Method of assessing lymph node disease using iron oxide nanoparticles in magnetic resonance imaging

Abstract

The present invention relates to a method for assessing lymph node disease by magnetic resonance imaging using a contrast agent. The contrast agent includes biologically functional magnetic nanoparticles coated with a polymer outside an iron core and conjugated with an antibody having specificity to a tumor type to be evaluated. An image generated from such magnetic resonance imaging using a contrast agent shows a heterogeneous low intensity or a heterogeneous configuration in the region where binding by the antibody is present.

Description

BACKGROUND

[0001] Magnetic resonance imaging (MRI) is a medical imaging technique that uses a magnetic field and computer-generated radio waves to create detailed images of the body's organs and tissues. During magnetic resonance imaging, a magnetic field causes water molecules within a mammal's body to temporarily rearrange. Radio waves cause these aligned atoms to produce faint signals, which are used to create cross-sectional MRI images.

[0002] MRI with contrast or imaging agents uses a contrast medium injected into the body to enhance image quality. This MRI contrast agent changes the relaxation time of atomic nuclei within the body tissue. MRI scans with contrast highlight specific parts of soft tissue by increasing the signal-to-noise ratio, and can be used to identify the presence of abnormalities within the tissue, such as tumors.

[0003] Contrast agents for MRI can be detected indirectly by their ability to interfere with water proton relaxation and change the MRI signal intensity. MR contrast agents work primarily by changing the Tl (longitudinal or spin-lattice relaxation time), T2 (spin-spin or transverse relaxation time), or T2* (out-of-phase spin-spin) properties of the imaged tissue. The physical presence of SPIONs causes a field perturbation of the external magnetic field, which can then be indirectly detected by MRI. SPION magnetic moments align with the MRI's magnetic field and become magnetized. When the magnetic field is removed, the magnetic moments return to a random, non-magnetized state. This ability to become magnetized and return to a non-magnetized state is called superparamagnetism.

[0004] SPIONs are dual contrast agents, changing both Tl tissue properties and T2 / T2* tissue properties. SPIONs decrease the Tl relaxation properties of the imaged tissue, which produces a positive MRI contrast on Tl-weighted images. Gadolinium-based contrast agents (GBCAs) also produce contrast by decreasing Tl relaxation. SPIONs also cause the spins of neighboring protons in tissue that has absorbed SPIONs to dephase more quickly, and cause a decrease in T2 and T2* MRI relaxation rates. This T2 / T2* decrease appears as a dark signal or low-intensity contrast on T2 and T2* weighted images, producing negative MRI contrast images.

[0005] The most commonly used contrast-enhancing compound is based on gadolinium. For large blood vessels, such as the aorta and its branches, the dosage can be as low as 0.1 mmol / kg body weight. For more delicate vasculature, higher concentrations are typically used. At higher concentrations, the T2 shortening effect of gadolinium is stronger, resulting in a lower brightness of gadolinium than the surrounding body tissue. However, at this concentration, gadolinium can be more toxic to the body tissue.

[0006] MRI is widely used in medical diagnostics and is an important diagnostic tool for cancer. Without the use of contrast agents, MRI provides images for morphological assessment of possible tumors. With the use of contrast agents, MRI provides images that are more easily assessed for the presence of abnormalities in the tissue.

[0007] Following a new breast cancer diagnosis, lymph node staging is performed, which involves a combination of clinical assessment and radiological imaging. Accurate lymph node staging is an essential component of breast cancer patient management, as treatment depends on the evaluation of specific characteristics of the patient’s primary tumor, the status of the lymph nodes, and distant metastatic disease (NCCN Guidelines, 4thEdition, 2022). While regional lymph node assessment is critical, different practice patterns and imaging modalities can be employed depending on available resources and institutional experience. Furthermore, regardless of the imaging modality, either negative imaging or radiological findings related to the presence of abnormalities, pathological confirmation through needle biopsy or through surgical resection of the sentinel lymph node (SLN) remains the gold standard.

[0008] The two most commonly used radiological modalities for assessing lymph node metastasis are axillary ultrasound and Gd-enhanced magnetic resonance imaging (MRI), and the combined diagnostic sensitivity and specificity of MRI are 75% to 80% and 89% to 91%, respectively, while for ultrasound they are 49% to 87% and 55% to 97%, respectively (Beenken 2003, Choi 2017). Given the importance of accurate lymph node assessment, there is still room for improvement in radiological modalities. While ultrasound has the advantages of convenience, patient comfort, and low cost compared to MRI, its practicality is dependent on the experience of the operator, which is the reason for the large variability in sensitivity and specificity. Ultrasound is also limited in its ability to scan the local regional disease burden (Cody 2012, Saksena 2021). On the other hand, MRI has less dependence on operator experience and can scan the entire lymph node region, but the findings that suggest regional lymph node disease are not tumor-specific and are merely surrogate indicators of possible involvement by a tumor, such as size and morphological changes. While it can be ensured that there is no enlarged regional lymph node disease, the presence of abnormal lymph nodes requires further evaluation by ultrasound and percutaneous biopsy.

[0009] Since the landmark publication of the American College of Surgeons Oncology Group (ACOSOG) Z0011 trial (Giulianio 2011, Giulianio 2017) in 2011, accurate lymph node assessment has become increasingly important. This trial led to a substantial reduction in axillary surgery, as the authors demonstrated that women who received breast-conserving therapy and met the inclusion criteria could omit axillary lymph node dissection (ALND) even if the sentinel lymph node was positive. Since these and other data were published, there has been growing interest in omitting axillary surgery and the sentinel lymph node biopsy (SLNB) procedure (Reiner 2018).

[0010] In these changing circumstances, there is an increased need to improve the accuracy of non-invasive imaging methods (Jatoi 2021, Leenders 2019). Contrast-enhanced non-invasive and molecular targeted, tumor-specific methods as widely accepted imaging modalities (such as MRI) would add value to the existing lymph node staging approaches and help the evolving practice of surgical downstaging and subsequent clinical decision making.

[0011] The present disclosure meets these and other needs in the art. SUMMARY

[0012] The present disclosure relates to agents and methods related to the use of imaging agents to evaluate target tissue, particularly to assess axillary lymph nodes and to identify cancerous (e.g., HER2-positive) cells or tissue in axillary lymph nodes. Exemplary methods utilize a solution of superparamagnetic iron oxide nanoparticles (i.e., biofunctional magnetic nanoparticles) imaging agent, by magnetic resonance imaging.

[0013] According to certain embodiments, a method for assessing lymph node disease by magnetic resonance imaging is provided, the method comprising: introducing a biofunctional magnetic nanoparticle solution into the subareolar or peritumoral region of a human subject diagnosed with breast cancer or suspected of having breast cancer (e.g., HER-2 positive breast cancer) by parenteral administration (e.g., including intravenous administration, intraperitoneal administration, peritumoral administration, subcutaneous administration, or intramuscular administration); performing magnetic resonance imaging on the region of interest of the human subject, the axillary region including at least one lymph node; assessing heterogeneous low intensity of the image of the at least one lymph node; and assessing lymph node disease of the at least one lymph node, wherein the biofunctional magnetic nanoparticle solution comprises a nanoparticle structure, each structure comprising an iron core surrounded by an organic coating layer, a polymer layer, at least one stealth-generating compound bound to the polymer coating layer, and a targeting ligand suitable for binding to a target molecule indicating the lymph node disease. In certain embodiments, the polymer comprises poly(maleic anhydride-alt-octadecene). In addition, in certain embodiments, the targeting ligand is conjugated to the nanoparticle structure at the polymer coating layer. Typically, the target molecule is capable of specifically binding to a protein or cell associated with cancer. Typically, the cancer is breast cancer. In certain embodiments, the target molecule is a HER2 protein or another protein differentially expressed on cancer cells, or their domains or regions. Additionally, in common embodiments, the targeting ligand includes an anti-HER2 protein (such as trastuzumab) or their functional portion or binding domain.

[0014] According to generally encompassed embodiments, identification of heterogeneous low intensity in the lymph node further indicates that the subject tissue is malignant.

[0015] According to certain embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided, the method comprising: introducing a solution of biofunctional magnetic nanoparticles into the inframammary or peritumoral region of a human subject diagnosed with or suspected of having breast cancer (e.g., HER-2 positive breast cancer) by parenteral administration (e.g., including intravenous administration, subcutaneous administration, or intramuscular administration); performing magnetic resonance imaging of an axillary region of interest of the human subject, the axillary region comprising at least one lymph node; assessing the heterogeneous low intensity of the image of the at least one lymph node; and assessing axillary lymph node disease of the at least one lymph node, wherein the solution of biofunctional magnetic nanoparticles comprises nanoparticle structures, each structure comprising an iron core surrounded by an organic coating layer, a polymer layer, at least one stealth generating compound bound to the polymer coating layer, and a targeting ligand adapted to bind a target molecule indicative of the axillary lymph node disease. In certain embodiments, the polymer comprises poly(maleic anhydride-alt-octadecene). Further, in certain embodiments, the targeting ligand is conjugated to the nanoparticle structure at the polymer layer. Typically the target ligand is capable of specifically binding to a protein or cell associated with cancer. Typically, the cancer is breast cancer. In certain embodiments, the target molecule is a HER2 protein, or a domain or region thereof. Further, in common embodiments, the targeting ligand comprises an anti-HER2 antibody (such as trastuzumab) or a functional portion or binding domain thereof.

[0016] According to commonly included embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein each of the biofunctional magnetic nanoparticles within the solution of nanoparticles has a diameter of 10 nanometers to 150 nanometers. Further, according to common embodiments, the nanoparticles within any particular solution have a polydispersity index (PDI) of <0.2. Further, according to certain embodiments, the diameter of each of the nanoparticles within the solution of nanoparticles is typically uniform or relatively uniform within any particular solution. Further, according to certain embodiments, the diameter of each of the nanoparticles within the solution of nanoparticles is typically 55 nanometers to 90 nanometers within any particular solution.

[0017] Typically, according to other embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the solution of biofunctional magnetic nanoparticles is a nanoparticle aqueous solution in 0.9% NaCl containing 0.05% polysorbate 20. In certain embodiments, the solution comprises an isotonic solution that supports product stability and human safe injection.

[0018] Generally, according to embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the polyethylene glycol (PEG) polymer is methoxypolyethylene glycol 2000 and methoxypolyethylene glycol 10000. In certain embodiments, the PEG polymer is in a size range of about 500 Da to about 20,000 Da.

[0019] According to other embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the organic coating layer is any organic acid, for example, oleic acid.

[0020] Generally, according to embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the polymeric coating comprises dextran.

[0021] According to common embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the antibody is an antibody or functional binding fragment thereof capable of specifically binding to a HER2 protein, such as trastuzumab or a functional binding fragment thereof.

