Methods for binding molecules to tumor-associated macrophages and their use.

Abstract

To provide methods for treatment and diagnosis of cancer.SOLUTION: There are described herein, molecules which preferentially bind to and target tumor associated macrophages (TAMs), and methods of treating cancer in a subject, including the step of administering pharmaceutical compositions thereof to the subject. In some embodiments, the TAM binding molecule is a cyclic peptide. The cyclic TAM binding peptide comprises a) CRVLRSGSC, or b) CRVLRSGSC with at least one conservative amino acid substitution.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related applications This application claims the interests of U.S. Provisional Application No. 62 / 717,656, filed on August 10, 2018, which is incorporated herein by reference in whole.

[0002] Description of federally funded research This invention was carried out with government support under the grant of R21EB022652 by the National Institutes of Health (NIH). The government has certain rights to this invention. [Background technology]

[0003] Macrophages are distributed across all major organs and play a central role in normal immune homeostasis and disease progression, such as cancer, and are important regulators within the tumor microenvironment. In many tumors, anti-inflammatory macrophages (substitutively activated or M2 subtype), also known as tumor-associated macrophages (TAMs), are responsible for creating an immunosuppressive tumor microenvironment, which hinders the immune system's recognition and elimination of tumor cells. [Overview of the project]

[0004] This specification describes molecules that preferentially bind to and target tumor-associated macrophages (TAMs), pharmaceutical compositions thereof, and methods for treating and diagnosing cancer and immunosuppressive tumor microenvironments. Furthermore, pharmaceutical compositions comprising tumor-associated macrophage (TAM) binding molecules conjugated to a portion and delivery agents are provided, and methods relating to the use of such pharmaceutical compositions for the treatment of cancer, wherein the pharmaceutical composition is cytolytic to TAMs, removing, reducing and / or neutralizing TAMs, or repolarizing TAMs from an M2 phenotype to an M1 phenotype, and methods relating to the use of such pharmaceuticals for cancer detection are provided, and methods relating to the use of such pharmaceuticals for the detection of TAMs or tumor microenvironments are provided.

[0005] In one embodiment, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises a TAM-binding molecule. In some embodiments, the pharmaceutical composition comprises a TAM-binding molecule conjugated to a portion. In some embodiments, the pharmaceutical composition comprises a TAM-binding molecule conjugated to a portion and a binder. In some embodiments, the TAM-binding molecule binds to the retinoid X receptor beta on TAM. In some embodiments, the TAM-binding molecule is a peptide, ligand, antibody, non-IG domain, or small molecule entity. In some embodiments, the TAM-binding molecule is an antibody or its antigen-binding fragment. In some embodiments, the antibody is an IgG, IgA, or IgM antibody. In some embodiments, the antibody is a single-domain antibody. In some examples, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In other examples, the antigen-binding fragment is Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, or a bispecific antibody. In other embodiments, the TAM-binding molecule is a peptide. In some examples, the TAM-binding peptide is cyclic. In other embodiments, the cyclic TAM-binding peptide comprises a) CRVLRSGSC, or b) CRVLRSGSC with at least one conserved amino acid substitution. In some embodiments, the moiety conjugated to the TAM-binding molecule is a therapeutic or diagnostic agent. In some embodiments, the moiety is a therapeutic agent, where the therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a protein, a peptide, an antibody, a growth inhibitor, a nucleic acid, or an antihormone agent. In other embodiments, the therapeutic agent is a cytotoxic agent, where the cytotoxic agent is a ribosome-inactivating protein, a histone deacetylase (HDAC) inhibitor, a tubulin inhibitor, an alkylating agent, an antibiotic, an antitumor agent, an antiproliferative agent, an antimetabolite, a topoisomerase I or II inhibitor, a hormone agonist or antagonist, an immunomodulator, a DNA subgroove conjugate, or a radioactive agent. In other embodiments, the moiety is a diagnostic agent that is a label. In some embodiments, the diagnostic agent is a label, where the label is a fluorescent label, a chromogenic label, or a radioactive label. In some embodiments, the pharmaceutical composition consists of a TAM-binding molecule directly conjugated to the moiety. In other embodiments, the pharmaceutical composition consists of TAM-bonded molecules partially indirectly conjugated via a linker.In some cases, the delivery agent is a liposome, microsphere, nanoparticle, microemulsion, microcapsule, polymer matrix, hydrogel, or viral vector.

[0006] In other embodiments, methods for treating cancer are provided. The methods generally include the step of administering the above-mentioned pharmaceutical composition to a subject. In some embodiments, the TAM-binding molecule is cytolytic against tumor cells. In some embodiments, the TAM-binding molecule inhibits tumor growth. In some embodiments, methods for treating cancer are provided, where cancer is selected from the group consisting of brain cancer, kidney cancer, ovarian cancer, prostate cancer, lymphoma, breast cancer, colon cancer, lung cancer, squamous cell carcinoma of the head and neck, and melanoma. In some embodiments, a method is performed in which the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, or orally, intramuscularly, or intracranially. In some embodiments, a method is performed in which the pharmaceutical composition is administered in combination with a second therapeutic agent. In further embodiments, the method is performed in combination with a second therapeutic agent, where the second therapeutic agent is a cancer chemotherapeutic agent, radiotherapy, a cytotoxic agent, another antibody, an NSAID, a corticosteroid, a nutritional supplement (e.g., an antioxidant), or a combination thereof.

[0007] In another embodiment, a method is provided for reducing the number of TAMs in the tumor microenvironment of a subject with cancer. This method generally comprises the above-mentioned pharmaceutical composition, wherein the pharmaceutical composition is cytolytic with respect to TAMs.

[0008] In another embodiment, a method is provided for eliminating immunosuppression within the tumor microenvironment of a subject with cancer. This method generally comprises the above-mentioned pharmaceutical composition, wherein the pharmaceutical composition removes, reduces and / or neutralizes TAM.

[0009] In another embodiment, a method is provided for repolarizing TAM from an M2 phenotype to an M1 phenotype in a subject with cancer. This method generally comprises the above-mentioned pharmaceutical composition, wherein the pharmaceutical composition repolarizes TAM from an M2 phenotype to an M1 phenotype.

[0010] In another embodiment, a method for detecting cancer in a subject is provided. This method, in whole, involves administering the subject the above-mentioned pharmaceutical composition.

[0011] In another embodiment, a method is provided for detecting TAMS within the tumor microenvironment of a subject with cancer. This method generally includes the step of administering the above-mentioned pharmaceutical composition to the subject.

