a target molecule, cell or tissue. In various embodiments, the targeting domain comprises a nucleic acid, peptide, antibody, antibody binding domain, small molecule, organic molecule, inorganic molecule, glycan, sugar, hormone, and the like that targets the delivery vehicle to a target location, tissue or cell where the therapeutic agent is desired. In some embodiments, the targeting domain is an antibody, or a binding portion, variant, or fragment thereof. An antibody may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879- 5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the targeting domain of a composition of the invention comprises an antibody fragment. In one embodiment, the targeting domain comprises an antibody fragment that comprises a scFv.

In some embodiments, the targeting domain is covalently attached to the composition comprising the delivery vehicle, such as through a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is attached to the delivery vehicle by biotinavidin interactions. In some embodiments, the delivery vehicle comprises biotin. In some embodiments, the targeting domain comprises biotin. In some embodiments, the delivery vehicle comprises avidin. In some embodiments, the targeting domain comprises avidin. In some embodiments, the targeting domain is attached to the delivery vehicle by pegylation. In some embodiments, the targeting domain is pegylated. In some embodiments, the delivery vehicle is pegylated. In some embodiments, the targeting domain is an additive in the delivery vehicle.

In some embodiments, the composition comprises a delivery vehicle conjugated to a targeting domain that binds a cell surface molecule of a target cell of interest (e.g., a cancer cell, etc.), thereby directing the composition to the target cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the endothelial cell is present in the vasculature of a tumor. In some embodiments, the endothelial cell is present in the vasculature of a cancer cell from primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, nonHodgkin’s lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinoma such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like. In some embodiments, the endothelial cell is present in the vasculature of a tumor from clear cell renal cell carcinoma (ccRCC), angiomyolipoma, leiomyosarcoma, Wilm’s tumor, chromophobe RCC, papillary RCC, invasive urothelial carcinoma, squamous cell carcinoma, breast cancer, colon adenocarcinoma, or pheochromocytoma. In some embodiments, the endothelial cell is present in the vasculature of a ccRCC tumor.

In some embodiments, the targeting domain preferentially, or specifically, binds to a cell surface molecule on a target cell, such as an endothelial cell or a cancer cell, etc. In some embodiments, the targeting domain preferentially, or specifically, binds to MCAM. In some embodiments, MCAM is present on the cell surface of an endothelial cell. In some embodiments, MCAM is present on the cell surface of an endothelial cell that is present in the vasculature of a tumor. In some embodiments, MCAM is present on the cell surface of an endothelial cell that is present in the vasculature of a cancer cell from primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinoma such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like. In some embodiments, MCAM is present on the cell surface of an endothelial cell that is present in the vasculature of a tumor from ccRCC, angiomyolipoma, leiomyosarcoma, Wilm’s tumor, chromophobe RCC, papillary RCC, invasive urothelial carcinoma, squamous cell carcinoma, breast cancer, colon adenocarcinoma, or pheochromocytoma. In some embodiments, MCAM is present on the cell surface of an endothelial cell that is present in the vasculature of a ccRCC tumor.

Therapeutic Agent

In various embodiments, the composition of the invention comprises a therapeutic agent. Therapeutic agents include any therapeutic agent that has a therapeutic effect on a target tissue or target cell, such as a cancer cell. Therapeutic agents include, without limitation, chemotherapeutic agents, toxins, radioactive isotopes, kinase inhibitors, immunomodulators, hormone blockers, etc.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to, abitrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracyclines, azacitidine, azathioprine, bicnu, blenoxane, busulfan, bleomycin, camptosar, camptothecins, carboplatin, carmustine, cerubidine, chlorambucil, cisplatin, cladribine, cosmegen, cytarabine, cytosar, cyclophosphamide, cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elspar, epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine, gemzar, hycamtin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide, ifex, irinotecan, lanvis, leukeran, leustatin, matulane, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mithramycin, mutamycin, myleran, mylosar, navelbine, nipent, novantrone, oncovin, oxaliplatin, paclitaxel, paraplatin, pentostatin, platinol, plicamycin, procarbazine, purinethol, ralitrexed, taxotere, taxol, teniposide, thioguanine, tomudex, topotecan, valrubicin, velban, vepesid, vinblastine, vindesine, vincristine, vinorelbine, VP- 16, and vumon.

In some embodiments, the therapeutic agent is a toxin. Toxins include toxins of animal, plant or microbial origin. Exemplary toxins include Pseudomonas exotoxin, ricin, abrin, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, and Pseudomonas endotoxin.