[0022] Generally, according to embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the polymeric coating external to the organic coating is poly(maleic anhydride-alt-octadecene) (POMA). In certain embodiments, the POMA is functionalized with carboxylate groups. In these embodiments, the functionalized POMA is suitable for forming covalent bonds with amine groups on other substances, such as proteins containing lysines with amine groups, through EDC chemistry. While POMA is generally preferred polymer, other polymers can also be included.

[0023] According to commonly included embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein one lymph node is assessed or more than one lymph node is assessed.

[0024] Generally, according to other embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the magnetic resonance imaging is performed using a Tl imaging sequence as described herein.

[0025] Generally, according to embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the magnetic resonance imaging is performed using a T2 imaging sequence as described herein.

[0026] According to commonly included embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the human subject holds their breath while the magnetic resonance imaging of the axillary region is performed.

[0027] Generally, according to an embodiment, a method of assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein a solution of biofunctional magnetic nanoparticles is injected into the peritumoral region of a tumor present in a human subject.

[0028] According to a general embodiment, a method for evaluating axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the magnetic resonance imaging is performed using a 1.5T clinical scanner or a 3T clinical scanner.

[0029] Generally, according to an embodiment, a method of assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, further comprising assessing heterogeneity in the image of at least one lymph node.

[0030] According to a common embodiment, a method for evaluating axillary lymph node disease by magnetic resonance imaging is provided as described above, further comprising evaluating the morphology of at least one lymph node for suspicion of a tumor.

[0031] Generally, according to other embodiments, a method of assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, further comprising biopsy of the lymph node if assessed to be diseased.

[0032] Generally, according to embodiments, a method of assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, further comprising performing magnetic resonance imaging on the human subject 24 hours after the first imaging, and reassessing the axillary lymph node disease of at least one lymph node. In certain embodiments, magnetic resonance imaging is performed on the human subject one hour after administration, one day after administration, two days after administration, three days after administration, four days after administration, five days after administration, six days after administration, or within the range of one to three days after administration, or within the range of one to seven days after administration.

[0033] Generally, according to an embodiment, a method for evaluating axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein a target molecule is capable of specifically binding to a protein or cell associated with cancer.

[0034] According to a common embodiment, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the target molecule is the HER2 protein, or a domain or region thereof.

[0035] Generally, according to an embodiment, a method of assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, further comprising assessing heterogeneity in the image of at least one lymph node.

[0036] Generally, according to embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, further comprising treating the subject for the health condition, which can involve administering a drug, biopsy, surgery, or not performing a biopsy or surgery.

[0037] According to common embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein identifying a heterogeneous low intensity in the lymph node further indicates that the subject's tissue is malignant.

[0038] Generally, according to embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein identifying a heterogeneous structure in the lymph node further indicates that the subject's tissue is malignant.

[0039] Also included herein are drugs for magnetic resonance imaging formulated for use in evaluating and / or treating a health condition characterized by the presence of a protein or cell associated with cancer, the method comprising: introducing a solution of biofunctional magnetic nanoparticles into a subject, wherein the solution of biofunctional magnetic nanoparticles is comprised of a plurality of biofunctional magnetic nanoparticles, each nanoparticle comprising an iron core coated with oleic acid, one or more lipids or polymers, and one or more targeting ligands adapted to bind a target molecule; allowing the solution of biofunctional magnetic nanoparticles to bind the target molecule, if present, wherein the target molecule is indicative of a health condition of the subject; performing magnetic resonance imaging on the subject to obtain a magnetic resonance image or image file representative of a target tissue of the subject; evaluating the magnetic resonance image or image file to determine a heterogeneous low intensity region representative of the presence of biofunctional magnetic nanoparticles bound to the target molecule in the target tissue, whereby identifying the heterogeneous low intensity region in the target tissue indicates the presence of the target molecule in the subject's tissue. Generally, the method further comprises treating the subject for the health condition, which can involve administering a drug, biopsy, surgery, or not performing a biopsy or surgery. In certain embodiments, the polymer comprises poly(maleic anhydride-alt-octadecene). Further, in certain embodiments, the targeting ligand is conjugated to the nanoparticle structure at the polymer coating layer. Generally, the target molecule is capable of specifically binding to a protein or cell associated with cancer. Generally, the cancer is breast cancer. In certain embodiments, the target molecule is a HER2 protein, or a domain or region thereof.

[0040] According to commonly included embodiments, a method for assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein identifying a heterogeneous low intensity in the target tissue or lymph node further indicates that the subject's tissue is malignant.

[0041] Generally, according to embodiments, a method of assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, further comprising evaluating the heterogeneity of the image of the at least one lymph node.

[0042] According to embodiments generally included, a method of assessing axillary lymph node disease by magnetic resonance imaging is provided as described above, wherein the identification of heterogeneity in the at least one lymph node is further indicative of the lymph node being malignant.

[0043] These and other embodiments, features, and advantages will become apparent to those of ordinary skill in the art upon review of the following description of various exemplary embodiments of the present disclosure in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Biologically functional magnetic nanoparticles used as drug substance in a solution of biologically functional magnetic nanoparticles are shown according to embodiments.

[0045] Figure 2 Pre- and post-contrast MR images of a normal lymph node are shown. The lymph node shows a uniform dark signal in the post-contrast MR image, indicating that the lymph node is normal.

[0046] Figure 3 Pre- and post-contrast MR images of a pathologically enlarged lymph node are shown. The lymph node shows a heterogeneous low intensity in the post-contrast MR image, indicating that the lymph node is diseased. Some cortex is slightly darker than the pre-contrast image, showing a uniform low intensity, indicating that this area is not pathologically affected.

[0047] Figure 4 A graphic depiction of a lymph node with certain characteristics is depicted. The left image is a pre-contrast lymph node. The right image is a pattern of observation of homogeneous low intensity after administration of an imaging agent on the same lymph node.

[0048] Figure 5 A graphic depiction of a different lymph node with certain characteristics is depicted. The left image is a pre-contrast lymph node. The right image is a pattern of observation of heterogeneous low intensity (“spotted”) after administration of an imaging agent on the same lymph node.

[0049] Figure 6 A graphic depiction of a lymph node with a pattern of observation of heterogeneous structure with partial irregular darkening after administration of an imaging agent is depicted.

[0050] Figure 7 A graphic depiction of a lymph node with a pattern of observation of both heterogeneous structure with partial irregular darkening and heterogeneous low intensity (“spotted”) after administration of an imaging agent is depicted.

[0051] Figure 8Pre- and post-contrast MR images of a lymph node are shown, demonstrating heterogeneous low intensity on post-contrast T2-weighted images.

[0052] Figure 9 Pre- and post-contrast MR images of a lymph node are shown, demonstrating heterogeneous structure (with irregular darkening) on post-contrast T2-weighted images.

[0053] Figure 10 Pre- and post-contrast MR images of a lymph node are shown, demonstrating both heterogeneous low intensity and heterogeneous structure on post-contrast T2-weighted images.

[0054] Figure 11 Pre- and post-contrast MR images of a lymph node are shown, demonstrating no contrast agent uptake, with an adjacent lymph node demonstrating homogeneous low intensity.

[0055] Figure 12 Relaxometry measurements and ICP-MS analysis of BT474 and MCF7 cells exposed to an imaging agent comprising an anti-HER2 targeting ligand are depicted.

[0056] Figure 13 Superparamagnetic relaxometry signal of high HER2-expressing cells, low HER2-expressing cells, and negative HER2-expressing cells exposed to an exemplary imaging agent specific for HER2 are depicted.

[0057] Figure 14 Superparamagnetic relaxometry signal of high HER2-expressing cells, medium HER2-expressing cells, and low HER2-expressing cells exposed to an imaging agent comprising an anti-HER2 targeting ligand are depicted.

[0058] Figure 15 Superparamagnetic relaxometry signal from a titration of high HER2-expressing cells and no HER2-expressing cells exposed to an imaging agent comprising an anti-HER2 targeting ligand are depicted.

[0059] Figure 16 Superparamagnetic relaxometry signal from BT474 cells, lymphocytes, and peripheral blood mononuclear cells exposed to an imaging agent comprising an anti-HER2 targeting ligand are depicted.

[0060] Figure 17 Superparamagnetic relaxometry signal from a HER2-positive cell implant exposed to an imaging agent comprising an anti-HER2 targeting ligand, a pegylated nanoparticle control, and a free antibody competitor are depicted.

[0061] Figure 18 Superparamagnetic relaxometry signal and staining of BT474 and MCF7 cell tumors exposed to an imaging agent comprising an anti-HER2 targeting ligand administered by different routes of administration are depicted.

[0062] Figure 19 Time course flow of imaging agent in lymph nodes as measured by MRX is depicted.

[0063] Figure 20 Presence of imaging agent in resected sentinel lymph nodes after 24 hours is depicted by lymph node color (top panel: dark brown / black) and Prussian blue iron stain.

[0064] Figure 21 Lymph node sections depicting presence of imaging agent in tumor cells and lymph node mouse tissue as shown by Prussian blue iron stain.

[0065] Abbreviations

[0066] CDR: complementarity determining region

[0067] HER-2: human epidermal growth factor receptor 2

[0068] MR: magnetic resonance

[0069] MRI: magnetic resonance imaging

[0070] MRX: magnetic relaxation measurement DETAILED DESCRIPTION

[0071] As used herein, "a" or "an" means "at least one" or "one or more"

[0072] As used herein, the term "and / or" can mean "and", it can mean "or", it can mean "exclusive-or", it can mean "one", it can mean "some, but not all", it can mean "neither", and / or it can mean "both", although the context can dictate a more specific meaning

[0073] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or immunologically reacts with a particular antigen, here HER2. Antibodies comprise complementarity determining regions (CDRs), also known as hypervariable regions, in both the light chain and heavy chain variable domains. The more conserved portions of the variable domains are referred to as framework (FR). As known in the art, the amino acid positions / boundaries defining the hypervariable regions of an antibody can vary according to context and various definitions known in the art. Some positions within the variable domains can be considered mixed hypervariable positions, as these positions can be considered within the hypervariable region under one set of criteria, and outside the hypervariable region under another set of criteria. One or more of these positions can also exist in an extended hypervariable region. The variable domains of the naturally occurring heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. See Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987). As used herein, unless otherwise indicated, numbering of immunoglobulin amino acid residues is in accordance with the Kabat et al. system of numbering immunoglobulin amino acid residues.

[0074] Antibodies and / or binding fragments that make up an anti-HER2 antibody typically comprise a heavy chain comprising a variable region (V H ) having three complementarity determining regions ("CDRs"), referred to herein (in N→C order) as V H CDR#1, V H CDR#2, and V H CDR#3, and a light chain comprising a variable region (V L ) having three complementarity determining regions, referred to herein (in N→C order) as V L CDR#1, V L CDR#2, and V L CDR#3. Amino acid sequences of exemplary CDRs are provided herein, as are amino acid sequences of V H regions and V L regions of heavy and light chains of exemplary anti-HER2 antibodies and / or binding fragments that can be included in an antigen binding moiety. Specific embodiments of anti-HER2 antibodies include, but are not limited to, those comprising these exemplary CDRs and / or VH and / or V L Embodiments of antibodies and / or binding fragments of sequences, and antibodies and / or binding fragments that compete for binding to HER2 with such antibodies and / or binding fragments.

