Methods for assessing nodal pathology using iron oxide nanoparticles in magnetic resonance imaging

Abstract

The present invention relates to a method for evaluating nodal lesions by magnetic resonance imaging using a contrast agent comprising biofunctionalized magnetic nanoparticles with an iron core coated on the outside with a polymer and conjugated to an antibody specific for the tumor type being evaluated. Images generated from magnetic resonance imaging using the contrast agent show heterogeneous hypointensity or heterogeneous architecture in areas where antibody binding is present.

Description

[Background technology]

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

[0002] MRI with contrast or imaging agents uses a contrast medium injected into the body to improve image quality. These MRI contrast agents alter the relaxation times of nuclei in body tissue. Contrast-enhanced MRI scans highlight specific areas of soft tissue by increasing the signal-to-noise ratio, which is useful for identifying possible abnormalities in tissue, such as tumors.

[0003] MRI contrast agents are indirectly detected by their ability to perturb water proton relaxation and alter MRI signal intensity. MR contrast agents function primarily by altering the T1 (longitudinal or spin-lattice relaxation time), T2 (spin-spin or transverse relaxation time), or T2* (dephased spin-spin) properties of the tissue being imaged. The physical presence of SPIONs creates a magnetic field perturbation in the external magnetic field that can be indirectly detected by MRI. The magnetic moment of the SPIONs aligns with the MRI field and becomes magnetized. When the field is removed, the magnetic moment returns to a random, unmagnetized state. This ability to switch back and forth is called superparamagnetism.

[0004] SPIONs are dual contrast agents, altering both T1 and T2 / T2* tissue properties. SPIONs decrease the T1 relaxation properties of the tissue being imaged, resulting in positive MRI contrast in T1-weighted images. Gadolinium-based contrast agents (GBCAs) also produce contrast by decreasing T1 relaxation. SPIONs also accelerate the dephasing of adjacent proton spins in tissues that take up the SPIONs, causing a decrease in the T2 and T2* MRI relaxation rates. This decrease in T2 / T2* appears as dark or low-intensity contrast in T2- and T2*-weighted images, resulting in negative MRI contrast images.

[0005] The most commonly used compounds for contrast enhancement are gadolinium-based. For large vessels, such as the aorta and its branches, the dose can be as low as 0.1 mmol / kg body weight. Higher concentrations are often used for finer vasculature. At much higher concentrations, gadolinium's T2 shortening effect is significant, making it appear less bright than surrounding body tissue. However, such concentrations are more toxic to body tissue.

[0006] MRI has a wide range of applications in medical diagnostics and is an important cancer diagnostic tool. When used without contrast, MRI produces images that can be used for the morphological evaluation of potential tumors. When contrast is used, MRI produces images that can be easily evaluated for the presence of abnormalities in tissue.

[0007] Following a new breast cancer diagnosis, lymph node staging is performed using a combination of clinical evaluation and radiographic imaging. Accurate lymph node staging is an essential component of breast cancer patient management, as treatment depends on patient-specific characteristics of the primary tumor, lymph node status, and assessment of distant metastatic disease (NCCN Guidelines, 4th Edition, 2022). Regional lymph node evaluation is important, but practice patterns and imaging modalities used vary based on available resources and institutional experience. Furthermore, pathological confirmation by either needle biopsy or surgical removal of a sentinel lymph node (SLN) remains the gold standard, regardless of whether the image is negative or radiographically concerning.

[0008] The two most commonly used radiographic modalities for assessing lymph node metastasis are axillary ultrasound and Gd-enhanced magnetic resonance imaging (MRI). MRI has an integrated diagnostic sensitivity and specificity of 75%–80% and 89%–91%, respectively, compared with 49%–87% and 55%–97% for ultrasound (Beenken 2003, Choi 2017). Given the importance of accurate lymph node assessment, there are still opportunities for improvement in radiographic modalities. Ultrasound offers advantages over MRI in terms of simplicity, patient comfort, and cost savings, but its usefulness is dependent on operator experience, which contributes to the wide variability in sensitivity and specificity. Ultrasound is also limited in its ability to scan the extent of locoregional disease burden (Cody 2012, Saksena 2021). On the other hand, MRI is less dependent on operator experience and can scan the entire lymph node basin, but findings suggestive of regional lymph node disease are not tumor-specific and are merely surrogate indicators of possible tumor invasion, such as changes in size and morphology.The absence of enlarged regional lymph node disease can be reassuring, but the presence of abnormal lymph nodes warrants further evaluation with ultrasound and percutaneous biopsy.

[0009] Accurate lymph node assessment has become even more important since the publication of the landmark American College of Surgeons Oncology Group (ACOSOG) Z0011 trial (Giulianio 2011, Giulianio 2017) in 2011. This trial initiated a dramatic shift toward de-escalation of axillary surgery, as the authors demonstrated that women undergoing breast-conserving therapy who met selection criteria could omit axillary lymph node dissection (ALND), even if their sentinel nodes were positive. Since the publication of these and other data, interest in omitting axillary surgery and sentinel lymph node biopsy (SLNB) procedures has continued to grow (Reiner 2018).