[0012] In another embodiment, a method for detecting the tumor microenvironment of a subject is provided. This method generally includes the step of administering the above-mentioned pharmaceutical composition to the subject. References

[0013] All publications, patents, and patent applications referenced herein are incorporated herein by reference to such an extent as they are specifically and individually indicated by each individual publication, patent, and patent application. [Brief explanation of the drawing]

[0014] Novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments. The detailed description utilizes the principles of the invention and is accompanied by the accompanying drawings and:

[0015] Table 1 summarizes the tumors tested for in vivo migration and colonization of CRV. [Figure 1A] Figure 1A depicts the binding of FAM peptides in vitro to different cell lines. Cells were incubated with FAM-CRV or FAM-GGS (10 μM) at 4°C for 1 hour and washed with PBS. Fluorescence was measured by flow cytometry. Higher binding of FAM-CRV compared to FAM-GGS was observed on J774, RAW, and THP-1 differentiated macrophages, but not on 4T1 tumor cells. [Figure 1B]Figure 1B shows a depiction of FAM peptide bonds. Representative UV-illuminated images of FAM-CRV migration and colonization within various tissues in mice with 4T1 breast cancer. 100 μg of FAM-CRV in 100 μL of PBS was intravenously injected into 4T1 mice for 1 hour of circulation. Tissues were collected after transcardiac perfusion with PBS. [Figure 2A] Figure 2A depicts immunofluorescence staining of tissue with FAM peptide and CD31 at different migration and settlement times (5 min, 15 min, and 60 min). Tissue sections of 4T1 orthotopic tumor-bearing mice injected with FAM-CRV or FAM-GGS peptide were stained with anti-FITC (green), anti-CD31 antibody (red), and DAPI (blue). Migration and settlement results of FAM-CRV or FAM-GGS within the tumor at different time points. Top row: View of the entire tumor. Bottom row: Magnified view of the area marked with a white square in the top row. [Figure 2B] Figure 2B depicts immunofluorescence staining of tissues with FAM peptide and CD31 at different migration and settlement times (5 min, 15 min, and 60 min). Tissue sections of 4T1 orthotopic tumor-bearing mice injected with FAM-CRV or FAM-GGS peptide were stained with anti-FITC (green) and anti-CD31 antibody (red) and DAPI (blue). Distribution of FAM-CRV in other organs. The FAM-CRV signal washed out over time. [Figure 3]Figure 3 depicts the association of FAM-CRV with tumor macrophages. A) Immunofluorescence staining of 4T1 mouse tissue for FAM-CRV and macrophage markers. Tissue sections were stained with anti-FITC (green), anti-CD11b, anti-F4 / 80 or anti-CD68 antibody (red) and DAPI (blue). Scale figure: 100 μm. BD) Binding of FAM-CRV or FAM-GGS with cells isolated from various organs of ex vivo 4T1 mice. Cells were incubated at 4°C for 1 hour with primary and secondary antibodies against FAM-CRV or FAM-GGS, CD11b, CD68, and F4 / 80. Cells were then washed and analyzed by flow cytometry. B: Selective binding of FAM-CRV to tumor cells compared with blood and spleen cells. C: Binding of FAM-CRV or FAM-GGS to tumor cells. D: Cell populations positive for CD11b and F4-80 within FAM-CRV-positive cells. E) Flow cytometry analysis of cells isolated from 4T1 tumors after in vivo FAM-CRV migration and colonization. Tumors were obtained 15 or 60 minutes after intravenous administration of FAM-CRV. Cells were incubated with CD11b and F4 / 80 primary and secondary antibodies at 4°C for 1 hour, washed, and analyzed by flow cytometry. [Figure 4] Figure 4 depicts the evaluation of FAM-CRV binding with macrophages in atherosclerotic plaques. A) In vivo migration and colonization of FAM-CRV, FAM-ARA (negative control), or FAM-LyP-1 (positive control) in ApoE- / - mice with atherosclerotic plaques in the aorta. 100 μg of FAM-CRV, FAM-LyP1, or FAM-ARA was intravenously injected for 1 hour of circulation. The aorta (middle), kidney (right), and liver (left) were resected and imaged with a UV illuminator. B) Ex vivo staining with FAM-CRV or FAM-GGS isolated from the aorta with plaque. Cells were incubated at 4°C for 1 hour and then analyzed by flow cytometry. [Figure 5]Figure 5 depicts the evaluation of RXRB as a CRV-binding receptor. A) High FAM-CRV binding with human immobilized recombinant RXRB protein compared to FAM-GGS. B) Arrest of CRV-binding expression with RAW inhibited by RXRB at 4°C for 1 hour. C) Immunohistochemical staining of RXRB on 4T1 tumors with in vivo migration and immobilization of FAM-CRV or FAM-GGS. RXRB IHC staining colocalized with FAM-CRV and CD11b from immunofluorescence staining. D) Ex vivo CRV binding with cells isolated from 4T1 tumors. Cells were stained with FAM-CRV or FAM-GGS, CD11b, CD68, F4 / 80, and RXRB primary and secondary antibodies at 4°C for 1 hour. Cells were washed and analyzed by flow cytometry. G) Staining of cells after in vivo FAM-CRV migration and immobilization. Tumors were obtained 60 minutes after intravenous administration of FAM-CRV. Cells were incubated with RXRB, CD11b, and F4 / 80 primary and secondary antibodies at 4°C for 1 hour. Cells were washed and analyzed by flow cytometry. [Figure 6] Figure 6 depicts immunofluorescence staining with RXRB antibody and in vivo FAM-CRV migration and immobilization of 4T1 mouse tissue. Tissue sections of mice with orthotopic 4T1 tumors were stained with anti-FITC (fuchsia), anti-RXRB antibody or control rabbit IgG (green), anti-CD11b, CD68, F4 / 80 or CD31 antibody (red), and DAPI (blue). The last column shows combined images from control IgG-receptor animals. [Figure 7] Figure 7 depicts CRV-pSiNP migration and colonization. A) Fluorescence imaging of tissue from 4T1 mice after 1 hour of NP circulation. Top row: Mice injected with pSiNP. Bottom row: Mice injected with CRV-pSiNP. B) Flow cytometry analysis of cells isolated from 4T1 tumors after in vivo CRV-pSiNP or pSiNP migration and colonization. Tumors were obtained 60 minutes after intravenous administration. Cells were incubated with CD11b and CD68 primary and secondary antibodies at 4°C for 1 hour, washed, and analyzed by flow cytometry. [Modes for carrying out the invention]

[0016] Macrophages and the Tumor Microenvironment In vivo, the tumor microenvironment is a complex environment that encompasses a multitude of cell types, including tumor cells, vascular cells such as endothelial cells, and stromal cells such as fibroblasts. Furthermore, in vivo, these cells are exposed to blood flow and various biological delivery conditions. In vivo, the microvascular cells within the tumor are influenced by blood flow and communicate with tumor cells and non-tumor cells through physical and diffusional factors. Additionally, the tumor vasculature is abnormal, characterized by disordered branching, low flow, and leaky capillaries, and thus functions as a major delivery barrier to anti-cancer therapies targeting tumor cells. The interaction between tumor cells, endothelial cells, and stromal cells affects each cell type and enhances angiogenesis and tumor cell proliferation. This crosstalk can be an important factor in determining the responsiveness of tumor cells to anti-cancer agents.