[0075] Antibodies can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multichain antibodies or single-chain antibodies, surrogate antibodies (including surrogate light chain constructs), single domain antibodies, camelized antibodies, scFv-Fc antibodies, and the like. They can be of any isotype or derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), IgM, or IgY. In some embodiments, the anti-HER2 antibody is an IgG (e.g., IgGl, IgG2, IgG3, or IgG4). Antibodies can be of human or non-human origin. Examples of non-human origin include, but are not limited to, mammalian origin (e.g., simian, rodent, goat, and rabbit) or avian origin (e.g., chicken).

[0076] Fab fragments contain the constant domain of the light chain and the first constant domain of the heavy chain (CH2). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH2 domain, including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of disulfide bonds at the hinge cysteines of the F(ab')2 pepsin digest product. Additional chemical conjugation of antibody fragments is known to those of ordinary skill in the art. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody.

[0077] An "Fv" fragment is the minimum fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain bound in a tight, non-covalent association (V H -V L The dimeric V H -V L H3CDRs interact to define an antigen binding site on the surface of the dimer. In general, the six CDRs confer antigen binding specificity to the antibody. However, in some instances, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and bind antigen, although at a lower affinity than the whole binding site.

[0078] A "single-chain Fv" or "scFv" antibody binding fragment comprises the VH and V L domains, where the domains are present in a single polypeptide chain. Typically, Fv polypeptides further comprise a polypeptide linker between the V H and V L domains, where the domains are present in a single polypeptide chain. Typically, Fv polypeptides further comprise a polypeptide linker between the V

[0079] As used herein, "targeting ligand" refers to the broadest sense of antibody used herein and in the art, or another moiety adapted to specifically bind a predetermined target molecule.

[0080] As used herein, "MR imaging" refers to magnetic resonance imaging or MRI. The terms MRI and "MR imaging" are used interchangeably herein.

[0081] As used herein, "heterogeneous low intensity" refers to a speckled molecular signature that appears in a heterogeneous low intensity pattern in an MRI image, which the inventors have found to be indicative of sufficient imaging agent in vivo specifically bound to a target molecule detectable by MRI, for example. Figure 4 An expected illustration of the term heterogeneous low intensity as observed in an exemplary target tissue is depicted. Other types of target tissue in a subject are specifically contemplated for imaging according to the methods described herein, including lymph nodes as well as in addition to lymph nodes.

[0082] As used herein, "heterogeneous architecture" refers to a feature in an MRI image having a mottled appearance of light and dark, which the inventors hypothesize to be indicative of sufficient imaging agent in vivo bound to a target molecule detectable by MRI, for example. Figure 6 An expected illustration of the term "heterogeneous architecture" as observed in an exemplary target tissue is depicted.

[0083] As used herein, "imaging agent" refers to a biologically functionalized magnetic nanoparticle solution composed of iron oxide nanoparticles, typically between 10 and 150 nanometers in diameter, functionalized with a targeting ligand, and having a polydispersity index (PDI) < 0.2, formulated for safe in vivo use in humans. The imaging agent is typically referred to as the complete solution or the functionalized nanoparticles contained therein. Nanoparticles, including magnetic nanoparticles, organic acids, polymers, and targeting ligands, as prepared and functionalized as described herein, are also referred to as biologically functionalized magnetic nanoparticles.

[0084] Cancer diagnosis and staging requires a combination of clinical classification and invasive pathological assessment. For breast cancer patients, cancer staging requires determination of whether primary tumor cells have spread to lymph nodes. Lymph nodes can be clinically suspected of metastasis according to palpation or imaging techniques such as ultrasound, PET, CT, or MRI, and require biopsy for pathological confirmation.

[0085] For many breast cancer patients, current imaging methods do not reliably detect microscopic and macroscopic metastases, and lymph node assessment involves surgical removal of 2 to 4 sentinel lymph nodes (SLN) and complete removal of axillary lymph nodes (ALN) based on the extent of tumor infiltration determined by laboratory pathologic analysis. While only about 25% of clinically node-negative breast cancer patients develop nodal involvement, current standard of care still requires surgical intervention to confirm the absence of metastatic spread for all patients diagnosed as node-negative by standard of care imaging. This means that the majority of patients unnecessarily undergo surgery, resulting in avoidable morbidity, such as lymphedema, intercostobrachial nerve pain, local cellulitis or infection, and limiting treatment options in the event of cancer recurrence. Non-invasive methods to confirm nodal involvement have been found to significantly improve patient care by avoiding the expense and risk of surgical biopsy for the majority of patients, and eliminating the serious concomitant morbidity associated with lymph node resection.

[0086] While not intending to be bound by any operating theory, it has been found that:

[0087] • the imaging agent described herein is specific for HER2-expressing cancer cells;

[0088] • the imaging agent can be used to provide a tumor-specific contrast agent in MRI and improve its detection sensitivity and specificity;

[0089] • the imaging agent does not have significant non-specific binding to non-tumor cells, such as isolated human cadaveric lymph node cells and peripheral blood mononuclear cells (PBMC);

[0090] • in a mouse model, lymph nodes take up the imaging agent when administered by intraperitoneal and peritumoral injection; and

[0091] • to obtain the best signal from the imaging agent, a period of up to or more than 16 hours from imaging agent administration to surgical measurement is preferred.

[0092] A single dose of 30 mg (or 20 mg) of imaging agent (based on iron oxide weight) is evaluated according to the protocol described herein. The selected dose is based on the assumption that at least 1% of the material (200 μg to 300 μg of imaging agent) is capable of distributing to lymph nodes by peritumoral injection of the breast tumor. Overall, the selected dose translates to a significantly lower dose of iron oxide per body weight (0.3 mg / kg to 0.5 mg / kg) compared to non-clinical studies (according to FDA guidance "Estimating the Maximum Safe Starting Dose in Initial

[0093] In the FIH study, a single dose of 30 mg (or 20 mg) of the imaging agent (based on iron oxide weight) was evaluated. Overall, the selected dose translation (according to FDA guidance "Estimating the Maximum Safe Starting Dose in Initial-Human-Subject Trials, Including Biosafety Guidelines"6) was a significantly lower dose of iron oxide per body weight (0.3 mg / kg to 0.5 mg / kg) as compared to the nonclinical studies.

[0094] The present disclosure relates to biologically functional magnetic nanoparticle solutions and their use as test reagents in magnetic resonance imaging. Provided herein are drug substances formulated as solutions for injection of a subject (e.g., by parenteral administration, including intravenous administration, subcutaneous administration, or intramuscular administration, e.g., subareolar administration, peritumoral administration, etc.) comprising biologically functional magnetic nanoparticles.

[0095] According to the methods of the present disclosure, a human subject suspected of or diagnosed with breast cancer is evaluated for lymph node disease or lymph node metastasis, including axillary lymph node disease. According to such methods, the human subject receives an injection of an imaging agent according to the embodiments described herein by, for example, parenteral administration, including intravenous administration, subcutaneous administration, or intramuscular administration, e.g., subareolar administration or peritumoral region administration. Thereafter, the human subject receives MR imaging of the region of interest, e.g., the axillary region. The MR imaging can be a Tl -weighted imaging sequence or a T2 / T2* -weighted imaging sequence, and the resulting images are evaluated by a radiologist. The methods of the present invention provide an imaging-based diagnosis without the need for biopsy, thereby reducing the need for additional hospital visits and surgery that are currently the standard of care.

[0096] The imaging agent according to embodiments herein is a solution of biologically functional magnetic nanoparticles, where the nanoparticles have a core of iron coated with an organic coating such as oleic acid, which is subsequently coated with a polymer such as poly(maleic anhydride-alt- octadecene), and then conjugated with a polyethylene glycol polymer and an antibody specific to the type of cancer present in the human subject, such as the anti-HER-2 antibody trastuzumab for a HER-2 breast cancer subject. Other organic substances such as dextran can be used for coating. Other polymers can be used for coating and / or conjugation. Other antibodies specific to different tumors are also contemplated. The imaging agent is provided in solution form for parenteral administration such as by injection. In MR imaging, the nanoparticles in the imaging agent that are loaded with the targeting ligand bind to the tissue of interest, providing an image that can be distinguished from non- interested tissue according to certain features of the image. These features include areas of heterogenous low intensity in the tissue of interest in the MRI image that are evaluated by the radiologist. The exemplary targeting ligand used in the studies described herein is an anti-HER-2 antibody known to be specific to the HER-2 protein associated with the presence of breast cancer. In axillary lymph nodes where the cancer has metastasized, there is high value in using an imaging agent that is able to provide a distinguishable imaging difference, because the lymph nodes are small and accuracy is low relying on morphological assessment alone.

[0097] According to certain embodiments described herein, after receiving an injection of an imaging agent as provided herein, the human subject receives MR imaging to evaluate the axillary lymph nodes to which the imaging agent will flow, as well as other areas. In a normal lymph node, the image of the lymph node shows a uniform dark signal, as shown in Figure 2 In a diseased lymph node of a HER-2 breast cancer patient, the image of the lymph node shows a heterogenous low intensity, as shown in Figure 3

[0098] By reading the MR image of the axillary lymph node injected with the imaging agent according to the embodiments, the radiologist can determine whether the lymph node is present with HER-2 breast cancer tissue according to the identification of areas of heterogenous low intensity in the MRI image of the tissue of interest.

[0099] ​According to one exemplary embodiment, the imaging plan baseline MRI assessment is performed prior to imaging agent administration and at one or two additional time points after imaging agent administration, as follows: Visit 2, Day 1 - prior to imaging agent injection; Visit 3, Day 2 within 18 hours to 30 hours, and Visit 5, Day 4 within a 66 hour to 78 hour time window, or Visit 3, Day 2 / 3, within an 18 hour to 78 hour time window after imaging agent injection. Imaging agent administration is performed by a surgeon or radiologist after completion of the baseline MRI scan on Day 1. Administration of the imaging agent can be performed in a radiology or other suitable setting. As peritumoral injection can involve additional interventional ultrasound (US) guidance, appropriate ultrasound equipment should be provided to support administration.

[0100] In this plan, it has been found that matching the resected or biopsied lymph node(s) to the post-administration imaged lymph node(s) can be used to compare the post-administration MRI scan to the pathological findings of the resected or biopsied lymph node for tumor presence. To this end, the use of a clip compatible with magnetic resonance inserted on the same day as the MRI is required to locate the suspected lymph node, and the use of imaging modalities available on site (e.g., ultrasound). The clip insertion can be performed at any time after signing the informed consent and prior to the post-administration MRI.