[0010] In this changing landscape, there is an even greater need to improve the accuracy of non-invasive imaging methods (Jatoi 2021, Leenders 2019). Non-invasive, molecularly targeted, tumor-specific approaches that function as contrast enhancement to widely accepted imaging modalities such as MRI will not only add value to existing lymph node staging methods, but will also contribute to the advancement of surgical de-escalation and subsequent clinical decision-making.

[0011] The present disclosure addresses these and other needs in the art. Summary of the Invention

[0012] The present disclosure relates to reagents and methods for evaluating target tissues, particularly axillary nodes, including the use of imaging agents for identifying cancerous (e.g., HER2-positive) cells or tissues in the axillary nodes. An exemplary method is performed by magnetic resonance imaging utilizing a superparamagnetic iron oxide nanoparticle (i.e., biofunctionalized magnetic nanoparticle) solution imaging agent.

[0013] According to certain embodiments, a method for assessing nodal disease by magnetic resonance imaging is provided, the method comprising: introducing a biofunctionalized magnetic nanoparticle solution into the subareolar or peritumoral region of a human subject diagnosed with or suspected of having breast cancer (e.g., HER-2-positive breast cancer) via parenteral administration, e.g., intravenous, intraperitoneal, peritumoral, subcutaneous, or intramuscular; performing magnetic resonance imaging of a region of interest in the human subject, the axillary region including at least one node; evaluating the image of the at least one node for heterogeneous hypointensity; and assessing nodal disease in the at least one node. The biofunctionalized magnetic nanoparticle solution comprises nanoparticle structures, each comprising an iron core surrounded by a layer of organic coating, a polymer layer, at least one stealth generating compound bound to the polymer coating layer, and a targeting ligand adapted to bind to a target molecule indicative of nodal disease. In certain embodiments, the polymer comprises poly(maleic anhydride-alt-octadecene). In certain embodiments, the targeting ligand is conjugated to the nanoparticle structure via the polymer coating layer. In many cases, this target molecule can specifically bind to cancer-related proteins or cells.In most cases, cancer is breast cancer.In certain embodiments, the target molecule is HER2 protein or another protein that is differentially expressed on cancer cells, or its domain or region.In most cases, the targeting ligand comprises anti-HER2 protein such as trastuzumab or its functional part or binding domain.

[0014] According to most included embodiments, the identification of heterogeneous hypointensity in lymph nodes further indicates the malignancy of the target tissue.

[0015] According to certain embodiments, a method for evaluating axillary node disease by magnetic resonance imaging is provided, the method comprising: introducing a biofunctionalized magnetic nanoparticle solution into the subareolar 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., intravenous, subcutaneous, or intramuscular, and performing magnetic resonance imaging of an axillary region of interest in the human subject, the axillary region including at least one lymph node; evaluating the image of the at least one lymph node for heterogeneous hypointensity; and evaluating the at least one lymph node for axillary node disease, wherein the biofunctionalized magnetic nanoparticle solution comprises nanoparticle structures, each structure comprising an iron core surrounded by a layer of organic coating, a polymer layer, at least one stealth generating compound bound to the polymer coating layer, and a targeting ligand adapted to bind to a target molecule indicative of axillary node disease. In certain embodiments, the polymer comprises poly(maleic anhydride-alt-octadecene). In certain embodiments, a targeting ligand is conjugated to the nanoparticle structure via a polymer layer. Often, the targeting ligand can specifically bind to a protein or cell associated with cancer. In most cases, the cancer is breast cancer. In certain embodiments, the target molecule is the HER2 protein, or a domain or region thereof. In most embodiments, the targeting ligand comprises an anti-HER2 antibody, such as trastuzumab, or a functional portion or binding domain thereof.

[0016] According to many embodiments, a method for evaluating axillary node lesions by magnetic resonance imaging is provided, as described above, wherein the biofunctional magnetic nanoparticles in the nanoparticle solution each have a diameter of 10 to 150 nanometers. Also, according to most embodiments, in any particular solution, the nanoparticles have a polydispersity index (PDI) of less than 0.2. Also, according to certain embodiments, in any particular solution, the diameter of each of the nanoparticles in the nanoparticle solution is often uniform or relatively uniform. Also, according to certain embodiments, in any particular solution, the diameter of each of the nanoparticles in the nanoparticle solution is often 55 to 90 nanometers.

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

[0018] Often, according to embodiments, a method of evaluating axillary node lesions by magnetic resonance imaging is provided as described above, wherein the polyethylene glycol (PEG) polymers are methoxypolyethylene glycol 2000 and methoxypolyethylene glycol 10000. In certain embodiments, the PEG polymers are within a size range of about 500 Da to 20,000 Da or up to about 20,000 Da.