[0017] Cells in the tumor microenvironment experience oxygen and nutrient deprivation. Hypoxic stress causes changes in the microenvironment after altering the metabolism of tumor cells and macrophages within the tumor. The changes in the microenvironment alter the phenotype and metabolism of macrophages to induce tumor-promoting reprogramming. Nutrient stress also elicits autophagy to ensure cell survival or induce cell death. The death of tumor cells attracts macrophages and is a communication system that regulates their phenotype. Depending on the form of tumor cell death, macrophage polarization ranges from pro-inflammatory activation to anti-inflammatory / immunosuppressive activation.

[0018] Chronic inflammation contributes to cancer growth. The presence and activation of chronic innate immune cell types (e.g., neutrophils, macrophages, and mast cells) promote cancer growth. Therefore, it is clear that some subpopulations of chronically activated innate cells promote the growth and / or survival of neoplastic cells. Depending on their polarization state, immune cells can exert either anti-tumor functions (e.g., helper T cell 1 (Th1) vs Th17 subpopulations of type I CD4(+) T cells) or tumor maintenance functions (e.g., vs type II NKT cells, M1 macrophages vs M2 macrophages, and N1 neutrophils vs N2 neutrophils). Chronically activated and polarized immune cells (e.g., M2 macrophages and N2 neutrophils) produce or possess numerous chemokines, cytokines, growth factors, and proteases, which result in tissue remodeling, angiogenesis, cell proliferation, genomic instability, and the spread of neoplastic cells to ectopic tissues.

[0019] Macrophages are a type of white blood cell in the immune system that, in a process called phagocytosis, engulf and digest cellular debris, foreign substances, microorganisms, cancer cells, and substances on their surface that do not possess the types of proteins specific to healthy somatic cells. Macrophages increase inflammation and stimulate the immune system. They also play an important anti-inflammatory role and can reduce the immune response by releasing cytokinins. Macrophages that promote inflammation are called M1 macrophages, while macrophages that reduce inflammation and promote tissue repair are called M2 macrophages. Macrophages are the dominant immune cell population in most solid tumors. Tumor-associated macrophages (TAMs) are a type of macrophage. TAMs are thought to be responsible for tumor growth and progression and acquire the M2 phenotype. TAMs can modulate tumor progression. Therapeutic strategies to reduce TAM numbers or manipulate the TAM phenotype are targets of cancer treatment. Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and possess essential nuclear and cytoplasmic functions as transcription factors. RXR is a type of nuclear receptor activated by 9-cis-retinoic acid, which is considered endogenously related. RXR is also activated by 9-cis-13,14-dihydroretinoic acid, which may be the primary endogenous mammalian RXR-selective agonist. There are three retinoin X receptors (RXRs), namely RXR-alpha (RXRA), RXR-beta (RXRB), and RXR-gamma (RXRG), encoded by the RXRA, RXRB, and RXRG genes, respectively. After catalytic activation, RXRs are heterodimerizing partners for members of the type II subfamily of nuclear receptors that regulate the transcription of numerous target genes. RXRs can heterodimerize with nuclear receptors of the type I subfamily, including CAR, FXR, LXR, PPAR, PXR, RAR, TR, and VDR. The RXR heterodimer binds to the hormone response element, which is complexed with the inhibitory complement protein, in the absence of a ligand. Binding of the agonist ligand to RXR results in the dissociation of the inhibitory complement and the recruitment of the coactivator protein.This promotes the transcription of downstream target genes into mRNA and ultimately proteins. RXR can modulate macrophages in inflammatory and metabolic disorders, and there is potential to directly modulate RXR signaling for the treatment of macrophage-related pathologies. Cell surface expression of RXRB is specific to TAM, and RXRB is attributed to the selective binding of CRV and other considered molecules to tumor macrophages.

[0020] Molecules that target TAM Peptide CRV (CRVLRSGSC) is a cyclic macrophage-targeting peptide having disulfide bonds between terminal cysteine ​​residues. CRV selectively migrates to and colonizes tumors, where it binds to TAMs within the tumor. CRV recognizes and binds to RXRB on the surface of TAMs within the tumor. CRV recognizes only TAMs and does not recognize macrophages in atherosclerotic plaques.

[0021] CRVs can be bound to therapeutic agents. These therapeutic agents may contain antibodies (e.g., IgG, IgA, or IgM).

[0022] CRVs can be modified to produce related peptides that are also considered to act as TAM molecules. Examples of such modifications include the substitution, deletion, or addition of one or more amino acids. Conservative substitutions include amino acid substitutions in which a given amino acid is replaced with another amino acid having similar characteristics, and further include aliphatic amino acid substitutions of alanine, valine, leucine, and isoleucine, as well as substitutions of hydroxyl residues serine and threonine, exchanges of acidic residues asparateto and glutamate, substitutions of amide residues asparagine and glutamine, exchanges of basic residues lysine and arginine, and substitutions between aromatic residues phenylalanine and tyrosine. In some embodiments, CRVs are modified by one or more amino acid conservative substitutions. In other embodiments, CRVs are modified by one amino acid conservative substitution.

[0023] Other TAM-binding molecules that can act as RXRB-binding molecules on the cell surface to selectively bind to TAM include antibodies (e.g., IgG, IgA, or IgM), antigen-binding fragments, peptides, ligands, non-IG domains, or small molecules. The TAM-binding molecules may bind to RXRB at different positions on the RXRB molecule. If the TAM-binding molecule is an antibody, it may be a single-domain antibody, a chimeric antibody, a humanized antibody, a human antibody, and / or a monoclonal antibody. If the TAM-binding molecule is an antigen-binding fragment, it may be Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, or a bispecific antibody. If the TAM-binding molecule is a peptide, it may be cyclic. If the TAM-binding molecule is cyclic, it may be CRV or another peptide.