In some embodiments, the therapeutic agent is a radioactive isotope. Therapeutic radionuclides include, but are not limited to, ⁿⁱIn, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁷⁷Br, ^113mIn, ⁹⁵RU, ⁹⁷RU, ¹⁰³RU, ¹⁰⁵RU, ¹⁰⁷Hg, ²⁰³Hg, ^121mTe, ¹²²mTe, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹TI, ²²⁵ Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In some embodiments, the therapeutic agent is an immunomodulator. Immunomodulators include, but are not limited to, a cytokine, a lymphokine, a monokine, a stem cell growth factor, a lymphotoxin (LT), a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, a transforming growth factor (TGF), such as TGF-a or TGF-0, insulin-like growth factor (IGF), erythropoietin, thrombopoietin, a tumor necrosis factor (TNF) such as TNF-a or TNF-P, a mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM- CSF), an interferon such as interferon-a, interferon-P, or interferon-y, SI factor, an interleukin (IL) such as IL-1, IL-lcc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 IL-21 or IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, and LT.

In some embodiments, the therapeutic agent is a hormone blocker. Hormone blockers include, but are not limited to, agents that block ER receptors (e.g. tamoxifen) or that block the production of estrogen, such as an aromatase inhibitor (e.g. anastrozole, or letrozole).

In some embodiments, the therapeutic agent is a receptor tyrosine kinase (RTK) inhibitor or a tyrosine kinase inhibitor (TKI). In some embodiments, the therapeutic agent is VEGFR or VEGFR2 TKI. In various embodiments, the therapeutic agent is at least one selected from the group consisting of Deucravacitinib, Avapritinib, Capmatinib, Pemigatinib, Ripretinib, Selpercatinib, Selumetinib, Tucatinib, Entrectinib, Erdafitinib, Fedratinib, Pexidartinib, Upadacitinib, Zanubrutinib, Baricitinib, Binimetinib, Dacomitinib, Fostamatinib, Gilteritinib, Larotrectinib, Lorlatinib, Acalabrutinib, Brigatinib, Midostaurin, Neratinib, Alectinib, Cobimetinib, Lenvatinib, Osimertinib, Ceritinib, Nintedanib, Afatinib, Ibrutinib, Trametinib, Axitinib, Bosutinib, Cabozantinib, Ponatinib, Regorafenib, Tofacitinib, Crizotinib, Ruxolitinib, Vandetanib, Pazopanib, Lapatinib, Nilotinib, Dasatinib, Sunitinib, Sorafenib, Erlotinib, Gefitinib, Imatinib, and Tivozanib. In some embodiments, the therapeutic agent is at least one selected from the group consisting of Sunitinib, Tivozanib, and Axitinib. Formulation

The compositions of the invention can be prepared for administration to a subject, for example by injection, spray, implantation, or the like. In some embodiments, the compositions of the invention are prepared with therapeutically effective amounts of a therapeutic agent in a pharmaceutical preparation in a pharmaceutically acceptable carrier.

In some embodiments, the composition of the invention is prepared for administration to a patient or subject. The composition may be dispersed in fluid for injection or formulated as an aerosol spray for introduction near the target.

In some embodiments, the composition of the invention is prepared as a slurry or emulsion suitable for injection, administration via an aerosol spray, or introduction via a catheter.

In addition to the composition and a pharmaceutically acceptable carrier, various other agents may be added to the formulations as desired. In some embodiments, one or more surfactants are included in the formulation. In other embodiments, no surfactants are added to the microbubble formulation. Other additives that may be present include pH-modifying agents, preservatives, labeling compounds and/or image enhancing compounds, salts, and the like.

Delivery and Administration

In various embodiments, the composition of the invention is administered into or near a target mass, tissue, cell, or tumor, or other site of interest. In some embodiments, the composition is administered, by way of non-limiting examples, by injection or spray. In some embodiments, the composition of the invention is administered to the blood, bile, urine, or cerebral spinal fluid. In some embodiments, composition of the invention is administered to an organ. In some embodiments, composition of the invention is administered via an orifice of the body. Orifices include any opening such as the mouth, nose, eyes, vagina, urethra, and ears. In some embodiments, composition of the invention is administered through the skin.

In some embodiments, the composition of the invention is introduced directly at the target site, such as by direct injection into a target tissue or mass. In other embodiments, composition of the invention is administered at a location that is remote to the target site (e.g., into the bloodstream via injection) and are allowed to accumulate or concentrate at the targeted site.