[0101] During imaging, the subject is typically supine on the spine coil, offset from the center, so that the side (location) of the breast pathology is centered in the magnet. For example, if the right breast (diseased side) has a suspected lesion, the subject should be offset to the left side of the spine coil so that the right axilla is centered in the spine coil, or as close as possible depending on the subject’s size. The highest density body array coil available is placed in the subject’s axillary region on the diseased side. The subject’s arm on the diseased side is preferably placed tightly against the body to eliminate any gap between the arm and the body. The other arm can also be placed against the body, or preferably placed overhead. Additional immobilization sponges can be used to help limit the subject’s motion.

[0102] The following combination of image sequences is typically used at each MRI acquisition (estimated total MRI acquisition time is approximately 20 minutes):

[0103] • Localizer

[0104] • Positioning of lymph node 1 uses:

[0105] • Tl gradient echo

[0106] • T2 fast spin echo (TSE)

[0107] • Tl multi-station gradient echo (VIBE) for nanoparticle detection

[0108] ○ T2* gradient recalled echo (GRE) for nanoparticle detection is feasible

[0109] Additional sequences may be performed to meet site practice standards, and optional imaging parameters may be applied at the site's discretion, as long as documented.

[0110] The field strength is preferably typically 1.5 T. Only when necessary, as a second option, a field strength of 3 T and an adapted acquisition scheme can be used.

[0111] Contrast materials

[0112] An exemplary imaging agent for MR imaging according to the present invention is a biofunctional magnetic nanoparticle solution, also referred to herein as an imaging agent. The exemplary biofunctional magnetic nanoparticles in the imaging agent are composed of iron oxide nanoparticles coated with a surfactant (e.g., oleic acid) and a layer consisting of carboxyl groups functionalized with a polymer poly(maleic anhydride-alternative-octadecene) (POMA). Such coated nanoparticles are conjugated to a polyethylene glycol polymer and the anti-HER-2 antibody trastuzumab. The exemplary imaging agent is typically composed of an optionally filtered aqueous solution of nanoparticles dissolved in 0.9% NaCl containing 0.05% polysorbate-20.

[0113] In general, exemplary imaging agents include injectable formulations consisting of SPIONs conjugated to anti-HER2 antibodies. NPs consist of a spherical iron oxide core (24 nm to 28 nm in diameter) coated with an amphiphilic polymer (polymaleic anhydride-alternative-1-octadecene [POMA]) that serves as a linker for the covalent conjugation of trastuzumab (anti-HER2 antibody) and polyethylene glycol (PEG). Core iron oxide NPs and POMAc-functionalized NPs are manufactured by nanoComposix (San Diego, CA).

[0114] The final antibody-conjugated NP drug substance is produced in the form of a colloidal solution containing NP (approximately 10 mg / mL based on iron oxide) dissolved in sterile (0.9%) saline and 0.05% polysorbate 20 solution (to ensure stability). The final imaging agent formulation is the same as the drug substance solution. According to GMP manufacturing guidelines, the imaging agent raw material is transferred to PCIPharma Services (Melbourne, Australia) and sterile filtered (0.22 μm filter) into 2 mL sterile vials (Crystal Zenith vials, West Pharma) containing 1.3 mL of imaging agent solution and covered with sterile 13 mm stoppers and aluminum caps. Sterility analysis is performed by a GMP analytical laboratory (Eurofins) in Australia.

[0115] Figure 1The primary structure of POMAc-NP conjugated with polyethylene glycol polymer and trastuzumab is shown. The primary structure comprises an iron oxide core coated with an oleic acid layer, which is in turn coated with a poly(maleic anhydride-alt-octadecene) layer, methoxypolyethylene glycol 2000, methoxypolyethylene glycol 10000, and trastuzumab conjugated to the poly(maleic anhydride-alt-octadecene) layer.

[0116] The biologically functional magnetic nanoparticle solution used in the present invention is an aqueous, dark brown to black colloidal solution of biologically functional magnetic nanoparticles as shown in Figure 1

[0117] To date, the largest dose volume delivered to the human body is 3 mL of imaging agent at 7.5 mg / mL Fe equivalent, i.e., 22.5 mg Fe equivalent dose of imaging agent. This dose volume and dose strength is also referred to as a 3 mL calculated dose of 10 mg / mL Fe3O4 equivalent of imaging agent, or a maximum dose of 30 mg Fe3O4 (calculated) or 30 mg imaging agent.

[0118] The imaging agent of the present invention, as an MRI contrast agent and with tumor targeting properties, has the potential to improve the diagnostic accuracy of clinical lymph node assessment when used alone or as a complement to the current imaging methods for HER-2 positive breast cancer patients, providing a rationale for evaluating the use of the imaging agent of the present invention in this specific patient population.

[0119] Example Study

[0120] In vitro cell binding and sensitivity of imaging agent HER2 test reagent

[0121] Study objective: Conduct a cell binding study to validate the sensitivity of the imaging agent to target HER2 positive tumor cells.

[0122] Methods: This study was conducted using the BT474 breast cancer cell line, which is known to express high levels of HER2, and MCF7 cells, which are known to be low or negative for HER2. Cells were seeded in 6-well cell culture plates 24 hours prior to the addition of 100 μg (based on iron oxide) of imaging agent. Following a 24 hour incubation period, cells were washed to remove unbound imaging agent and harvested for SPMR measurement. ​

[0123] Results: Titration experiments (cell numbers ranged from 0.25 x 106to 2 x 106) showed that the test reagent could be detected in BT474 cells at the lowest titration of 0.25 x 106( Figure 5 , left panel). The results were confirmed by inductively coupled plasma mass spectrometry (ICP-MS) analysis to measure the amount of iron present in each titration ( Figure 5 , right panel).

[0124] Conclusion: The imaging agent can specifically bind to HER2 positive cells (BT474). These signals were further validated using ICP-MS by measuring the amount of iron present.

[0125] In vitro specificity and selectivity of the imaging agent containing the anti-HER2 targeting ligand

[0126] Study objective: To perform an in vitro cell binding study to assess the specificity of the imaging agent to target high HER2 expressing tumor cells, low HER2 expressing tumor cells, and negative HER2 expressing tumor cells.

[0127] Methods: The binding of one high HER2 expressing cell line (BT474) was compared to two low or non-expressing cell lines (MCF10, MCF7, OVCAR) and one HER2 positive but Herceptin non-responsive cell line (JIMT). In this study, 100 μg of the imaging agent was incubated with 1 x 106of each type of cell for 24 hours and binding was assessed by detection of SPMR signal.

[0128] Results: The imaging agent was able to produce a clearly measurable signal by SPMR for high HER2 expressing cancer cells (BT474), but not for low or negative HER2 expressing cells (MCF10, JIMT, OVCAR, and MCF7) Figure 6 .

[0129] Conclusion: This study shows that the imaging agent has high specificity and selectivity for targeting HER2 positive tumor cells.

[0130] Cellular selectivity and specificity study

[0131] Study objective: To perform a cell binding study to assess the specificity of the imaging agent to target high HER2 expressing tumor cells, medium HER2 expressing tumor cells, low HER2 expressing tumor cells, or negative HER2 expressing tumor cells with and without exposure to free anti-HER2 antibody (competition).

[0132] Methods: In this study, 100 μg of imaging agent was incubated with 1 x 106cells from multiple HER2 positive (IHC 3+) cell lines with varying degrees of HER2 expression (SKBR3, BT474, HCC1954), as well as a moderately expressing (IHC 2+) cell line (ZR75) and a low or non-expressing cell line (MCF7) for 24 hours. The study included a competition study arm, in which all cells were exposed to free anti-HER2 antibody (100-fold excess compared to Herceptin on the NPs) prior to incubation with the imaging agent. Binding was assessed by detection of SPMR.

[0133] Results: The results also demonstrated that the imaging agent and SPMR signal could be competed off by free antibody Figure 7 ).

[0134] Conclusion: The imaging agent has high specificity and selectivity for binding to cells with HER2 expression.

[0135] Additional cell selectivity and specificity studies

[0136] Study objectives: Additional cell binding studies were performed to assess the effect of tumor cell titration on the level of imaging agent binding.

[0137] Methods: High HER2 expressing cell lines (SKBR3, BT474, and HCC1954) and a non-HER2 expressing cell line (MDA-MB231) were titrated with 100 μg of imaging agent and incubated for 24 hours. The number of cells evaluated were 2.5 x 106, 5.0 x 106, and 10 x 106cells, respectively. Binding was assessed by detection of SPMR signal, which was confirmed by iron content measurement using ICP-MS.

[0138] Results: Titration of high HER2 expressing cell lines and non-HER2 expressing cell lines with the imaging agent demonstrated a unique and direct correlation between the number of cells and the SPMR signal Figure 8 , left panel). These results were further confirmed by the corresponding iron content measured using ICP-MS Figure 8 , right panel).

[0139] Conclusion: The SPMR signal is directly related to the number of imaging agent-labeled cells, and this signal is directly related to the amount of imaging agent present in the sample.

[0140] In vitro non-specific interaction of the imaging agent with lymphocytes and peripheral blood mononuclear cells

[0141] Study Goal: Since the intended use of the imaging agent is to detect HER2 positive tumor cells in lymph nodes, a study was conducted to determine if the imaging agent binds non-specifically to lymphocytes and PBMCs that would interfere with the SPMR signal.

[0142] Methods: Human cadaveric lymph nodes were purchased from a commercial vendor and lymph node cells were isolated. 10 x 106isolated lymphocytes were incubated with 100 μg of imaging agent for 24 hours after isolation. The imaging agent was also incubated with 1 x 106HER2 positive cells (BT474) and co-cultured with both cell types (1 x 106BT474 and 10 x 106lymphocytes). In addition, the imaging agent was incubated with 10 x 106PBMCs. Binding was assessed by detection of SPMR signal.

[0143] Results: Lymphocytes incubated with the imaging agent produced SPMR signal, indicating some degree of non-specific interaction with the lymphocytes. HER2 expressing cells incubated with the imaging agent also produced SPMR signal, which was approximately 3 times higher than the non-specific signal produced by the lymphocyte culture ( Figure 9 , left panel). PBMCs incubated with the imaging agent did not produce SPMR signal, indicating no interaction of the imaging agent with the PBMCs ( Figure 9 , right panel).

[0144] Conclusions: In summary, these results indicate that the imaging agent is unlikely to produce significant false positive signals due to non-specific interaction with non-tumor cells in lymph nodes and blood.

[0145] Tumor detection and specificity of the imaging agent in a xenograft tumor model in mice

[0146] Study Goal: To determine if the imaging agent can target HER2+ tumors in vivo and produce a signal sufficient for SPMR detection.

[0147] Methods: 3 x 106HER2 positive (BT474) cells were implanted subcutaneously in the flank region of female athymic nude mice. After 6-10 weeks, palpable tumors of size ranging from 0.125 cm3to 1 cm3were generated. Multiple different routes of administration were used to deliver 400 μg of imaging agent, including intraperitoneal administration, peritumoral administration, and tail vein injection. Polyethylene glycolated NPs (same structure as the imaging agent, but without the anti-HER2 antibody) were used as a control vehicle. In vivo competition studies were also performed by pre-injecting 1 mg of free anti-HER2 antibody via tail vein 24 hours prior to imaging agent delivery. Mice were euthanized 24 hours after administration. Tumors and other major organs were excised and ex vivo SPMR measurements were performed on the excised tissues, and iron content was measured using ICP-MS.