[0019] According to another embodiment, there is provided a method for assessing axillary node pathology by magnetic resonance imaging as described above, wherein the organic coating layer is any organic acid, for example oleic acid.

[0020] Often, according to embodiments, a method of assessing axillary node pathology by magnetic resonance imaging is provided as described above, wherein the polymer coating comprises dextran.

[0021] According to most embodiments, a method of assessing axillary 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 HER2 protein, such as trastuzumab or a functional binding fragment thereof.

[0022] In many cases, according to embodiments, a method for evaluating axillary node lesions by magnetic resonance imaging is provided as described above, wherein the polymer coating outside the organic coating is poly(maleic anhydride-alt-octadecene) (POMA). In certain embodiments, the POMA is functionalized with carboxylate groups. In such embodiments, the functionalized POMA is adapted to form covalent bonds with amine groups on other materials, such as proteins (containing lysines with amine groups), via EDC chemistry. While POMA is often the preferred polymer, other polymers may be included.

[0023] According to most included embodiments, a method for assessing axillary 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] In most cases, according to other embodiments, a method of evaluating axillary node lesions by magnetic resonance imaging is provided as described above, wherein performing the magnetic resonance imaging uses a T1 imaging sequence as described herein.

[0025] In many cases, according to embodiments, a method of evaluating axillary node pathology by magnetic resonance imaging is provided as described above, wherein performing the magnetic resonance imaging uses a T2 imaging sequence as described herein.

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

[0027] In most cases, according to embodiments, a method for assessing axillary node lesions by magnetic resonance imaging is provided as described above, wherein a biofunctional magnetic nanoparticle solution is injected into the peritumoral region of a tumor present in a human subject.

[0028] According to many embodiments, a method for assessing axillary node pathology by magnetic resonance imaging is provided as described above, wherein performing the magnetic resonance imaging is performed using a 1.5T or 3T clinical scanner.

[0029] In most cases, according to embodiments, there is provided above a method of evaluating axillary node pathology by magnetic resonance imaging, further comprising evaluating an image of at least one lymph node for heterogeneous architecture.

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

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

[0032] In many cases, according to various embodiments, there is provided a method for assessing axillary node disease by magnetic resonance imaging, as described above, further comprising performing magnetic resonance imaging on the human subject 24 hours after the first imaging to reassess the axillary node disease of at least one lymph node. In certain embodiments, the magnetic resonance imaging of the human subject is performed 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or in the range of 1-3 days or 1-7 days after administration.

[0033] In most cases, according to embodiments, a method of assessing axillary node lesions by magnetic resonance imaging is provided as described above, wherein the target molecule is capable of specifically binding to a protein or cell associated with cancer.

[0034] According to most embodiments, a method of assessing axillary 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] In most cases, according to embodiments, there is provided above a method of evaluating axillary node pathology by magnetic resonance imaging, further comprising evaluating an image of at least one lymph node for heterogeneous architecture.

[0036] In many cases, according to embodiments, a method of evaluating axillary node pathology by magnetic resonance imaging is provided as described above, the method further comprising subjecting the subject to treatment for a condition, the treatment involving administration of a medication, biopsy, surgery, or forgoing biopsy or surgery.

[0037] According to most embodiments, a method for assessing axillary node disease by magnetic resonance imaging is provided as described above, wherein identification of heterogeneous hypointensity in lymph nodes further indicates malignancy of the tissue of interest.

[0038] Often, according to embodiments, a method for assessing axillary node disease by magnetic resonance imaging is provided as described above, wherein identification of heterogeneous architecture in lymph nodes further indicates the malignancy of the target tissue.

[0039] Also included herein is a drug for use in magnetic resonance imaging formulated for use in assessing and / or treating a health condition characterized by the presence of a protein or cell associated with cancer, the method including: introducing a biofunctionalized magnetic nanoparticle solution into a subject, the biofunctionalized magnetic nanoparticle solution comprising a plurality of biofunctionalized 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 to a target molecule; allowing the biofunctionalized magnetic nanoparticle solution to bind to the target molecule, if present, wherein the target molecule is indicative of the health condition of the subject; performing magnetic resonance imaging of the subject to obtain a magnetic resonance image or image file representing the target tissue of the subject; and evaluating the magnetic resonance image or image file for areas of heterogeneous hypointensity in the target tissue indicative of the presence of the biofunctionalized magnetic nanoparticles bound to the target molecule, thereby identifying that the areas of heterogeneous hypointensity in the target tissue are indicative of the presence of the target molecule in the target tissue. In many cases, the method further includes subjecting the subject to treatment for the condition, which may involve administering a drug, biopsy, surgery, or forgoing biopsy or surgery. In certain embodiments, the polymer includes poly(maleic anhydride-alt-octadecene). Also, in certain embodiments, a targeting ligand is conjugated to the nanoparticle structure in the polymer coating layer. In many cases, the target molecule can specifically bind to a protein or cell associated with cancer. In most cases, the cancer is breast cancer. In certain embodiments, the target molecule is HER2 protein, or a domain or region thereof.