[0024] Modifications to the TAM-binding molecule are considered. These modifications include conjugation with a supplementary moiety. This moiety may be a therapeutic or diagnostic agent used clinically or for research purposes.

[0025] These modifications can further constitute a delivery system that allows TAM-binding molecules to be delivered to target the tumor or tumor microenvironment. The advantages of this delivery system may include reduced administration frequency to the patient, more uniform drug effects, reduced drug side effects, and reduced variability in circulating drug concentrations.

[0026] The aforementioned portion, the TAM-binding molecule, or both can be retained within the delivery agent. The delivery agent may be a liposome, microsphere, nanoparticle, microemulsion, microcapsule, polymer matrix, hydrogel, or viral vector.

[0027] Liposomes are non-toxic, non-hemolytic, and non-immunogenic even after repeated injection; they are biocompatible and biodegradable, and can be designed to circumvent clearance mechanisms (reticular endothelial system (RES), renal clearance, chemical or enzymatic inactivation, or other undesirable effects). Lipid-based, ligand-coated nanocarriers can house their deliverables in a hydrophobic shell or hydrophilic interior, depending on the nature of the drug being delivered.

[0028] Microspheres can contain a wide range of drugs, including small molecules, proteins, and nucleic acids, and are easily administered via injection needles. Overall, microspheres are biocompatible, offer high bioavailability, and allow for sustained release over long periods. Disadvantages of microspheres include difficulties in large-scale production, inactivation of drugs during manufacturing, and insufficient control of drug release rates.

[0029] Nanoparticle-based drug delivery systems are considered an effective method for delivering drugs. Some of the advantages of using nanoparticles include regulated release of deliveries into the cytoplasm and avoidance of oncological drug resistance.

[0030] Microemulsions are thermodynamically stable, isotropic, transparent (or translucent) systems of oil, water, and surfactant, often combined with a co-surfactant containing droplets with an overall size in the range of 20–200 nm. Depending on their structure, these microemulsions can be classified as oil-in-water (o / w), water-in-oil (w / o), or co-continuous systems, characterized by extremely low interfacial tension between the oil and water phases. These systems are currently of great technical and scientific interest to researchers due to their potential to incorporate a wide range of drug molecules (hydrophilic and hydrophobic) due to the presence of both lipophilic and hydrophilic domains. These adaptable delivery systems offer protection from oxidation and enzymatic hydrolysis, improve the solubilization of lipophilic drugs, and thus enhance their bioavailability. Microemulsions are suitable for oral and intravenous delivery systems, as well as sustained-release and targeted delivery (e.g., via eye, tooth, lung, vagina, and topical methods). Microemulsions are used to improve the oral bioavailability of various poorly soluble drugs. In some applications, microemulsions are used to address challenges such as the delivery of chemotherapeutic agents to newly generated cells and oral delivery of insulin. Microcapsules, which have a diameter of 1–1000 μm and can be precisely constructed internally or externally, can be used for drug delivery. Microcapsules offer a variety of important advantages as a drug delivery system (e.g., effective protection against degradation of the encapsulated active drug (e.g., enzymatic degradation), the possibility of precisely controlling the release rate of the encapsulated drug over periods ranging from hours to months, ease of administration (compared to alternative extraintestinal controlled-release drug delivery methods such as macro-sized implants)), and the ability to provide desired, pre-programmed drug release profiles that meet the patient's treatment needs.

[0031] Drugs can be embedded in a polymer matrix or co-crystallized in a polymer template. Polymers are a drug delivery technology that provides controlled release, circulating administration, and tunable release of hydrophilic and hydrophobic drugs at constant doses over extended periods. Polymer matrices may be tailored to specific cargoes and designed to perform distinct biofunctions.

[0032] Hydrogels are three-dimensional crosslinked networks of water-soluble polymers. They can be made from virtually any water-soluble polymer, thereby encompassing a wide range of chemical compositions and numerous physical properties. Furthermore, hydrogels can be formulated in various physical forms, including slabs, microparticles, nanoparticles, coatings, and films. As a result, hydrogels are commonly used in clinical practice and experimental medicine for a wide range of applications, including tissue engineering and regenerative medicine, diagnostics, cell fixation, biomolecule or cell isolation, and barrier materials for regulating bioadhesion.

[0033] Viral delivery vectors are a type of nanomaterial that can be used as drug delivery materials. An effective vector must be able to effectively deliver a drug payload to a specific target and subsequently deliver it.

[0034] The above conjugation can be achieved directly or indirectly, with or without the presence of a linker molecule. This linker molecule may be a pH-sensitive linker, a disulfide linker, a peptide linker, a beta-gluronide linker, a redox-reactive linker, a hydrazone linker, a hydrophilic linker, an azo linker, or another type of linker. The linker can respond to a stimulus to initiate drug release. The stimulus may be internal or external. The stimulus may be local. The stimulus may be pH, an enzyme, light, heat, or another stimulus.

[0035] When a TAM-binding molecule is conjugated to the therapeutic portion, the therapeutic agent may be a cytotoxic agent (e.g., ribosome-inactivating protein, histone deacetylase inhibitor, tubulin inhibitor, alkylating agent, antibiotic, antitumor agent, antiproliferative agent, antimetabolites, type I or type II topoisomerase inhibitor, hormone agonist or antagonist, immunomodulator, DNA subgroove binding agent, or radiopharmaceutical), a chemotherapeutic agent (e.g., alkylating agent, anthracycline, taxane, epothilon, type I or type II topoisomerase inhibitor, histone deacetylase inhibitor, kinase inhibitor, nucleotide analog, peptide antibiotic, platinum-based agent, retinoid, vinca alkaloid or its mobile and stationary form, or other chemotherapeutic agent), a protein, peptide, antibody, growth inhibitor, nucleic acid, or antihormone agent.