[0148] Results: BT474 tumor cells exposed to the imaging agent produced greater SPMR signal than tumor cells exposed to the control vehicle ( Figure 10 ). In addition, pre-injected free antibody was more competitive than the imaging agent, producing little to no SPMR signal.

[0149] Absorption of the imaging agent by the xenograft tumor was further confirmed by measuring iron content using ICP-MS. Quantitative analysis of SPMR signal and ICP-MS indicated that there was an average of about 2-5 μg of imaging agent in the tumor, while the control vehicle was below the ICP-MS detection limit.

[0150] Conclusion: The imaging agent can bind to tumor cells in vivo and produce a specific signal that can be measured by the MRX instrument.

[0151] Additional tumor detection and specificity of the imaging agent in a bilateral flank xenograft tumor model in mice in vivo

[0152] Study objectives: As a follow-up study, a bilateral flank tumor model was used to further demonstrate that the imaging agent can bind to HER2-positive tumor cells in vivo, but not to HER2-negative tumors, and produce a specific signal through various delivery routes.

[0153] Methods: A bilateral flank tumor model was generated by implanting 3 x 106BT474 (HER2-positive) and 1 x 106MCF7 (HER2 low expression or non-expression, MCF7 is a faster growing cell than BT474) cancer cells in each flank of female athymic nude mice. Multiple different routes of administration (including intraperitoneal administration, peritumoral administration, and tail vein injection) were used to deliver 400 μg of the imaging agent. PEGylated NPs (same structure as the imaging agent, but without the anti-HER2 antibody) were used as a control vehicle. Mice were euthanized 24 hours after administration. Tumors and other major organs were excised and ex vivo SPMR measurements were performed on the excised tissues, in addition to Prussian blue and anti-Herceptin staining to localize the imaging agent within the tumor.

[0154] Results: All routes of administration tested produced significantly higher SPMR signal in the BT474 tumors than in the MCF7 tumors ( Figure 11 , left panel). The presence of the imaging agent was localized primarily outside of the tumor, as shown by Prussian blue staining (blue staining) and anti-Herceptin staining (brown staining) Figure 11 , right panel).

[0155] Conclusion: These results show that the specificity of the imaging agent for targeting HER2-positive tumor cells is maintained in vivo and can be detected by SPMR measurements. Clearance of the imaging agent is primarily through the liver and spleen, as expected for NPs in the similar size range in the scientific literature.

[0156] Distribution of the imaging agent in the lymph nodes of mice

[0157] Study objective: Design and conduct a mouse-based study to assess the time required for the imaging agent to drain from the lymph nodes when injected into the distal mammary pad.

[0158] Methods: 433 μg of the imaging agent was injected into the right nipple / areola of the fourth abdominal mammary pad of nine female athymic nude mice. Axillary and inguinal lymph nodes were excised at 24 hours, 48 hours, and 72 hours post-injection (3 mice per time point). Visual inspection of the excised lymph nodes was performed at 24 hours post-injection to determine the presence of the imaging agent within the lymph nodes, and Prussian blue staining was performed to locate the position of the imaging agent within the lymph nodes. Excised lymph nodes were measured ex vivo using the MRX instrument. Iron content within each lymph node was also measured using ICP-MS to confirm the MRX results. Figure 12

[0159] Results: The target ALN was significantly darker, brown / black in color, compared to the inguinal lymph nodes on the same side and the ALN on the other side of the body. Figure 13 Figure 14 The presence of the imaging agent in the ALN was confirmed by Prussian blue staining, which showed that the imaging agent was primarily localized in the lymphatic tissue sinuses, indicating proper drainage.

[0160] ICP-MS further confirmed the presence of the imaging agent in the ALN by detecting the element iron. On average, 6 μg Fe was detected in the ALN and 1 μg Fe in the inguinal lymph nodes by ICP-MS in the same injection in mice.

[0161] Most of the imaging agent had drained from the lymph nodes after 72 hours, especially the inguinal lymph nodes closest to the injection site. Figure 12

[0162] Conclusion: The imaging agent drains more effectively from the lymph nodes within 72 hours, thereby minimizing the potential non-specific signal on MRI / MRX readouts.

[0163] MRI imaging using the imaging agent - first cohort

[0164] The imaging agent was used in an in vivo MRI study in six (6) patients as described below.

[0165] ​​​Human participants were selected based on the following criteria: participants were diagnosed with HER-2 positive primary breast cancer, planned to undergo preoperative clinical assessment of axillary lymph node disease, followed by core needle biopsy or pathologic confirmation. Participants with a routine axillary assessment of lymph node suspicious for disease were enrolled in the study. Exclusion criteria were known inflammatory breast cancer and prior axillary surgery, including sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND) or prior radiation therapy to the ipsilateral breast of the primary breast cancer. Participants with a likelihood of lymph node metastasis and planned to undergo sentinel lymph node biopsy (SLNB) and / or axillary lymph node dissection (ALND) (Group 1), or planned to undergo suspicious lymph node biopsy (Group 2), or had already undergone suspicious lymph node biopsy (Group 3) were selected for the study, according to the investigator’s judgment.

[0166] All eligible participants received a single dose of 22.5 mg Fe equivalent of drug substance injected into the subareolar interstitial tissue or the area near and around the primary tumor on study day 1. Baseline MRI assessment was performed within 3 days prior to the administration of the imaging agent (the imaging agent is a solution of biofunctional magnetic nanoparticles described herein) and a second MRI was performed within 18 to 30 hours after administration of the imaging agent and prior to any neoadjuvant therapy, and a third MRI was performed within 66 to 78 hours. Whole lymph node or lymph node tissue from core needle biopsy was obtained for histopathological assessment. These samples were also used for ex vivo MRX measurements.

[0167] The study included screening, baseline, imaging, and follow-up periods. Participants were screened between day -28 and day -1 to determine if they were eligible for the study. Each participant was given a baseline period on the day after the baseline MRI scan or on day 1 of the imaging period. Post-administration MRI scans were performed and pathology samples were obtained during the imaging period. Data collection and assessments included safety assessments and safety laboratories; image collection, storage, transmission, and central image reading; lymph node specimen harvesting, ex vivo MRX measurements, participant’s clinical care pathology, study sample transfer, study sample pathology, and central pathology reading.

[0168] Post-administration safety and imaging evaluations (including MRI and biopsy) were performed at visit 2 (day 1) and visits 3 through 5 (18 to 78 hours after administration). Two post-administration MRI imaging scans were performed, one at approximately 24 hours and another at approximately 72 hours. Additional safety evaluations were performed at visit 6 (day 7 ± 2 days) and the end of study visit (day 28 ± 3 days).

[0169] Evaluation of results. While not intending to be bound by any particular theory of operation, contrast agents for MRI can be detected indirectly by their ability to interfere with water proton relaxation and change the MRI signal intensity. The imaging agent changes the Tl (spin lattice or longitudinal relaxation) and T2 (spin-spin or transverse relaxation) or T2* (out of phase spin-spin) properties of the local tissue being imaged, resulting in image contrast. The nuclei are designed for high magnetic relaxivity, changing the T2 contrast. For normal lymph nodes, the imaging agent is taken up by the resident macrophages, resulting in a relatively uniform T2 low intensity (dark) contrast. When tumor cells metastasize to the lymph nodes, they replace the macrophages (in whole or in part), thus, the homogenous low intensity normally seen is not present in the areas of the lymph node where tumor cells are present. However, because the imaging agent contains molecularly targeted nanoparticles (different from the particles discussed in the literature), it has been found that, according to the embodiments herein, the specific binding between the target on the tumor and the ligand on the imaging agent results in a heterogeneous low intensity, where the nanoparticles bind to the tumor cells that have invaded the lymph node. When comparing the pre-administration and post-administration MRI images, the non-targeted particles show no change in intensity for the tumor-involved lymph nodes, however, it has been found that the solution described herein shows a change in signal intensity for both the tumor-involved lymph nodes and the normal lymph nodes in the pre-administration and post-administration MRI images. However, the amount of change in signal intensity for the tumor-involved lymph nodes is significantly different and distinguishable from the change in the normal lymph nodes, which allows for the differentiation of the tumor-involved lymph nodes from the normal lymph nodes on the post-administration MRI images.

[0170] MRI measurements were performed using a 1.5 T clinical scanner or a 3 T clinical scanner. The MRI examination involved having the participant lie supine on a spine coil, off-center so that the breast diagnosed with cancer was closer to the center of the magnet. A high-density body array coil was placed in the axillary region of the breast to be imaged. All image acquisitions were performed in the axial orientation. Tl, T2, and T2* imaging sequences were used.

[0171] It is expected that the primary effect of the imaging agent will be based on T2 and T2* relaxation, and thus on T2 / T2* weighted sequences. In addition, it is expected that the contrast enhancement will also be based on Tl weighted images. All sequences except the T2* sequence were acquired by providing the participant with explicit breath-hold instructions. The approximate imaging time for the MR imaging sequences described above was about 20 minutes. To ensure consistency, it is expected that the imaging scanner and protocol acquired pre-administration and post-administration will be similar. All images were shipped to the central radiology laboratory by secure file transfer or secure shipping of CD containing the image data.

[0172] The central imaging laboratory reviewed MRI scans from the six participant cohorts. Lymph nodes were assessed using conventional radiological measurements, such as size and morphology on pre-administration images, changes in contrast intensity between pre- and post-administration MRI scans, and discriminative factors on post-administration images, such as homogeneous versus heterogeneous low-intensity patterns. Radiologists used these image features to grade lymph nodes as "suspicious," "normal," or "indeterminate" on both pre- and post-administration scans.

[0173] Tissue from lymph nodes imaged with imaging agents was collected as formalin-fixed specimens. Whenever possible, MRX measurements were performed before the tissue was processed for pathology.

[0174] MRX Results. MRX measurements were performed ex vivo in the MRX laboratory using preclinical instrumentation to determine whether an MRX signal could be detected in participant lymph nodes and to inform future clinical instrument parameters. In one participant, significant MRX signal (3x to 10x the LOQ) was measured in 8 of 9 samples (3 lymph nodes sectioned into 9 samples) (LOQ approximately 2.5 μg iron). Core needle biopsy samples did not produce a measurable MRX signal. Core needle biopsies represent 2% to 5% of the entire lymph node, which is not large enough to provide information on the sensitivity of MRX for clinical in vivo use.

[0175] Histopathology was evaluated using hematoxylin and eosin (H&E), HER-2, and Prussian blue (iron) staining. Five participants had samples available for pathological staining. Four participants had Prussian blue staining in their lymph nodes, confirming the presence of iron particles. One participant had undetectable iron levels in their sample. The same participant did not show any evidence of the imaging agent on post-MR images. Lymphatic flow problems or injection technique were suspected. Four participants showed HER-2-positive lymph node metastases, and one participant was tumor-negative.