[0040] According to most included embodiments, a method for assessing axillary node disease by magnetic resonance imaging is provided as described above, wherein identification of heterogeneous hypointensity in the target tissue or lymph node further indicates malignancy of the target tissue.

[0041] In most cases, according to embodiments, there is provided above a method of evaluating axillary node pathology by magnetic resonance imaging, further comprising evaluating an image of at least one lymph node for heterogeneous architecture.

[0042] According to most included embodiments, there is provided a method for assessing axillary node disease by magnetic resonance imaging as described above, wherein identification of heterogeneous architecture in at least one lymph node is further indicative of lymph node malignancy.

[0043] These and other embodiments, features, and advantages will become apparent to those skilled in the art upon review of the following more detailed description of various exemplary embodiments of the present disclosure in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0044] [Figure 1] 1 shows biofunctionalized magnetic nanoparticles used as active pharmaceutical ingredients in biofunctionalized magnetic nanoparticle solutions according to embodiments. [Figure 2] Pre- and post-contrast MR images of a normal lymph node are shown. In the post-contrast MR image, the lymph node appears uniformly dark, suggesting its normal state. [Figure 3] Pre- and post-contrast MR images of a pathologically enlarged lymph node are shown. In the post-contrast MR image, the lymph node exhibits heterogeneous hypointensity, suggesting that it is diseased. Portions of the cortex are slightly darker than in the pre-contrast image and are homogeneously hypointense, indicating that this area is not pathologically involved. [Figure 4] Shown are images of lymph nodes with specific characteristics: (Left) a lymph node before contrast; (Right) a homogeneous hypointense pattern observed in the same lymph node after administration of an imaging agent. [Figure 5] Shown are different lymph node images with specific characteristics: (Left) a lymph node before contrast; (Right) a heterogeneous hypointense ("patchy") pattern observed after administration of an imaging agent to the same lymph node. [Figure 6] FIG. 1 shows an image of a lymph node in which a heterogeneous architectural pattern with irregular darkening was observed after administration of an imaging agent. [Figure 7] Figure 1 shows a lymph node in which both a heterogeneous architecture with irregularly darkened areas and a heterogeneous pattern of hypointensity ("patchy") were observed after administration of an imaging agent. [Figure 8] Pre- and post-contrast MR images of the lymph node are shown, with the post-contrast T2-weighted image showing heterogeneous hypointensity. [Figure 9] Pre- and post-contrast MR images of the lymph node are shown, with the post-contrast T2-weighted image showing heterogeneous architecture (and irregular darkening). [Figure 10] Pre- and post-contrast MR images of the lymph node are shown, with the post-contrast T2-weighted image showing both heterogeneous hypointensity and heterogeneous architecture. [Figure 11] Pre-contrast MR images of the lymph node are shown, along with post-contrast MR images showing no contrast agent uptake and homogeneous hypointensity of the adjacent lymph node. [Figure 12] 1 shows magnetic relaxometry and ICP-MS analysis of BT474 and MCF7 cells exposed to imaging agents containing anti-HER2 targeting ligands. [Figure 13] 1 shows superparamagnetic relaxometry signals from HER2-high, HER2-low, and HER2-negative cells exposed to an exemplary imaging agent specific for HER2. [Figure 14] 1 shows superparamagnetic relaxometry signals from HER2 high, HER2 medium, and HER2 low expressing cells exposed to an imaging agent containing an anti-HER2 targeting ligand. [Figure 15] 1 shows superparamagnetic relaxometry signals from titrations of HER2-high and HER2-non-expressing cells exposed to an imaging agent containing an anti-HER2 targeting ligand. [Figure 16] 1 shows superparamagnetic relaxometry signals from BT474 cells, lymphocytes, and peripheral blood mononuclear cells exposed to imaging agents containing anti-HER2 targeting ligands. [Figure 17] 1 shows superparamagnetic relaxometry signals from HER2-positive cell implants exposed to an imaging agent containing an anti-HER2 targeting ligand, a PEGylated nanoparticle control, and a free antibody competitor. [Figure 18] 1 shows superparamagnetic relaxometry signals and staining of BT474 and MCF7 cell tumors exposed to imaging agents containing anti-HER2 targeting ligands via different routes of administration. [Figure 19] Figure 1 shows the time course of excretion of imaging agents in lymph nodes as measured by MRX. [Figure 20] Shown is the presence of imaging agent in excised target accessory lymph nodes after 24 hours, as indicated by node color (top image: dark brown / black) and Prussian blue iron staining. [Figure 21] 1 shows lymph node sections demonstrating the presence of tumor cells and imaging agent by Prussian blue iron staining in lymph node mouse tissue. DETAILED DESCRIPTION OF THE INVENTION

[0045] abbreviation CDR: Complementarity determining region

[0046] HER-2: Human epidermal growth factor receptor 2

[0047] MR: magnetic resonance

[0048] MRI: Magnetic Resonance Imaging

[0049] MRX: Magnetic relaxometry

[0050] As used herein, "a" or "an" means "at least one" or "one or more."