[0036] When a TAM-binding molecule is conjugated to the portion that is a diagnostic agent, the diagnostic agent may be a label, and the label may be a fluorescent label, a color-developing label, or a radioactive label. The label is intended for use in diagnostic imaging (e.g., PET or MRI imaging, or imaging protocols incorporating PET or MRI). [Examples]

[0037] Example 1: CRV specifically binds to macrophages in vitro. RAW (a mouse macrophage cell line derived from tumors, RAW264.7), J774 (a mouse macrophage cell line derived from tumors, J774A.1), THP-1 differentiated macrophages (human macrophages differentiated from the human monocyte cell line THP-1), and 4T1 (a mouse mammary cancer cell line) were cultured in DMEM supplemented with 10% FCS. 1 × 10 6The cells were incubated in 300 μL of complete growth medium in an Eppendorf tube with FAM-CRV or the control peptide FAM-GGS (10 μM). After incubation at 4°C for 1 hour, the peptide-containing medium was removed by centrifugation, and the cells were washed twice with PBS. Subsequently, 100 μL of PFA (4% buffer) was added to the cells for fixation, and flow cytometry data were obtained using FACSCanto (BD Biosciences, San Jose). The experiment was repeated three times on different days.

[0038] FAM-CRV showed significantly higher binding to RAW, J774, and THP-1 differentiated macrophages than the control peptide FAM-GGS (Figure 1A). In contrast, the binding of FAM-CRV to the 4T1 breast cancer cell line was very limited. These results demonstrate that CRV specifically binds to macrophages in vitro.

[0039] Example 2: In vivo binding of CRV to macrophages To investigate whether CRVs can bind to macrophages in vivo, FAM-CRVs were intravenously injected into tumor-bearing mice. For this purpose, human breast cancer cells of 4T1 and MCF10CA1a, and mouse PDAC cells in KRAS ink were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin 100 U / mL, and streptomycin 100 μg / mL. Py8119 cells were cultured in Ham's F12K Medium containing 5% FCS, amphotericin B 2.5 μg / mL, gentamicin 50 μg / mL, and MITO+. Human cell lines were certified by the DNA Analysis Core Facility at Sanford Burnham Prebys Medical Discovery Institute (La Jolla, California), and KRAS ink cell lines were certified by DDC Medical (Fairfield, Ohio). All cells tested were negative for mycoplasma contamination. To generate 4T1 tumors, 1 × 10⁶ cells were used. 6Tumor cells (suspended in 100 μL of PBS) were orthotopically injected into the mammary fat body of normal BALB / c mice. 2 × 10⁶ cells were used to generate MCF10CA1a tumors. 6 Tumor cells (suspended in 100 μL of PB) were orthotopically injected into the mammary fat body of female BALB / c athymoid nude mice. 1 × 10⁶ cells were injected to generate Py8119 tumors. 6 Tumor cells (suspended in 100 μL of PBS) were orthotopically injected into the mammary fat body of C57BL6 mice. 1 × 10⁶ KRAS-infused PDAC tumors were generated. 6 The cells were injected into female BALB / c mice. 1 × 10⁶ cells were used to generate H1975 tumors. 6 The cells (suspended in 100 μL of PBS) were injected. (What type of mouse was used? Where was the injection performed?) All animal experiments were approved by the Animal Research Committee of the Sanford Burnham Prebys Medical Discovery Institute.

[0040] The in vivo distribution of fluorescein-conjugated peptides (FAM-CRV, FAM-GGS, or FAM-ARA) was examined after intravenous injection of 100 μL of peptide solution (1 mg / mL PBS) into the tail vein of mice. The peptides were circulated for 1 hour, and transcardiac perfusion was performed with PBS. Tissues were collected, fixed with 4% formaldehyde buffer, and subsequently immersed overnight in 30% sucrose in PBS.

[0041] The tumor models tested in this study are summarized in Table 1. FAM-CRV migration and colonization were positive for orthotopic 4T1 breast cancer, orthotopic MCF10CA1a breast cancer, subcutaneous kras-INK pancreatic cancer, subcutaneous KPC pancreatic cancer, and subcutaneous H1975 lung cancer. Compared to other organs, tumors exhibited strong fluorescence signals under UV illumination.

[0042] [Table 1]

[0043] Representative images of FAM-CRV migration and colonization over 1 hour in a 4T1 mammary cancer mouse model are shown in Figure 1B. FAM-CRV was primarily observed in tumors and kidneys, with moderate to low accumulation observed in the liver and spleen, which have high volumes of monocytes / macrophages. Similar biodistribution was observed in other CRV-positive models. These results suggest that FAM-CRV preferentially migrates and colonizes tumors over other macrophage-rich organs.

[0044] Example 3: Translocation and establishment of CRV within the tumor FAM-CRV was intravenously injected into tumor-bearing mice. For this purpose, 4T1 human breast cancer cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The human cell line was certified by the DNA Analysis Core Facility at the Sanford Burnham Prebys Medical Discovery Institute (La Jolla, California), and the cell line was negative for mycoplasma contamination. To generate 4T1 tumors, 1 × 10⁶ cells were used. 6 Tumor cells (suspended in 100 μL of PBS) were orthotopically injected into the mammary fat body of normal BALB / c mice. All animal experiments were approved by the Animal Research Committee of the Sanford Burnham Prebys Medical Discovery Institute.

[0045] The in vivo distribution of fluorescein-conjugated peptides (FAM-CRV, FAM-GGS, or FAM-ARA) was examined after intravenous injection of 100 μL of peptide solution (1 mg / mL PBS) into the tail vein of mice. The peptides circulated for 1 hour, and transcardiac perfusion was performed with PBS. Tissues were collected, fixed with 4% formaldehyde buffer, and subsequently immersed overnight in 30% sucrose in PBS. The tissues were finally frozen in OCT embedding medium (Tissue-Tek) and sliced ​​for immunofluorescence staining.

[0046] Tissue sections were blocked for 1 hour in 1% bovine serum albumin with 0.1% Triton X-100 and incubated with appropriate primary and secondary antibodies. Blood vessels were visualized by staining of tissue sections with a monoclonal antibody against CD-31. The primary antibody was rat anti-mouse CD31 (BD Biosciences). The secondary antibody was Invitrogen's 594 donkey anti-rat IgG. After washing with PBS, sections were embedded in DAPI-containing mounting medium (Vector Laboratories, Burlingame, California) and examined with a Zeiss LSM 710 NLO confocal microscope.

[0047] Immunofluorescence staining was performed on all tissue sections for FAM-CRV and CD31. Representative images from a 4T1 breast cancer model are shown in Figure 2. CRV migrated and settled in the tumor and diffused within it. Within the tumor, the peptide could leave the blood vessels within 5 minutes after administration because a very small FAM-CRV signal was co-localized by CD31 staining (Figure 2A). At different migration and settlement times (5 min, 15 min, and 60 min), the FAM-CRV signal gradually increased within the tumor. This indicated that the peptide penetrated into the stroma via the blood vessels. In comparison, the FAM-labeled control peptide GGS (GGSGGSKG) did not produce a fluorescence signal within the tumor at any time point. Interestingly, FAM-CRV accumulated in other organs such as the liver, spleen, and lymph nodes after 5 minutes of circulation, but was rapidly washed out after 1 hour.