[0176] Final results. Four (4) of the six (6) participants were evaluable for MRI concordance with pathology at the per-participant level. In three participants, the central radiologist's post-administration MRI evaluation was consistent with the pathological confirmation of lymph node metastasis. The radiologist reported that suspicious lymph nodes were found in one participant with negative pathology (pre- and post-administration). Of the two participants who could not be evaluated, one participant did not have a pathological specimen and the other participant did not show any signs of particle migration into the lymph nodes.

[0177] Accordingly, the imaging agent disclosed herein was present in the lymph nodes after subareolar administration. Histopathological examination of the excised lymph node tissue confirmed the presence of tumor cells and the imaging agent in the lymph nodes. Normal lymph nodes could be distinguished from suspected lymph nodes by comparison of pre- and post-administration MR images as shown by the different post-administration intensity patterns, as expected for the non-specific uptake of the imaging agent in normal lymph nodes versus the specific binding between the HER-2 targeted imaging agent and the HER-2 receptor in the tumor-containing lymph nodes. These data indicate that the combination of standard morphological assessment (size and shape) with the observable changes in MRI contrast using the nanoparticle solution described herein improves radiological assessment and thus conventional axillary clinical assessment and treatment selection.

[0178] MRI imaging using the imaging agent - second cohort

[0179] The imaging agent was used in an in vivo MRI study performed on an additional seven (7) patients as described below.

[0180] Human participants were selected based on the following criteria: participants were diagnosed with HER-2 positive primary breast cancer and planned to undergo preoperative clinical assessment of axillary lymph node disease followed by either core needle biopsy or pathologic confirmation. Participants with a conventional axillary assessment of lymph node suspected disease were enrolled in the study. Exclusion criteria were known inflammatory breast cancer and prior axillary surgery, including sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND) or prior radiation therapy to the ipsilateral breast of the primary breast cancer. Participants with a likelihood of lymph node metastasis and planned to undergo sentinel lymph node biopsy (SLNB) and / or axillary lymph node dissection (ALND) (Group 1) or planned to undergo suspicious lymph node biopsy (Group 2), or who had already undergone suspicious lymph node biopsy (Group 3) were selected for inclusion in the study at the discretion of the investigator.

[0181] All eligible participants received a single dose of 22.5 mg Fe equivalent of drug substance injected into the subareolar interstitial tissue or the area near and around the primary tumor on study day 1. Baseline MRI assessment was performed within 3 days prior to imaging agent administration (the imaging agent was a biologically functional magnetic nanoparticle solution described herein) and a second MRI was performed within 18 hours to 78 hours after imaging agent administration and prior to any neoadjuvant therapy. MR compatible clip insertion was introduced under ultrasound guidance prior to imaging. Whole lymph nodes were obtained or lymph node tissue was obtained from core needle biopsy of clipped lymph nodes for histopathological assessment. Samples from patient 7 and patient 8 were also used for ex vivo MRX measurements.

[0182] The study includes screening, baseline, imaging, and follow-up periods. Participants are screened between Day -28 and Day -1 to determine if they are eligible for the study. Each participant is dosed on Day 1 of the baseline period or after the baseline MRI scan is obtained. In each patient, an MRI scan is performed after administration during the imaging period and a pathology sample is obtained. Pathology samples are obtained from the lymph node under ultrasound guidance from the clip insertion. Data collection and assessments include safety assessments and safety laboratories; image acquisition, storage, transmission, and central image reading; lymph node specimen harvesting, ex vivo MRX measurements, participant clinical care pathology, study sample transfer, study sample pathology, and central pathology reading.

[0183] Post-administration safety and imaging evaluations (including MRI and biopsy) are performed at Visit 2 (Day 1) and Visit 3 (18 hours to 72 hours after administration). One post-administration MRI imaging scan is performed within the 18 hours to 72 hours time window after administration of the imaging agent. Additional safety evaluations are performed at Visit 4 (Day 7 ± 2 days) and the End of Study Visit (Day 28 ± 3 days) and a telephone follow-up (Day 90 ± 14 days).

[0184] Evaluation of results. In certain lymph nodes, the homogeneous low intensity typically observed in a completely normal lymph node was present in a portion of the lymph node, while the remainder of the lymph node had a heterogeneous low intensity. The homogeneous low intensity region was considered (or assumed) to belong to normal lymph node tissue with nanoparticle flow or non-specific uptake. The heterogeneous low intensity (spotted) region was indicative of ligand binding to tumor cells. The region that did not change appearance after contrast was indicative of no nanoparticle flow. The appearance of irregular darkened regions within the lymph node, while the remainder of the lymph node remained unchanged in appearance or was of homogeneous low intensity, was indicative of a heterogeneous configuration from non-specific uptake of the large normal region of the lymph node, combined with specific binding to accessible tumor cells and non-flowing regions within the lymph node. It was postulated that the administration route of the nanoparticles through the lymphatic flow was the same as the route of tumor invasion, enabling the appearance of a partially irregular darkened heterogeneous configuration with or without a spotted pattern after administration, which included non-specific uptake by macrophages in regions of the lymph node not invaded by the tumor, and specific binding of the particles to the tumor in tumor regions or interfaces or regions that touched the tumor. These differences in particle uptake within the same lymph node were indicative of the presence of normal lymph node tissue and abnormal lymph node tissue, thereby indicating that the suspected lymph node was invaded by the tumor. Some lymph nodes did not show any particle uptake, presumably due to occlusion of the lymphatic vessels leading to them by high tumor burden. However, the presence of low intensity in surrounding adjacent lymph nodes was used to assess the lymph node status at the patient level.

[0185] Final Results. Four (4) of seven (7) participants were evaluable for MRI vs. pathology concordance at each lymph node level. In 4 participants, the post-administration MRI assessment of the clipped and biopsied lymph nodes by the central radiologist was concordant with the pathologic confirmation of lymph node metastasis in the gross needle biopsy specimen from the same lymph node. In 3 participants not evaluable, 2 participants showed no evidence of granules flowing out of the injection site, and 1 participant had an unreadable image due to MRI susceptibility artifact.

[0186] Figure 8 An example of post-administration MRI assessment of a lymph node showing heterogeneous low intensity (spotted) appearance is shown. The radiologist assessed this lymph node as suspicious for tumor and was therefore confirmed by pathology.

[0187] Figure 9 An example of post-administration MRI assessment of a lymph node showing heterogeneous architecture (partially irregular darkening) is shown. The radiologist assessed this lymph node as suspicious for tumor and was therefore confirmed by pathology.

[0188] Figure 10 An example of post-administration MRI assessment of a lymph node showing both heterogeneous architecture (partially irregular darkening) and heterogeneous low intensity (spotted) is shown. The radiologist assessed this lymph node as suspicious for tumor and was confirmed as a highly suspicious for tumor abnormal enlarged lymph node by routine clinical assessment methods, and pathology showed the patient had 15 metastatic lymph nodes.

[0189] Figure 11 An example of post-administration MRI assessment of a lymph node without contrast uptake is shown. The MRI scan pre- and post-contrast the lymph node appears the same. However, the adjacent lymph node appears homogenously low intensity post-contrast, indicating that this adjacent lymph node has contrast uptake. It is likely that the tumor has completely invaded the lymph node and that no contrast can enter the lymph node, but the contrast still flows to the adjacent normal lymph node, giving it a homogenously low intensity appearance. In this patient, routine clinical assessment showed 3 enlarged lymph nodes highly suspicious for tumor, one of which had a positive pathology result from lymph node biopsy.

[0190] Accordingly, the imaging agent disclosed herein is present in the lymph node after injection administration in the subareolar or peritumoral region. Histopathological examination of the excised lymph node tissue confirms the presence of tumor cells and the imaging agent in the lymph node. By comparison of pre- and post-administration MR images, as shown by the different post-administration intensity patterns, normal lymph nodes can be distinguished from suspicious lymph nodes, as expected by the non-specific uptake of the imaging agent in normal lymph nodes and the specific binding between the HER2-targeted imaging agent and the HER2 receptor in the tumor-containing lymph node.

[0191] In a first embodiment, a method of evaluating a target tissue by magnetic resonance imaging, the method comprising: introducing a biofunctional magnetic nanoparticle solution into a subject, wherein the biofunctional magnetic nanoparticle solution is comprised of a plurality of biofunctional magnetic nanoparticle structures, each nanoparticle structure comprising an iron core coated with oleic acid, one or more lipids or polymers, one or more stealth generating compounds, and one or more targeting ligands adapted to bind to a target molecule; allowing the biofunctional magnetic nanoparticle solution to bind to the target molecule, if present, wherein the target molecule is indicative of a health condition of the subject; performing magnetic resonance imaging of the subject to obtain a magnetic resonance image or image file representative of a target tissue of the subject; and evaluating the magnetic resonance image or image file to determine a heterogeneous low intensity region in the target tissue representative of the presence of biofunctional magnetic nanoparticles bound to the target molecule, whereby the heterogeneous low intensity region is identified in the target tissue to be indicative of the presence of the target molecule in the subject tissue.

[0192] In a second embodiment, the first embodiment includes: within the nanoparticle solution, each of the biofunctional magnetic nanoparticle structures has a diameter of 10 nanometers to 150 nanometers.

[0193] In a third embodiment, the first embodiment includes: within the biofunctional magnetic nanoparticle solution, the diameter of each of the nanoparticle structures in the nanoparticle solution is uniform.

[0194] In a fourth embodiment, each of the first through third embodiments includes: the biofunctional magnetic nanoparticle solution is a nanoparticle aqueous solution in 0.9% NaCl containing 0.05% polysorbate 20.

[0195] In a fifth embodiment, each of the first through third embodiments includes: the biofunctional magnetic nanoparticle solution is an isotonic solution that supports product stability and human safe injection.

[0196] In a sixth embodiment, each of the first through third embodiments includes: the at least one stealth generating compound is a polyethylene glycol polymer.

[0197] In a seventh embodiment, each of the first through third embodiments includes: the at least one stealth generating compound includes PEG with a molecular weight between 500 Da and 20,000 Da.

[0198] In an eighth embodiment, each of the first through third embodiments includes: the at least one stealth generating compound includes methoxypolyethylene glycol 2000 and methoxypolyethylene glycol 10000.

[0199] In a ninth embodiment, each of the first through third embodiments includes: the organic coating comprises an organic acid, the organic acid comprising oleic acid.

[0200] In a tenth embodiment, each of the first through third embodiments includes: the organic acid is oleic acid.

[0201] In an eleventh embodiment, each of the first through third embodiments includes: the polymeric coating comprises dextran.

[0202] In a twelfth embodiment, each of the first through third embodiments includes: the targeting ligand is an anti-HER-2 antibody or a functional binding fragment thereof.

[0203] In a thirteenth embodiment, each of the first through third embodiments includes: the targeting ligand is trastuzumab or a functional binding fragment thereof.