In various embodiments, composition of the invention is administered as part of a pharmaceutical formulation that can include, for example, solvents or other carriers, additives (e.g., stabilizers and preservatives, colorants, surfactants, pH- modifiers, etc.), and/or one or more pharmaceutically active agents.

Methods of Treatment

In some embodiments, the invention is a method of treating a disease or disorder in a subject in need thereof, comprising administering the composition of the invention to a subject. In some embodiments, the invention is a method of treating a disease or disorder in a subject in need thereof, comprising administering the composition of the invention to a subject and applying energy to disrupt the structure (e.g., cavitation) of the delivery vehicle to allow the therapeutic agent to be released. Disruption of the structure of the delivery vehicle can be initiated by a variety of means. In some embodiments, such means involve the application of energy. In some embodiments, such energy is applied to the subject from outside of the body of the subject. In some embodiments, such energy is applied to the subject from inside of the body of the subject. Examples include application of directed ultrasound and radio waves. In some embodiments, electromagnetic (EM) energy of frequencies between 400 kHz and 10 MHz is suitable because it propagates through tissue without strong interactions (due to low electrical conductivity). In one example, standard ultrasound units are applied within or adjacent to the body with sufficient power to initiate cavitation of the pre-positioned delivery vehicle.

In some embodiments, the invention is a method of treating cancer, comprising administering to the subject an effective amount of a composition of the invention. In some embodiments, the composition comprises a delivery vehicle, a targeting domain, and at least one therapeutic agent. In some embodiments, the delivery vehicle comprises a microbubble. In some embodiments, the composition comprises a delivery vehicle comprising a targeting domain. In some embodiments, the delivery vehicle is targeted to a cancer cell. In some embodiments, the delivery vehicle is targeted to a cell surface molecule of a cancer cell. In some embodiments, the delivery vehicle is targeted to an endothelial cell. In some embodiments, the delivery vehicle is targeted to an endothelial cell that is part of the vasculature of a tumor. In some embodiments, the composition comprises a microbubble comprising an MCAM targeting domain. In some embodiments, the composition comprises a delivery vehicle comprising a therapeutic agent. In some embodiments, the composition comprises a delivery vehicle comprising an MCAM targeting domain and a therapeutic agent.

In some embodiments, the targeted cell is an endothelial cell associated with cancer and/or is part of the vasculature of a tumor. Cancers that are treatable using the compositions and methods of the invention include, but are not limited to, adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing’s family of tumors (e.g. Ewing’s sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Kaposi’s sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumors, male breast cancer, malignant mesothelioma, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, squamous cell carcinomas, sarcomas, melanoma skin cancer, non-melanoma skin cancers, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, ccRCC, angiomyolipoma, leiomyosarcoma, Wilm’s tumor, chromophobe RCC, papillary RCC, invasive urothelial carcinoma, breast cancer, or Pheochromocytoma.

Effective amounts of the delivery vehicle, or the therapeutic agent, of the present invention for the treatment of cancer, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but nonhuman mammals may also be treated, e.g. companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like. Treatment dosages can be titrated to optimize safety and efficacy.

In some embodiments, the therapeutically effective amount of the delivery vehicle, or the therapeutic agent, ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example, the therapeutically effective amount can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. Exemplary treatment regimens include a single administration or multiple administrations. Exemplary treatment regimens include administration once a day, once a week, once every two weeks, once a month, once every 2 months, once every 2-6 months, twice a day, twice a week, twice every two weeks, twice a month, twice every 2 months, twice every 2-6 months, three times a day, three times a week, three times every two weeks, three times a month, three times every 2 months, three times every 2-6 months etc. In various embodiments, intervals between administrations can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the therapeutic agent in the patient. Alternatively, therapeutic agents of the invention can be administered as a sustained release formulation. Dosage and frequency may vary depending on the half-life of the therapeutic agent in the patient.

In prophylactic administrations, a relatively low dosage may be administered at relatively infrequent intervals over a long period of time. Some patients may continue to receive treatment for the rest of their lives. In other therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease or disorder is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.

Such dosage forms encompass physiologically acceptable carriers that are inherently non-toxic and non-therapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and PEG. Carriers for topical or gel-based forms of polypeptides include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene- polyoxypropylene-block polymers, PEG, and wood wax alcohols. For all administrations, conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained-release preparations.

In some embodiments, the therapeutic agent is a conventional chemotherapeutic agent or other biological anti -cancer drug such as a checkpoint inhibitor (e.g., PD1 or PDL1 inhibitors) or a therapeutic monoclonal antibody (e g., Avastin, Herceptin).