[0048] Example 4: CRV targets macrophages within the tumor. To determine whether CRV targeted macrophages or other cell types within the tumor, tissue sections from 4T1 tumor-bearing mice were also stained with macrophage markers including CD11b, F4 / 80, and CD68. For this purpose, 4T1 human breast cancer cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The human cell lines were validated by the DNA Analysis Core Facility at the Sanford Burnham Prebys Medical Discovery Institute (La Jolla, California), and were negative for mycoplasma contamination. To generate 4T1 tumors, 1 × 10⁶ cells were used. 6 Tumor cells (suspended in 100 μL of PBS) were orthotopically injected into the mammary fat body of normal BALB / c mice. All animal experiments were approved by the Animal Research Committee of the Sanford Burnham Prebys Medical Discovery Institute.

[0049] The in vivo distribution of fluorescein-conjugated peptides (FAM-CRV, FAM-GGS, or FAM-ARA) was examined after intravenous injection of 100 μL of peptide solution (1 mg / mL PBS) into the tail vein of mice. The peptides circulated for 1 hour, and transcardiac perfusion was performed with PBS. Tissues were collected, fixed with 4% formaldehyde buffer, and subsequently immersed overnight in 30% sucrose in PBS. The tissues were finally frozen in OCT embedding medium (Tissue-Tek) and sliced ​​for immunofluorescence staining.

[0050] Tissue sections were blocked for 1 hour in 1% bovine serum albumin with 0.1% Triton X-100 and incubated with appropriate primary and secondary antibodies. Primary antibodies were rat anti-mouse CD11b (BD Biosciences), rat anti-mouse F4 / 80 monoclonal (BD Biosciences), and rabbit anti-fluorescein / Oregon Green (Invitrogen) polyclonal antibodies. Secondary antibodies were Invitrogen's Alexa Fluor488 goat anti-rabbit IgG and 594 donkey anti-rat IgG. After washing with PBS, sections were embedded in DAPI-containing mounting medium (Vector Laboratories, Burlingame, California) and examined under a Zeiss LSM 710 NLO confocal microscope.

[0051] Mice with tumor sizes of approximately 8 mm were euthanized by cervical dislocation under deep anesthesia (unresponsive to aveltin and toe pinch). Target (tumor) and control (e.g., liver, spleen) tissues were collected and further separated into single cells. Tumor cells were isolated using the MACS tumor dissociation kit. The cells were then incubated at 4°C for 1 hour with fluorescently labeled CRV or other cell markers (CD11b, CD68, F4 / 80, FAB, EpCAM, CD31). Positive cells were quantified using BD LSRFORTESSA, and the data were analyzed using FCS Express Version 3 (De Novo Software).

[0052] As shown in Figure 3A, the FAM-CRV signaling strongly overlapped with their macrophage markers. This suggested that the accumulation of FAM-CRV in tumors was primarily due to its association with tumor macrophages. Immunofluorescence staining was performed on other animal models, yielding similar results. For further validation, cells were isolated from tumors, spleens, livers, and blood of 4T1 tumor mice and incubated with FAM-CRV for ex vivo binding studies. In the 4T1 model, tumor cells exhibited much higher FAM-CRV binding compared to spleens and blood cells, thereby confirming the preferential migration and colonization of FAM-CRV in tumors (Figure 3B). In comparison, the control FAM-GGS peptide was unable to specifically label macrophages in tumors (Figure 3C). Approximately 15% of single tumor cells showed very high FAM-CRV signaling compared to other tumor cells, and these were designated as FAM-CRV positive. Over 50% of FAM-CRV-positive cells were stained positively for both CD11b and F4 / 80 (Figure 3D). Low CRV binding to blood and spleen cells further suggested that FAM-CRV recognized tumor blood vessels, extruded, and bound to macrophages within the tumor tissue, rather than binding to circulating monocytes / macrophages and migrating to and colonizing the tumor.

[0053] Example 5: FAM-CRV preferentially binds to tumor-associated macrophages. After in vivo migration and colonization of FAM-CRV (15 or 60 minutes after intravenous administration), flow cytometry analysis of isolated 4T1 tumor cells was further performed. For this purpose, FAM-CRV was intravenously injected into tumor-bearing mice. For this purpose, 4T1 human breast cancer cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin 100 U / mL, and streptomycin 100 μg / mL. The human cell line was certified by the DNA Analysis Core Facility at Sanford Burnham Prebys Medical Discovery Institute (La Jolla, California), and the cell line was negative for mycoplasma contamination. To generate 4T1 tumors, 1 × 10⁶ cells were used.6 Tumor cells (suspended in 100 μL of PBS) were injected locally into the mammary fat pads of normal BALB / c mice. All animal experiments were approved by the Animal Research Committee of the Sanford Burnham Prebys Medical Discovery Institute.

[0054] The fluorescein-conjugated peptide (100 μL, 1 mg / mL in PBS) was injected intravenously into the tail vein of tumor-bearing mice. The peptide circulated for 1 hour. The mice were then euthanized by cervical dislocation under deep anesthesia (Avertin, unresponsive to toe pinch). Tumors were collected and further separated into single cells, which were incubated with different cell markers (CD11b, CD68, F4 / 80, FAB, EpCAM, CD31) at 4°C for 1 hour. Positive cells were quantified by BD LSRFORTESSA, and the data were analyzed using FCS Express Version 3 (De Novo Software).

[0055] Tumor cells from animals with both circulation lengths showed higher FAM-CRV signals compared to non-injected control animals (Figure 3E). Approximately 20% of the FAM-CRV-positive cells were identified as macrophages, which was much less than the proportion in in vitro binding studies. The reason for the separation of many weakly bound FAM-CRVs may be the extensive staining and washing steps for the primary and secondary antibodies. All these binding results indicated that FAM-CRV could preferentially bind to tumor-associated macrophages.