[0204] In a fourteenth embodiment, each of the first through third embodiments includes: the polymeric coating external to the organic coating is poly(maleic anhydride-alt-octadecene).

[0205] In a fifteenth embodiment, each of the first through third embodiments includes: the magnetic resonance imaging is performed using a Tl imaging sequence.

[0206] In a sixteenth embodiment, each of the first through third embodiments includes: the magnetic resonance imaging is performed using a T2 imaging sequence.

[0207] In a seventeenth embodiment, each of the first through third embodiments includes: the biofunctional magnetic nanoparticle solution is injected into a peritumoral region of a tumor present in the human subject.

[0208] In an eighteenth embodiment, each of the first through third embodiments includes: the magnetic resonance imaging is performed using a 1.5 T clinical scanner or a 3 T clinical scanner.

[0209] In a nineteenth embodiment, each of the first through third embodiments further includes: the image of the target tissue is evaluated for heterogeneic architecture.

[0210] In a twentieth embodiment, each of the first through third embodiments further comprises: assessing whether the target tissue has a morphology that is suspicious for a tumor.

[0211] In a twenty-first embodiment, each of the first through third embodiments further comprises: performing magnetic resonance imaging on the human subject 24 hours after the first imaging, and reevaluating the target tissue.

[0212] In a twenty-second embodiment, each of the first through third embodiments comprises: one or more target ligands are capable of specifically binding to a protein or cell associated with cancer.

[0213] In a twenty-third embodiment, each of the first through third embodiments comprises: the target molecule is a HER2 protein, or a domain or region thereof.

[0214] In a twenty-fourth embodiment, each of the first through third embodiments further comprises: assessing the image of the target tissue for heteroarchitecture.

[0215] In a twenty-fifth embodiment, each of the first through third embodiments comprises: identifying a hetero-low intensity in the target tissue further indicates that the target tissue is malignant.

[0216] In a twenty-sixth embodiment, each of the first through third embodiments comprises: identifying a heteroarchitecture in the target tissue further indicates that the target tissue is malignant.

[0217] In a twenty-seventh embodiment, each of the first through third embodiments further comprises: performing a treatment for a health condition on the subject, the treatment comprising administering a drug, a biopsy, a surgery, or no biopsy or surgery.

[0218] In a twenty-eighth implementation, a method of assessing axillary lymph node disease by magnetic resonance imaging is provided, the method comprising: introducing a solution of biofunctional magnetic nanoparticles into a human subject diagnosed with or suspected of having breast cancer; performing magnetic resonance imaging of a region of interest of the human subject, the region comprising at least one lymph node; assessing the heterogeneous low intensity of the image of the at least one lymph node; and assessing axillary lymph node disease of the at least one lymph node, wherein the solution of biofunctional magnetic nanoparticles comprises nanoparticle structures, each structure comprising an iron core surrounded by an organic coating layer, and a polymeric coating layer in contact with the organic coating, a targeting ligand specific to a target molecule conjugated to the polymeric coating layer, the presence of which is indicative of the presence of a cancer cell or a protein associated with the presence of cancer, and at least one stealth generating compound bound to the polymeric coating layer.

[0219] In a twenty-ninth implementation, the twenty-eighth implementation comprises: within the solution of nanoparticles, each of the biofunctional magnetic nanoparticle structures has a diameter of 10 nanometers to 150 nanometers.

[0220] In a thirtieth implementation, the twenty-eighth implementation comprises: within the solution of biofunctional magnetic nanoparticles, the diameter of each of the nanoparticle structures in the solution of nanoparticles is uniform.

[0221] In a thirty-first implementation, the twenty-eighth implementation comprises: the polymeric coating external to the organic coating is poly(maleic anhydride-alt-octadecene).

[0222] In a thirty-second implementation, each of the twenty-eighth implementation through the thirty-first implementation comprises: the solution of biofunctional magnetic nanoparticles is a nanoparticle aqueous solution in 0.9% NaCl containing 0.05% polysorbate 20.

[0223] In a thirty-third implementation, each of the twenty-eighth implementation through the thirty-first implementation comprises: the solution of biofunctional magnetic nanoparticles is an isotonic solution that supports product stability and human safe injection.

[0224] In a thirty-fourth implementation, each of the twenty-eighth implementation through the thirty-first implementation comprises: the at least one stealth generating compound is a polyethylene glycol polymer.

[0225] In a thirty-fifth implementation, each of the twenty-eighth implementation through the thirty-first implementation comprises: the at least one stealth generating compound comprises a polyethylene glycol (PEG) polymer, including PEG with a molecular weight between 500 Da and 20,000 Da.

[0226] In a thirty-sixth embodiment, each of the twenty-eighth through thirty-first embodiments includes: the at least one stealth generating compound includes methoxypolyethylene glycol 2000 and methoxypolyethylene glycol 10000.

[0227] In a thirty-seventh embodiment, each of the twenty-eighth through thirty-first embodiments includes: the organic coating includes an organic acid.

[0228] In a thirty-eighth embodiment, each of the twenty-eighth through thirty-first embodiments includes: the organic acid is oleic acid.

[0229] In a thirty-ninth embodiment, each of the twenty-eighth through thirty-first embodiments includes: the polymeric coating comprises dextran.

[0230] In a fortieth embodiment, each of the twenty-eighth through thirty-first embodiments includes: the targeting ligand is an anti-HER-2 antibody or a functional binding fragment thereof.

[0231] In a forty-first embodiment, each of the twenty-eighth through thirty-first embodiments includes: the targeting ligand is trastuzumab or a functional binding fragment thereof.

[0232] In a fortieth embodiment, each of the twenty-eighth through thirty-first embodiments includes: one lymph node is evaluated.

[0233] In a forty-third embodiment, each of the twenty-eighth through thirty-first embodiments includes: more than one lymph node is evaluated.

[0234] In a forty-fourth embodiment, each of the twenty-eighth through thirty-first embodiments includes: the magnetic resonance imaging is performed using a Tl imaging sequence.

[0235] In a forty-fifth embodiment, each of the twenty-eighth through thirty-first embodiments includes: the magnetic resonance imaging is performed using a T2 imaging sequence.

[0236] In a forty-sixth embodiment, each of the twenty-eighth through thirty-first embodiments includes: the human subject holds their breath while the magnetic resonance imaging of the axillary region is performed.

[0237] In a forty-seventh embodiment, each of the twenty-eighth through thirty-first embodiments includes: the biofunctional magnetic nanoparticle solution is injected into a peritumoral region of a tumor present in the human subject.

[0238] In a forty-eighth implementation, each of the twenty-eighth implementation through the thirty-first implementation further includes performing magnetic resonance imaging using a 1.5T clinical scanner or a 3T clinical scanner.

[0239] In a forty-ninth implementation, each of the twenty-eighth implementation through the thirty-first implementation further includes performing a biopsy of the lymph node if assessed as diseased.

[0240] In a fiftieth implementation, each of the twenty-eighth implementation through the thirty-first implementation further includes performing an assessment of the heterogeneity of the image of the at least one lymph node.

[0241] In a fifty-first implementation, each of the twenty-eighth implementation through the thirty-first implementation further includes performing an assessment of whether the morphology of the at least one lymph node is suspicious for a tumor.

[0242] In a fifty-second implementation, each of the twenty-eighth implementation through the thirty-first implementation further includes performing magnetic resonance imaging of the human subject 24 hours after the first imaging and again assessing the axillary lymph node disease of the at least one lymph node.

[0243] In a fifty-third implementation, each of the twenty-eighth implementation through the thirty-first implementation includes identifying a heterogeneous low intensity in the at least one lymph node further indicating that the lymph node is malignant.

[0244] In a fifty-fourth implementation, each of the twenty-eighth implementation through the thirty-first implementation and the fifty-second implementation includes identifying a heterogeneous low intensity in the target tissue further indicating that the lymph node is malignant.

[0245] In a fifty-fifth implementation, there is provided a drug for use in magnetic resonance imaging, formulated for use in a method for valuing and / or treating a health condition characterized by the presence of a protein or cell associated with cancer, the method comprising: introducing a solution of biofunctional magnetic nanoparticles into a human subject diagnosed with or suspected of having breast cancer; performing magnetic resonance imaging of a region of interest of the human subject, the region comprising at least one lymph node; evaluating the image of the at least one lymph node for heterogeneous low intensity; and evaluating the at least one lymph node for axillary lymph node disease, wherein the solution of biofunctional magnetic nanoparticles comprises nanoparticle structures each comprising a core of iron surrounded by a layer of organic coating, and a layer of polymer coating in contact with the organic coating, a targeting ligand specific for a target molecule conjugated to the layer of polymer coating, and at least one stealth generating compound bound to the layer of polymer coating, the presence of the target molecule being indicative of the presence of a cancer cell or a protein associated with the presence of cancer.

[0246] In a fifty-sixth implementation, the fifty-fifth implementation includes: identifying the heterogeneous low intensity in the target tissue or lymph node further indicating that the subject tissue is malignant.

[0247] In a fifty-seventh implementation, each of the fifty-fifth implementation and the fifty-sixth implementation includes: evaluating the image of the at least one lymph node for heterogeneous architecture.

[0248] In a fifty-eighth implementation, the fifty-seventh implementation includes: identifying the heterogeneous architecture in the target tissue or lymph node further indicating that the subject tissue is malignant.

[0249] Other features and advantages of the present application will be apparent from the following detailed description, from the drawings, and from the claims.

[0250] The above examples are for illustrative purposes only and are not intended to limit the scope of the present application. Numerous variations of the above described examples are possible. Since modifications and changes to the above described examples will be apparent to one skilled in the art, the present application is intended to only be limited to the scope of the appended claims.

[0251] References

[0252] Beenken SW, Urist MM, Zhang Y, et al. Axillary lymph node status, but not tumor size, predicts locoregional recurrence and overall survival after mastectomy for breast cancer. Ann Surg 2003;237(5):732-738; discussion 738-739

[0253] Choi HY, Park M, Seo M, Song E, Shin SY, Sohn YM. Preoperative Axillary Lymph Node Evaluation in Breast Cancer: Current Issues and Literature Review. Ultrasound Q (2017) 33(1): 6-14.

[0254] H.S. Cody III, N. Houssami, Axillary management in breast cancer: What's new for 2012? The Breast 21, 2012, 411e415412

[0255] Giuliano AE, Hunt KK, Ballman KV, et al. Axillary dissection vs no axillary dissection in women with invasive breast cancer and sentinel node metastasis: a randomized clinical trial. JAMA 2011;305(6):569-575

[0256] Giuliano AE, Ballman KV, McCall L, et al. Effect of Axillary Dissection vs No Axillary Dissection on 10-Year Overall Survival Among Women with Invasive Breast Cancer and Sentinel Node Metastasis: The ACOSOG Z0011 (Alliance) Randomized Clinical Trial. JAMA 2017; 318(10): 918-926

[0257] Jatoi I, Kunkler IH. Omission of sentinel node biopsy for breast cancer: Historical context and future perspectives on a modern controversy. Cancer. 2021; vol. 127, no. 23, pp. 4376-4383.