[0051] As used in this document, the term "and / or" can mean "and," can mean "or," can mean "exclusive or," can mean "one," can mean "some but not all," can mean "neither," and / or can mean "both."

[0052] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen (here, HER2). Antibodies contain complementarity-determining regions (CDRs), also known as hypervariable regions, in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As is known in the art, the amino acid positions / boundaries that define the hypervariable regions of an antibody can vary depending on the context and the various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because they can be considered within a hypervariable region under one set of criteria but outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. Each naturally occurring heavy and light chain variable domain contains four FR regions, primarily in a β-sheet configuration, connected by three CDRs, which form loops that connect (and in some cases form part of) the β-sheet structure. The CDRs within each chain are held together in close proximity by the FR regions and, together 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, immunoglobulin amino acid residue numbering is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified.

[0053] The antibodies and / or binding fragments that make up the anti-HER2 antibodies are generally referred to herein as (in N→C order) V H CDR#1, V H CDR#2 and V H A variable region (V) having three complementarity determining regions ("CDRs") designated CDR#3 H ), and heavy chains containing (in N→C order) V L CDR#1, V L CDR#2 and V L A variable region (V) with three complementarity determining regions (CDRs) designated CDR#3 L Exemplary CDR amino acid sequences of the heavy and light chains of exemplary anti-HER2 antibodies and / or binding fragments that may be included in the antigen-binding portion, as well as V H and V L The amino acid sequences of the regions are provided herein. Particular embodiments of anti-HER2 antibodies may be derived from or contain these exemplary CDRs and / or V H and / or V L These include, but are not limited to, antibodies and / or binding fragments comprising the sequence, as well as antibodies and / or binding fragments that compete with such antibodies and / or binding fragments for binding to HER2.

[0054] Antibodies may be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi- or single-chain antibodies, surrobodies (including surrogate light chain constructs), single-domain antibodies, camelized antibodies, scFv-Fc antibodies, etc. They may be of or derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, or IgY. In some embodiments, the anti-HER2 antibody is an IgG (e.g., IgG1, IgG2, IgG3, or IgG4). Antibodies may be of human or non-human origin. Examples of non-human origin include, but are not limited to, mammalian (e.g., monkey, rodent, goat, and rabbit) or avian (e.g., chicken) origins.

[0055] Fab fragments contain the constant domain of the light chain and the first constant domain (CH2) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl 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 the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical coupling of antibody fragments is known to those skilled in the art. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more rapidly from the animal's circulation, and may have lower nonspecific tissue binding than intact antibodies.

[0056] An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (V H -V L The three CDRs of each variable domain are V H -V L It is in this configuration that they interact to define an antigen-binding site on the surface of the dimer. In many cases, the six CDRs confer antigen-binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0057] "Single-chain Fv" or "scFv" antibody binding fragments are fragments of the V of an antibody. H and V L Fv polypeptides generally comprise V domains, and these domains are present in a single polypeptide chain. H Domains and V L It further comprises a polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding.

[0058] As used herein, "targeting ligand" refers to an antibody or another moiety adapted to specifically bind to a predetermined target molecule in the broadest sense as that term is used herein and in the art.

[0059] As used herein, "MR imaging" refers to magnetic resonance imaging or MRI. The terms MRI and "MR imaging" are used interchangeably herein.

[0060] As used herein, "heterogeneous hypointensity" refers to a patchy molecular signature in a heterogeneous hypopattern in an MRI image. This has been found by the inventors to indicate that sufficient imaging agent has specifically bound to the target molecule to be detected by MRI (e.g., in vivo). Figure 4 shows an illustration of what is intended by the term heterogeneous hypointensity as viewed in an exemplary target tissue. Other types of target tissue in a subject, including lymph nodes and non-lymph nodes, are specifically contemplated for imaging by the methods described herein.

[0061] As used herein, "heterogeneous architecture" refers to a heterogeneous visual signature of light and dark in an MRI image. We hypothesize this indicates that sufficient imaging agent is bound to the target molecule to be detected by MRI (e.g., in vivo). Figure 6 shows an illustration of what is intended by the term "heterogeneous architecture" as viewed in an exemplary target tissue.