In some embodiments, the therapeutic agent is a chemical agent. Examples of chemical agents that are useful in the compositions and methods of the invention, include but are not limited to, abitrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracyclines, azacitidine, azathioprine, bicnu, blenoxane, busulfan, bleomycin, camptosar, camptothecins, carboplatin, carmustine, cerubidine, chlorambucil, cisplatin, cladribine, cosmegen, cytarabine, cytosar, cyclophosphamide, cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elspar, epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine, gemzar, hycamtin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide, ifex, irinotecan, lanvis, leukeran, leustatin, matulane, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mithramycin, mutamycin, myleran, mylosar, navelbine, nipent, novantrone, oncovin, oxaliplatin, paclitaxel, paraplatin, pentostatin, platinol, plicamycin, procarbazine, purinethol, ralitrexed, taxotere, taxol, teniposide, thioguanine, tomudex, topotecan, valrubicin, velban, vepesid, vinblastine, vindesine, vincristine, vinorelbine, VP-16, and vumon.

In some embodiments, the therapeutic agent is a tyrosine-kinase inhibitor, such as Imatinib mesylate (Gleevec, also known as STI-571), Gefitinib (Iressa, also known as ZD1839), Erlotinib (marketed as Tarceva), Sorafenib (Nexavar), Sunitinib (Sutent), Dasatinib (Sprycel), Lapatinib (Tykerb), Nilotinib (Tasigna), and Bortezomib (Velcade), Jakafi (ruxolitinib); a Janus kinase inhibitor, such as tofacitinib; an ALK inhibitor, such as crizotinib; a Bcl-2 inhibitor, such as obatoclax, venclexta, and gossypol; a FLT3 inhibitor, such as midostaurin (Rydapt), an IDH inhibitor, such as AG-221; a PARP inhibitor, such as Iniparib and Olaparib; a PI3K inhibitor, such as perifosine; a VEGF Receptor 2 inhibitor, such as Apatinib; AN- 152 (AEZS-108) doxorubicin linked to [D-Lys(6)]-LHRH; a Braf inhibitor, such as vemurafenib, dabrafenib, and LGX818; a MEK inhibitor, such as trametinib; a CDK inhibitor, such as PD-0332991 and LEE011; an Hsp90 inhibitor, such as salinomycin; a small molecule drug conjugate, such as Vintafolide; and a serine/threonine kinase inhibitor, such as Temsirolimus (Torisel), Everolimus (Afinitor), Vemurafenib (Zelboraf), Trametinib (Mekinist), and Dabrafenib (Tafinlar).

In some embodiments, the therapeutic agent is a cytokine or cytokine antagonist, such as IL-12, INF a, or anti-epidermal growth factor receptor, radiotherapy, irinotecan; a tetrahydrofolate antimetabolite, such as pemetrexed; an antibody against a tumor antigen, a complex of a monoclonal antibody and toxin, a T-cell adjuvant, a signal transduction inhibitor (e.g., Gleevec® or Herceptin®) or an immunomodulator, a cyclooxygenase-2 (COX-2) inhibitor, a steroid, and a TNF antagonist (e.g., Remicade® and Enbrel®), interferon-P 1 a (Avonex®), and interferon-31b (Betaseron®).

In some embodiments, the therapeutic agent is, or can be combined with, immune checkpoint therapy. Examples of immune checkpoint therapies include, but are not limited to, inhibitors of the binding of PD1 to PDL1 and/or PDL2. PD1 to PDL1 and/or PDL2 inhibitors are well known in the art. Examples of commercially available monoclonal antibodies that interfere with the binding of PD1 to PDL1 and/or PDL2 include nivolumab (Opdivo®, BMS-936558, MDX1106, commercially available from BristolMyers Squibb, Princeton NJ), pembrolizumab (Keytruda® MK-3475, lambrolizumab, commercially available from Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech/Roche, South San Francisco CA). Additional examples of PD1 inhibitory antibodies include but are not limited to durvalumab (MED14736, Medimmune/AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, Bristol Myers Squibb), and avelumab (MSB0010718C, Merck Serono/Pfizer) and SHR-1210 (Incyte). Additional antibody PD1 pathway inhibitors are described in U.S. Pat. No. 8,217,149 (Genentech, Inc) issued Jul. 10, 2012; U.S. Pat. No. 8,168,757 (Merck Sharp and Dohme Corp.) issued May 1, 2012, U.S. Pat. No. 8,008,449 (Medarex) issued Aug. 30, 2011, U.S. Pat. No. 7,943,743 (Medarex, Inc) issued May 17, 2011. Additionally, small molecule PD1 to PDL1 and/or PDL2 inhibitors are known in the art. See, e.g. Sasikumar, et al as WO2016142833A1 and Sasikumar, et al. WO2016142886A2, BMS-1166 and BMS-1001 (Skalniak, et al (2017) Oncotarget 8(42): 72167-72181).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out certain embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1