[0258] Leenders M, Kramer G, Belghazi K, et al. Can We Identify or Exclude Extensive Axillary Nodal Involvement in Breast Cancer Patients Preoperatively. J Onc. 2019, doi.org / 10.1155 / 2019 / 8404035

[0259] NCCN Guidelines for Breast Cancer 2022, Version 4.

[0260] Reiner T, Engel J, Schmidt M, Offerson BV, Smidt MI, Gentilini OD. Is axillary sentinel lymph node biopsy required in patients who undergo primary breast surgery? Breast Care (Basel). 2018; 13: 324-330

[0261] Saksena M, Jimenez R, Coopey S, et al. Axillary Ultrasound Evaluation in Breast Cancer Patients: A Multidisciplinary Viewpoint and Middle Ground; Journal of Breast Imaging, 2021, 672-675.

Claims

1. A method for evaluating a target tissue by magnetic resonance imaging, comprising: introducing a biofunctional magnetic nanoparticle solution into a subject, wherein the biofunctional magnetic nanoparticle solution is comprised of a plurality of biofunctional magnetic nanoparticle structures, each nanoparticle structure comprising an iron core coated with oleic acid, one or more lipids or polymers, one or more stealth-generating compounds, and one or more targeting ligands suitable for binding to a target molecule; allowing the biofunctional magnetic nanoparticle solution to bind to the target molecule if the target molecule is present, wherein the target molecule is indicative of a health condition of the subject; performing magnetic resonance imaging on the subject to obtain a magnetic resonance image or image file representing a target tissue of the subject; as well as The magnetic resonance image or image file is evaluated to determine heterogeneous low-intensity areas in the target tissue indicating the presence of biofunctional magnetic nanoparticles bound to the target molecule, whereby identification of the heterogeneous low-intensity areas in the target tissue indicates the presence of the target molecule in the subject's tissue. 2 . The method of claim 1 , wherein within the nanoparticle solution, each of the biofunctional magnetic nanoparticle structures has a diameter of 10 to 150 nanometers. 3 . The method of claim 1 , wherein within the biofunctional magnetic nanoparticle solution, the diameter of each of the nanoparticle structures in the nanoparticle solution is uniform.

4. The method according to any one of claims 1 to 3, wherein the biofunctional magnetic nanoparticle solution is an aqueous solution of nanoparticles containing 0.05% polysorbate 20 dissolved in 0.9% NaCl.

5. The method according to any one of claims 1 to 3, wherein the biofunctional magnetic nanoparticle solution is an isotonic solution that supports product stability and safe injection into the human body.

6. The method of any one of claims 1 to 3, wherein the at least one stealth-generating compound is a polyethylene glycol polymer.

7. The method of any one of claims 1 to 3, wherein the at least one stealth-generating compound comprises PEG having a molecular weight between 500 Da and 20,000 Da.

8. The method of any one of claims 1 to 3, wherein the at least one stealth-generating compound comprises methoxy polyethylene glycol 2000 and methoxy polyethylene glycol 10000.

9. The method of any one of claims 1 to 3, wherein the organic coating comprises an organic acid comprising oleic acid.

10. The method of claim 9, wherein the organic acid is oleic acid.

11. The method of any one of claims 1 to 3, wherein the polymer coating comprises dextran.

12. The method of any one of claims 1 to 3, wherein the targeting ligand is an anti-HER-2 antibody or a functional binding fragment thereof.

13. The method of any one of claims 1 to 3, wherein the targeting ligand is trastuzumab or a functional binding fragment thereof.

14. The method of any one of claims 1 to 3, wherein the polymeric coating external to the organic coating is poly(maleic anhydride-alt-octadecene).

15. The method of any one of claims 1 to 3, wherein magnetic resonance imaging is performed using a T1 imaging sequence.

16. The method of any one of claims 1 to 3, wherein magnetic resonance imaging is performed using a T2 imaging sequence.

17. The method of any one of claims 1 to 3, wherein the biofunctional magnetic nanoparticle solution is injected into the peritumoral region of a tumor present in a human subject.

18. The method of any one of claims 1 to 3, wherein the magnetic resonance imaging is performed using a 1.5T clinical scanner or a 3T clinical scanner.

19. The method of any one of claims 1 to 3, further comprising assessing a heterogeneous structure of the image of the target tissue.

20. The method of any one of claims 1 to 3, further comprising evaluating the morphology of the target tissue for the presence of a suspicious tumor.

21. The method of any one of claims 1 to 3, further comprising performing magnetic resonance imaging on the human subject 24 hours after the first imaging and re-evaluating the target tissue.

22. The method of any one of claims 1 to 3, wherein the one or more target ligands are capable of specifically binding to a protein or cell associated with cancer.

23. The method of any one of claims 1 to 3, wherein the target molecule is a HER2 protein, or a domain or region thereof.

24. The method of any one of claims 1 to 3, further comprising assessing the image of the target tissue for heterogeneous texture.

25. The method of any one of claims 1 to 3, wherein identification of heterogeneous low intensity in the target tissue further indicates that the target tissue is malignant.

26. The method of any one of claims 1 to 3, wherein identifying a heterogeneous structure in the target tissue further indicates that the target tissue is malignant.

27. The method of any one of claims 1 to 3, further comprising treating the subject for a health condition, the treatment comprising administering a drug, performing a biopsy, performing surgery, or not performing a biopsy or surgery.

28. A method of evaluating axillary lymph node disease by magnetic resonance imaging, the method comprising: introducing a biofunctional magnetic nanoparticle solution into a human subject diagnosed with or suspected of having breast cancer; performing magnetic resonance imaging of a region of interest in the human subject, the region including at least one lymph node; assessing heterogeneous hypointensity in the image of the at least one lymph node; as well as evaluating said at least one lymph node for axillary lymph node disease, The biofunctional magnetic nanoparticle solution comprises nanoparticle structures, each structure comprising an iron core surrounded by an organic coating layer, a polymer coating layer in contact with the organic coating, a targeting ligand specific for a target molecule conjugated to the polymer coating layer, and at least one stealth-generating compound associated with the polymer coating layer, wherein the presence of the target molecule indicates the presence of cancer cells or a protein associated with the presence of cancer.

29. The method of claim 28, wherein within the nanoparticle solution, each of the biofunctional magnetic nanoparticle structures has a diameter of 10 nm to 150 nm.

30. The method of claim 28, wherein within the biofunctional magnetic nanoparticle solution, the diameter of each of the nanoparticle structures in the nanoparticle solution is uniform.

31. The method of claim 28, wherein the polymeric coating overlying the organic coating is poly(maleic anhydride-alt-octadecene).

32. The method of any one of claims 28 to 31, wherein the biofunctional magnetic nanoparticle solution is an aqueous solution of nanoparticles containing 0.05% polysorbate 20 dissolved in 0.9% NaCl.

33. The method of any one of claims 28 to 31, wherein the biofunctional magnetic nanoparticle solution is an isotonic solution that supports product stability and safe injection into humans.

34. The method of any one of claims 28 to 31, wherein the at least one stealth-generating compound is a polyethylene glycol polymer.

35. The method of any one of claims 28 to 31, wherein the at least one stealth-generating compound comprises a polyethylene glycol (PEG) polymer, including PEG having a molecular weight between 500 Da and 20,000 Da.

36. The method of any one of claims 28 to 31, wherein the at least one stealth-generating compound comprises methoxy polyethylene glycol 2000 and methoxy polyethylene glycol 10000.

37. The method of any one of claims 28 to 31 , wherein the organic coating comprises an organic acid.

38. The method of claim 37, wherein the organic acid is oleic acid.

39. The method of any one of claims 28 to 31 , wherein the polymeric coating comprises dextran.

40. The method of any one of claims 28 to 31, wherein the targeting ligand is an anti-HER-2 antibody or a functional binding fragment thereof.

41. The method of any one of claims 28 to 31, wherein the targeting ligand is trastuzumab or a functional binding fragment thereof.

42. The method of any one of claims 28 to 31 , wherein one lymph node is assessed.

43. The method of any one of claims 28 to 31 , wherein more than one lymph node is assessed.

44. The method of any one of claims 28 to 31 , wherein magnetic resonance imaging is performed using a T1 imaging sequence.

45. The method of any one of claims 28 to 31, wherein magnetic resonance imaging is performed using a T2 imaging sequence.

46. ​​The method of any one of claims 28 to 31 , wherein the human subject holds his breath while performing magnetic resonance imaging of the axillary region.

47. The method of any one of claims 28 to 31, wherein the biofunctional magnetic nanoparticle solution is injected into the peritumoral region of a tumor present in a human subject.

48. The method of any one of claims 28 to 31, wherein the magnetic resonance imaging is performed using a 1.5T clinical scanner or a 3T clinical scanner.

49. The method of any one of claims 28 to 31 , further comprising biopsy of the lymph node if assessed as diseased.

50. The method of claim 28, further comprising assessing the image of the at least one lymph node for heterogeneous texture.

51. The method of any one of claims 28 to 31, further comprising evaluating the morphology of the at least one lymph node for the presence of a tumor.

52. The method of any one of claims 28 to 31 , further comprising performing magnetic resonance imaging on the human subject 24 hours after the first imaging and re-evaluating the at least one lymph node for axillary lymph node disease.

53. The method of any one of claims 28 to 31, wherein identifying heterogeneous low intensity in the at least one lymph node further indicates that the lymph node is malignant.

54. The method of claim 28, claim 29, claim 30, claim 31 or claim 52, wherein identifying a heterogeneous structure in the at least one lymph node further indicates that the lymph node is malignant.

55. A drug for use in magnetic resonance imaging, formulated for use in a method for evaluating and / or treating a health condition characterized by the presence of a protein or cell associated with cancer, the method comprising: introducing a biofunctional magnetic nanoparticle solution into a human subject diagnosed with or suspected of having breast cancer; performing magnetic resonance imaging of a region of interest in the human subject, the region including at least one lymph node; assessing heterogeneous hypointensity in the image of the at least one lymph node; as well as evaluating said at least one lymph node for axillary lymph node disease, The biofunctional magnetic nanoparticle solution comprises nanoparticle structures, each structure comprising an iron core surrounded by an organic coating layer, a polymer coating layer in contact with the organic coating, a targeting ligand specific for a target molecule conjugated to the polymer coating layer, and at least one stealth-generating compound associated with the polymer coating layer, wherein the presence of the target molecule indicates the presence of cancer cells or a protein associated with the presence of cancer.

56. The medicament of claim 55, wherein identification of heterogeneous low intensity in the target tissue or lymph node further indicates that the subject's tissue is malignant.

57. The medicament of claim 55 or claim 56, further comprising assessing the heterogeneous texture of the image of the at least one lymph node.

58. The medicament of claim 57, wherein identification of heterogeneous structures in the target tissue or lymph node further indicates that the subject's tissue is malignant.

Images