[0062] As used herein, "imaging agent" refers to a biofunctionalized magnetic nanoparticle solution formulated for safe in vivo use in humans, consisting of iron oxide nanoparticles, often 10-150 nanometers in diameter, functionalized with targeting ligands, and having a polydispersity index (PDI) of less than 0.2. The imaging agent is often referred to as the complete solution or the functionalized nanoparticles contained therein. The prepared functionalized nanoparticles described herein, including magnetic nanoparticles, organic acids, polymers, and targeting ligands, are also referred to as biofunctionalized magnetic nanoparticles.

[0063] Cancer diagnosis and staging require a combination of clinical classification and invasive pathological evaluation. In breast cancer patients, cancer staging requires determining whether primary tumor cells have spread to lymph nodes. Based on palpation or imaging techniques such as ultrasound, PET, CT, or MRI, lymph nodes may be clinically suspicious and biopsied for pathological confirmation.

[0064] In many breast cancer patients, current imaging methods cannot reliably detect micro- and macrometastases, and lymph node evaluation requires surgical removal of two to four sentinel lymph nodes (SLNs) and, depending on the extent of tumor involvement as determined by laboratory pathology analysis, complete axillary lymph node (ALN) dissection. Although only approximately 25% of clinically node-negative breast cancer patients have lymph node involvement, current standard of care still results in all patients with a node-negative diagnosis by standard of care imaging undergoing surgery to determine whether metastatic spread exists. This means that the majority of patients undergo unnecessary surgery, resulting in avoidable morbidity such as lymphedema, intercostobrachial neuralgia, local cellulitis, or infection, as well as limited treatment options if the cancer recurs. A non-invasive method for identifying lymph node involvement has been found to greatly improve patient care by avoiding the cost and risks of surgical biopsy procedures in the majority of patients and eliminating the significant complications associated with lymph node removal.

[0065] Without intending to be bound by any theory of operation, the following has been found. ● The imaging agents described herein are specific to HER2-expressing cancer cells; This imaging agent is useful as a tumor-specific contrast agent in MRI, improving detection sensitivity and specificity. - This imaging agent does not undergo significant non-specific binding to non-tumor cells, such as lymph node cells and peripheral blood mononuclear cells (PBMCs) isolated from human cadavers. The imaging agent is taken up by lymph nodes when administered by intraperitoneal and peritumoral injection in mouse models; and In order to obtain an optimal signal from this imaging agent, it is preferable that the period between administration of the imaging agent and surgical measurement is, for example, 16 hours or more.

[0066] According to the protocol described herein, a single dose of 30 mg (or 20 mg) of imaging agent (based on iron oxide weight) is evaluated. This is based on the assumption that a minimum of 1% of the material (200–300 μg of imaging agent) can be distributed to lymph nodes via intramammary peritumoral injection. Overall, the selected dose corresponds to a significantly reduced dose of iron oxide per body weight (0.3–0.5 mg / kg) compared to nonclinical studies (per the FDA guidance "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers").

[0067] The FIH study evaluated a single 30 mg (or 20 mg) dose of imaging agent (based on iron oxide weight). Overall, the selected dose corresponds to a significantly reduced dose of iron oxide per body weight (0.3–0.5 mg / kg) compared with nonclinical studies (per the FDA guidance, “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers”).

[0068] The present disclosure relates to biofunctionalized magnetic nanoparticle solutions and their use as examination reagents in magnetic resonance imaging. Provided herein is a drug substance formulated into a solution for injection into a subject (e.g., by parenteral administration, including intravenously, subcutaneously, or intramuscularly, e.g., subareolar, peritumoral, etc.), comprising biofunctionalized magnetic nanoparticles.

[0069] According to the methods of the present disclosure, a human subject with suspected or confirmed breast cancer is evaluated for nodal disease or nodal metastasis, including axillary nodal disease. According to such methods, the human subject receives an injection of an imaging agent according to an embodiment described herein, e.g., via parenteral administration, including intravenous, subcutaneous, or intramuscular, into, for example, the subareolar or peritumoral area. The human subject then undergoes MR imaging of the target area, e.g., the axillary region. The MR imaging can be a T1-weighted or T2 / T2*-weighted imaging sequence, and the resulting images are evaluated by a radiologist. The method provides an imaging-based diagnosis without the need for a biopsy, thereby reducing the need for additional clinic visits and surgery, which is the current standard of care.