[0056] Example 6: FAM-CRV does not specifically bind to macrophages within atherosclerotic plaques Since macrophages are present in other disease sites such as atherosclerotic plaques, it is interesting to know whether CRV recognizes other pathological tissues, including macrophages. Therefore, the in vivo migration and colonization of FAM-CRV was tested in ApoE- / - mice with atherosclerotic plaques in the aorta. Unlike LyP1, which migrates and colonizes both tumors and plaques via the p32 receptor and binds to macrophages, FAM-CRV did not migrate and colonize aortic plaques after systemic administration (Figure 4A), suggesting that CRV can distinguish between macrophages and TAMs in atherosclerotic plaques in vivo. Immunofluorescence staining of the aorta further showed no accumulation of FAM-CRV in the plaques. Single-cell suspensions were also obtained from the aortas of mice with atherosclerotic plaques, and in vitro binding studies confirmed that FAM-CRV does not specifically bind to macrophages in the plaques compared to FAM-GGS (Figure 4B). This characteristic makes CRV a desirable peptide for targeting macrophages for disease-specific applications.

[0057] Example 7: RXRB is responsible for CRV binding to macrophages. The differences between CRV and LyP-1 suggest that CRV likely binds to different receptors for TAM recognition. Therefore, the CRV receptor on macrophages was identified. To reduce complexity, the cell membrane fraction of RAW cells was isolated, and affinity chromatography was performed to isolate the putative CRV receptor. For this purpose, CRV was fixed on a column. Lysates of 4T1 tumor tissue and membrane proteins from RAW cells (isolated with the MemPERPlus kit from ThermoFisher) were used as independent sources for the two putative receptors. After washing with buffer and control peptide GGS, the putative receptors were eluted with excess free CRV peptide. Mass spectrometry then identified retinoid X receptor beta (RXRB) as the receptor candidate.

[0058] To confirm that RXRB binds to CRV, FAM-CRV binding with human recombinant RXRB protein immobilized on a plate was tested. Human recombinant RXRB protein (50 μL, 5 μg / mL) was immobilized overnight at 4°C in a high-binding 96-well plate. Bovine serum albumin was used as a control protein. The protein solution was removed and washed once with PBS. Subsequently, 1% BSA in PBS solution was added to the wells and incubated at room temperature for 1 hour to block any remaining available binding sites. After one wash with PBS, RXRB and BSA were incubated with FAM-CRV (100 μL, 1 μM) at room temperature for 1 hour. The wells were washed three times with PBS, and fluorescence was measured using a multiplate reader.

[0059] Compared to the control peptide FAM-GGS, there was significantly higher binding of the CRV peptide to RXRB (Figure 5A). Conversely, neither peptide bound to the control protein BSA. Both J774 and RAW cells showed high FAM-CRV binding along with high surface expression of RXRB, while tumor cell lines such as 4T1 and PPC1 exhibited very limited RXRB surface expression and low FAM-CRV binding. CRISPR was used to deactivate RXRB expression in RAW cells, and the binding of the CRV peptide to those designed cells was evaluated. As shown in Figure 5B, deactivation of RXRB expression reduced FAM-CRV binding with wild-type RAW cells by approximately 70%, which was quantified by flow cytometry after 1 hour incubation. These in vitro results indicated that RXRB is responsible for CRV binding with macrophages.

[0060] Example 8: RXRB surface bonding is important for CRV bonding Furthermore, immunohistochemical (IHC) staining for RXRB was performed on 4T1 tumor tissue. For this purpose, 4T1 human breast cancer cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The human cell line was certified by the DNA Analysis Core Facility at Sanford Burnham Prebys Medical Discovery Institute (La Jolla, California), and the cell line was negative for mycoplasma contamination. To generate 4T1 tumors, 1 × 10⁶ cells were used. 6 Tumor cells (suspended in 100 μL of PBS) were orthotopically injected into the mammary fat body of normal BALB / c mice. All animal experiments were approved by the Animal Research Committee of the Sanford Burnham Prebys Medical Discovery Institute.

[0061] Tissue sections were blocked for 1 hour with 1% bovine serum albumin containing 0.1% Triton X-100 and incubated with appropriate primary and secondary antibodies. Primary antibodies were rat anti-mouse CD11b (BD Biosciences), rat anti-mouse F4 / 80 monoclonal (BD Biosciences), and rabbit anti-fluorescein / Oregon Green (Invitrogen) polyclonal antibodies. Secondary antibodies, Alexa Fluor488 goat anti-rabbit IgG and 594 donkey anti-rat IgG, were from Invitrogen. After washing with PBS, sections were embedded in a DAPI-containing mounting medium (Vector Laboratories, Burlingame, California) and examined under a Zeiss LSM 710NLO confocal microscope.

[0062] This experiment demonstrated that RXRB co-localized with FAM-CRV, similar to macrophage markers (CD11b, F4 / 80, and CD68) (Figure 5C). Furthermore, it is noteworthy that RXRB staining (brown) was observed not only on the nucleus but throughout the cell, supporting the idea that RXRB surface expression is important for CRV binding.

[0063] Example 9: RXRB-positive cells showed enhanced CRV binding. To further investigate the correlation between CRV binding to macrophages and RXRB surface expression, ex vivo conjugation of CRV and RXRB antibodies to tumor cells isolated from a 4T1 tumor model was performed. Mice with tumor sizes of approximately 8 mm were euthanized by cervical dislocation under deep anesthesia (unresponsive to abeltin and toe pinch). Target (tumor) and control (e.g., liver, spleen) tissues were collected and further isolated into single cells. Tumor cells were isolated using the MACS Tumor Isolation Kit. The cells were then incubated with fluorescently labeled CRV or other cellular markers (CD11b, CD68, F4 / 80) at 4°C for 1 hour. Positive cells were quantified using BD LSRFORTESSA, and the data were analyzed using FCS Express Version 3 (De Novo Software).

[0064] Flow cytometry analysis showed that approximately 15% of cells from the tumor were RXRB-positive, and that FAM-CRV first bound to cells with high RXRB surface expression (Figure 5D). The majority of RXRB-positive cells (approximately 73%) were CD11b-positive and F4 / 80-positive (Figure 5E), indicating that they were primarily expressed by TAM. 73% of FAM-CRV-positive tumor cells were CD11b-positive and RXRB-positive. Staining of tumor cells after 1 hour of in vivo migration and immobilization of FAM-CRV further showed that RXRB-positive cells had higher CRV binding. However, due to FAM-CRV loss in numerous washing steps, the difference was reduced compared to the results of ex vivo binding (Figure 5D).