Tumor vasculature is histologically and molecularly different from normal vasculature. Using immunohistochemistry (IHC), it was confirmed that melanoma cell adhesion molecule (MCAM), a plasma membrane protein, was highly expressed on the endothelial cells of the tumor vasculature in mouse xenograft tumors generated with human clear cell renal cell carcinoma (ccRCC) cells (Figure 1).

CD31 IHC was used to identify blood vessels in both tumor and normal vasculatures. Interestingly, it was found that MCAM’s expression level in the microvessels of normal mouse organs, such as kidney, brain, bladder, colon, heart, intestine, lung, muscle, pancreas, skin or stomach, was significantly lower than that in the tumor vasculature in xenografts (Figure 2, Figure 3, Figures 11-13, and Figures 15-19). In human ccRCC tumors, the MCAM’s expression level was significantly higher than that in the normal kidneys, including the glomeruli (Figures 6-9). To investigate if MCAM expression was increased in early-stage ccRCC cancers, MCAM expression was examined in a tissue microarray of ccRCC from stage I to IV (Figure 4). MCAM was found to be expressed at high levels in all tumor stages.

MCAM was assessed to determine whether it can be used to enhance the retention of targeted ultrasound enhancing microbubbles (Figure 5). The microbubbles only travel in blood due to their large sizes, and the ones with conjugated antibody could bind MCAM protein on the surface of endothelial cells facing the lumen, which will help retain the microbubbles. Microbubbles that were conjugated with anti-MCAM antibody showed much greater retention in ccRCC tumors than the microbubbles conjugated with a control antibody. In mouse kidneys, neither kind of microbubbles showed significant retention (Figure 14). Thus, MCAM could serve as a unique marker for ccRCC tumor vasculature, and it can be potentially utilized for in vivo imaging of cancer, and/or targeted drug delivery to increase efficacy and reduce side effects.

Further emphasizing this conclusion, the normal mouse kidney had the lowest TMCAM/TCDSI ratio when compared to all the ratios from the xenograft tumors (Figure 20). Interestingly, since all the endothelial cells came from mice, this suggests that the high expression of MCAM in tumor vasculature is induced by the implanted human cancer cells. This can be seen in other mouse organs as well (Figure 21). This pattern is also mostly seen in humans (Figures 22-24). The vasculature in normal human organs, with the exception of cervix, ovary, uterus and breast, expressed low level of MCAM compared to the vasculature of human ccRCC and human adrenal gland tumor.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A composition comprising a delivery vehicle, a targeting domain, and at least one therapeutic agent.

2. The composition of claim 1, wherein the delivery vehicle is a microbubble.

3. The composition of claim 1, wherein the therapeutic agent is a chemotherapeutic agent.

4. The composition of claim 1, wherein the therapeutic agent is a receptor tyrosine kinase (RTK) inhibitor or a tyrosine kinase inhibitor (TKI).

5. The composition of claim 1, wherein the therapeutic agent is Sunitinib, Tivozanib, Axitinib, or a combination thereof.

6. The composition of claim 1, wherein the targeting domain specifically binds to melanoma cell adhesion molecule (MCAM).

7. The composition of claim 1, wherein the targeting domain is an antibody, or a binding portion, variant, or fragment thereof.

8. The composition of claim 6, wherein the MCAM is present on the cell surface of an endothelial cell that is present in the vasculature of a tumor.

9. The composition of claim 8, wherein the tumor is a tumor from clear cell renal cell carcinoma (ccRCC), Angiomyolipoma, Leiomyosarcoma, Wilm’s tumor, chromophobe RCC, papillary RCC, invasive urothelial carcinoma, squamous cell carcinoma, breast cancer, colon adenocarcinoma, or Pheochromocytoma.

10. A method of treating or preventing cancer in a subject in need thereof, comprising administering a composition of any one of claims 1-9.

11. The method of claim 10, wherein the cancer is ccRCC, Angiomyolipoma, Leiomyosarcoma, Wilm’s tumor, chromophobe RCC, papillary RCC, invasive urothelial carcinoma, squamous cell carcinoma, breast cancer, colon adenocarcinoma, or Pheochromocytoma.