[0070] The imaging agent according to embodiments herein is a biofunctionalized magnetic nanoparticle solution, in which the nanoparticles have an iron core coated with an organic coating such as oleic acid, which is then coated with a polymer such as poly(maleic anhydride-alt-octadecene), which is then conjugated with a polyethylene glycol polymer and an antibody specific to the type of cancer present in a human subject (e.g., an anti-HER-2 antibody, trastuzumab, in subjects with HER-2 breast cancer). Other organic materials, such as dextran, may also be used for the coating. Other polymers for coating and / or conjugation may also be used. Other antibodies specific to different tumors are also contemplated. The imaging agent is prepared as a solution for parenteral administration, such as injection. In MR imaging, the targeting ligand-loaded nanoparticles in the imaging agent bind to the tissue of interest, thereby producing an image that can be distinguished from non-target tissue based on specific image features. These features include heterogeneous hypointense areas in the tissue of interest in the MRI image, as assessed by a radiologist. An exemplary targeting ligand used in the studies described herein is an anti-HER-2 antibody specific for the HER-2 protein, which is known to be associated with the presence of breast cancer. Because lymph nodes are small and relying solely on morphological assessment is inaccurate, the use of imaging agents that can provide distinct imaging differences in axillary lymph nodes, where cancer has spread, is highly valuable.

[0071] According to certain embodiments described herein, a human subject is injected with an imaging agent provided herein and then undergoes MR imaging to evaluate, among other areas, the axillary lymph nodes into which the imaging agent drains. In normal lymph nodes, the lymph node image appears uniformly dark, as shown in Figure 2. In diseased lymph nodes of HER-2 breast cancer patients, the lymph node image shows heterogeneous hypointensity, as shown in Figure 3.

[0072] By reading MR images of axillary lymph nodes injected with an imaging agent according to embodiments, a radiologist can determine whether HER-2 breast cancer tissue is present in the lymph node based on identifying areas of heterogeneous hypointensity in the MRI images of the tissue of interest.

[0073] According to an exemplary embodiment, the imaging schedule is as follows: a baseline MRI assessment is performed before imaging agent administration and at one or two additional time points after imaging agent administration: Visit 2, Day 1, before imaging agent injection; Visit 3, Day 2, within 18-30 hours after imaging agent injection; and Visit 5, Day 4, within the 66-78 hour time frame, or Visit 3, Day 2 / 3, within the 18-78 hour time frame. Imaging agent administration is performed by a surgeon or radiologist after completion of the baseline MRI scan on Day 1. Imaging agent administration can be performed in a radiology department or other suitable setting. Peritumoral injections may require additional interventional ultrasound (US) guidance, so appropriate ultrasound equipment should be available to support administration.

[0074] In this schedule, it has been found useful to match the excised or biopsied lymph node(s) with the post-dose imaged lymph node(s) to compare the post-dose MRI scan and pathology results of the excised or biopsied lymph node(s) for the presence of tumor. This requires using an imaging modality available at the facility (e.g., ultrasound) to identify the location of suspicious lymph nodes with magnetic resonance compatible clips inserted on the same day as the MRI. Clip insertion can be performed any time after informed consent and before the post-dose MRI.

[0075] During imaging, the subject is often positioned supine on the spine coil, with the side of the breast cancer lesion at the center of the magnet. For example, if the suspected lesion is in the right breast (affected side), the subject should be shifted to the left side of the spine coil so that the right axilla is at the center of the spine coil, or as close as possible, depending on the subject's size. A surface array coil with the highest available density is placed over the axillary region of the affected side of the subject. The subject's arm on the affected side is preferably placed firmly at their side, with no space between the arm and the torso. The other arm may also be placed at their side, or may be placed above their head, if preferred. Additional immobilization sponges may be used to help limit the subject's movement.

[0076] The following combination of image sequences is often utilized during each MRI (estimated total MRI time is approximately 20 minutes): ● Localizer ● Localize lymph node 1 (using: ○ T1 gradient echo ○ T2 Turbo Spin Echo (TSE) T1 Multistation Gradient Echo (VIBE) for Nanoparticle Detection T2* gradient echo (GRE) for nanoparticle detection (if available)

[0077] Additional sequences may be performed to meet institutional practice standards, and optional imaging parameters may be applied at the discretion of the institution, as long as they are documented.

[0078] A magnetic field strength of 1.5 T is often preferred. As a second option, a magnetic field strength of 3 T may be used with tailored imaging protocols, but only if necessary.

[0079] Contrast agents An exemplary imaging agent used in MR imaging according to the present invention is a biofunctionalized magnetic nanoparticle solution, also referred to herein as the imaging agent. An exemplary biofunctionalized magnetic nanoparticle in the imaging agent is composed of iron oxide nanoparticles coated with a surfactant (e.g., oleic acid) and a layer composed of carboxyl groups functionalized with poly(maleic anhydride-alt-octadecene) (POMA) polymer. The coated nanoparticles are conjugated with a polyethylene glycol polymer and the anti-HER-2 antibody trastuzumab. The exemplary imaging agent generally consists of an optionally filtered aqueous solution of nanoparticles in 0.9% NaCl containing 0.05% polysorbate-20.

[0080] In general, exemplary imaging agents include injectable formulations composed of SPIONs conjugated to anti-HER2 antibodies. These NPs consist of iron oxide spherical cores (24-28 nm in diameter) coated with an amphiphilic polymer (polymaleic anhydride-alt-1-octadecene [POMA]) that serves as a linker for covalent conjugation of trastuzumab (an anti-HER2 antibody) to polyethylene glycol (PEG). Core iron oxide NPs and POMAc-functionalized NPs were manufactured by nanoComposix (San Diego, CA).