[0065] Example 10: The main organs have different levels of RXRB expression. The good correlation between RXRB and CRV has led to increased interest in investigating RXRB surface expression in different tissues, which may explain the in vivo distribution of CRV in vivo. Both RXRB antibody (or rabbit IgG as a control) and FAM-CRV were intravenously injected into the same mice (RXRB antibody: 4 hours circulation; FAM-CRV: 1 hour circulation), and RXRB presentation was evaluated after transcardiac perfusion. Tissue sections were blocked with 1% bovine serum albumin with 0.1% Triton X-100 for 1 hour and incubated with appropriate primary and secondary antibodies. Blood vessels were visualized by staining of tissue sections with monoclonal antibodies against CD-31. Primary antibodies were mouse anti-mouse CD31CD11b (BD Biosciences), mouse anti-mouse CD11b (BD Biosciences), mouse anti-mouse F4 / 80 monoclonal (BD Biosciences), and rabbit anti-fluorescein / Oregon Green (Invitrogen) polyclonal antibodies. Secondary antibodies, Alexa Fluer 488 goat anti-rabbit IgG and 594 donkey anti-mouse IgG, were from Invitrogen. After washing with PBS, sections were embedded in a DAPI-containing mounting medium (Vector Laboratories, Burlingame, California) and examined with a Zeiss LSM 710 NLO confocal microscope.

[0066] Immunofluorescence staining of tissue sections revealed that major organs had different levels of RXRB expression. RXRB was not observed in the heart and lungs, but could be detected in tumors, liver, spleen, and kidneys, while IgG control staining was not observed in any organ. FAM-CRV signaling could only be confirmed in tumors and kidneys after 1 hour of circulation. In tumors, where RXRB staining was highest among all organs, RXRB showed excellent co-localization with FAM-CRV and macrophage markers, best with CD11b, followed by CD68 and F4 / 80 (Figure 6). In the liver, RXRB expression was lower, mainly co-localized with CD31, and hardly co-localized with CD68. In the spleen, RXRB antibodies were present in some parts of the red medulla region, with little co-localization with CD68 and no co-localization with CD31. RXRB expression was very low and mainly co-localized with CD31 in lymph nodes and kidneys. This explains why FAM-CRV was rapidly washed out over time in these two organs. Clearly, the results of RXRB antibody staining were similar with and without the use of Triton X-100 reagent for cell permeabilization.

[0067] Example 11: RXRB is a surface marker with high specificity regarding tumor macrophages. 50 μg of rabbit anti-mouse RXRB antibody (GeneTex, diluted in PBS, total volume 100 μL) was intravenously injected into each mouse (n=3). 50 μg of rabbit IgG per mouse was injected into the control group (n=3). Three hours later, FAM-CRV (100 μg in 100 μL) was injected into all animals. After 1 hour of circulation of FAM-CRV, the animals were killed by transcardiac perfusion. Tissues were collected, fixed, and sliced ​​as described above.

[0068] When RXRB antibody was intravenously injected alone into 4T1 mice (and circulated for an additional 4 hours), the clear distribution of RXRB was similar to that with co-injection of FAM-CRV, indicating that the presence of FAM-CRV did not affect RXRB recognition by the antibody. In other words, the RXRB antibody did not block in vivo CRV binding to RXRB, and vice versa. This indicates that FAM-CRV bound to a different binding site as an RXRB antibody, which was consistent with what was observed in in vitro binding studies. These in vivo migration and colonization results demonstrated that RXRB is a good surface marker with high specific expression on tumor macrophages. Furthermore, it showed that RXRB antibody can preferentially migrate and colonize tumors via intravenous injection, penetrate blood vessels, bind to macrophages in extravascular regions, and function as a target agent.

[0069] Example 12: Migration and fixation of nanoparticles to tumors via CRV To investigate the translational potential of CRV-targeting approaches, the migration and implantation of nanoparticles into tumors via CRVs were evaluated. Extensive studies have shown porous silicon nanoparticles (pSiNPs) as therapeutic carriers with controlled loading and release of various types of drugs (24, 25). Their high efficiency of drug loading and time-gated imaging properties make them particularly desirable as drug carriers.

[0070] To validate the conceptual study, CRV-conjugated porous silicon nanoparticles (CRV-pSiNPs) were loaded with the red fluorescent probe, SR101, and the nanoparticles demonstrated tumor migration and adhesion effects in 4T1 tumor-bearing mice. 100-200 μL of a solution containing CRV peptide, CRV-pSiNPs, or a control nonspecific peptide was injected into the tail vein of tumor mice with a tumor size of approximately 8 mm. After 5 minutes, within 24 hours of circulation, the mice were anesthetized and imaged using imaging equipment (e.g., Xenogen IVIS, Lightools light table, multiphoton LSM). Where available, such as Xenogen IVIS, the mice were anesthetized with isoflurane during imaging (migration / adhesion: flow rate 0.8-1.5 l / min, isoflurane vaporizer 2-3%, maintenance: flow rate 0.4-0.8 l / min, isoflurane vaporizer 2-3%). The mice were then perfused with PBS. Tumors and control organs are collected and used for histological characterization and immunohistochemical evaluation.

[0071] Ex vivo IVIS imaging showed higher SR101 intensity in tumors of animals treated with CRV-pSiNP compared to control NPs (Figure 7A). Cells isolated from tumors with flow cytometry analysis were also analyzed. In the CRV-pSiNP group, 74% of CRV-pSiNP-positive cells were CD11b-positive and CD68-positive, compared to only 52.5% of control pSiNP-positive cells being CD11b-positive and CD68-positive macrophages. This suggests that CRV-targeted NPs mimic the ability of free peptides to migrate and settle, and that the CRV-pSiNP system may deliver drugs to tumor-associated macrophages, thus improving clinical outcomes. Therefore, CRV-pSiNPs can modulate macrophage polarity and be used to load drugs to evaluate the therapeutic effect of tumor treatment.

[0072] While preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Those skilled in the art will find numerous variations, modifications, and substitutions that do not depart from the present invention. It will be understood that various alternatives to the embodiments of the present invention may be utilized in the practice of the present invention as described herein. The following claims define the scope of the present invention and are intended to encompass methods and structures, and their equivalents, within the scope of these claims.

Claims

1. Use of an RXRB-binding molecule in the manufacture of a pharmaceutical product for targeted delivery of a drug to activated tumor-associated macrophages (TAMs) positive for cell surface RXRB expression in a subject, wherein the RXRB-binding molecule is an antibody or a peptide, and the peptide comprises a) CRVLRSGSC, or b) CRVLRSGSC having at least one conserved amino acid substitution.

2. The use according to claim 1, wherein the antibody is an IgG, IgA, or IgM antibody.

3. The use according to claim 1, wherein the antibody is a single-domain antibody.

4. The use according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

5. The use according to claim 1, wherein the antibody is a monoclonal antibody.

6. The use according to claim 1, wherein the peptide is cyclic.

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