[0081] The final antibody-conjugated NP drug substance was produced as a colloidal solution containing NP (approximately 10 mg / mL based on iron oxide) in sterile (0.9%) saline and 0.05% polysorbate 20 solution for stability. The final imaging agent formulation was identical to the bulk drug substance solution. The imaging agent bulk material was transferred to PCI Pharma 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 capped with sterile 13 mm stoppers and aluminum caps according to GMP manufacturing guidelines. Sterility analysis was performed by an Australian GMP analytical laboratory (Eurofins).

[0082] The main structure of POMAc-NPs conjugated with polyethylene glycol polymer and trastuzumab is shown in Figure 1. This main structure comprises an iron oxide core coated with a layer of oleic acid and then with a layer of poly(maleic acid-alt-octadecene), with methoxypolyethylene glycol 2000, methoxypolyethylene glycol 10000, and trastuzumab conjugated to the layer of poly(acid anhydride-alt-octadecene).

[0083] The biofunctionalized magnetic nanoparticle solution used in this invention is a dark brown to black aqueous colloidal solution of the biofunctionalized magnetic nanoparticles shown in Figure 1 and is supplied as a drug product for parenteral administration, including intravenous, subcutaneous, or intramuscular injection, e.g., subareolar or peritumoral. It is supplied as a 1 mL single-use colloidal solution formulation at a strength of 7.5 mg / mL Fe equivalent in sterile filled and finished vials. The imaging agent in the drug product is formulated as an aqueous nanoparticle solution in 0.9% NaCl containing 0.05% polysorbate 20. This product is stored at 2°C to 8°C.

[0084] The largest dose volume delivered to humans to date is 3 mL of 7.5 mg / mL Fe equivalent imaging agent, or a dose equivalent to 22.5 mg of Fe. This dose volume and dose strength is also expressed as a calculated dose of 3 mL of 10 mg / mL Fe3O4 equivalent, or a calculated maximum dose of 30 mg Fe3O4, or 30 mg imaging agent.

[0085] As an MRI contrast agent, and due to its tumor-targeting properties, this imaging agent has the potential to improve the diagnostic accuracy of clinical lymph node evaluation in patients with HER-2-positive breast cancer, either when used alone or in addition to current imaging modalities, providing the rationale for evaluating this imaging agent in this specific patient population.

[0086] Exemplary Tests In vitro cell binding and sensitivity of HER2 imaging reagents Study Objective: A cell binding study was performed to verify the sensitivity of the imaging agent to target HER2-positive tumor cells.

[0087] Methods: Studies were performed using the BT474 breast cancer cell line, known to express high levels of HER2, and MCF7 cells, known to be a HER2-low or HER2-negative cell line. Cells were seeded into 6-well cell culture plates 24 hours before the addition of 100 μg (iron oxide-based) of imaging agent. After the 24-hour incubation period, cells were washed to remove unbound imaging agent and harvested for SPMR measurements.

[0088] Results: Titration experiments (varying cell numbers from 0.25 x 10 to 2 x 10) showed that the test reagent was detectable in BT474 cells at the lowest titration of 0.25 x 10 (Figure 5, left panel). Results were confirmed by inductively coupled plasma mass spectrometry (ICP-MS) analysis to measure the amount of iron present at each titration (Figure 5, right panel).

[0089] Conclusion: This imaging agent can specifically bind to HER2-positive cells (BT474). These signals are further validated by measuring iron abundance using ICP-MS.

[0090] Specificity and selectivity of imaging agents containing anti-HER2 targeting ligands in vitro Study Objective: In vitro cell binding studies were performed to evaluate the specificity of imaging agents targeting HER2-high, HER2-low, and HER2-negative tumor cells.

[0091] METHODS: Binding to one HER2-high cell line (BT474) was compared with two cell lines with low or no expression (MCF10, MCF7, OVCAR) and one HER2-positive but non-responsive cell line (JIMT). 100 μg of imaging agent was incubated with 1 × 106 cells of each type for 24 hours, and binding was assessed by detection of SPMR signal.

[0092] Results: The imaging agent could produce clearly measurable signals by SPMR in HER2-high expressing cancer cells (BT474), but not in HER2-low expressing or HER2-negative cells (MCF10, JIMT, OVCAR, and MCF7) (Fig. 6).

[0093] Conclusion: This study demonstrated that this imaging agent has high specificity and selectivity for targeting HER2-positive tumor cells.

[0094] Cell selectivity and specificity testing Study Objective: Cell binding studies were performed to evaluate the specificity of imaging agents for targeting HER2 high-, medium-, and low-expressing, or negative tumor cells with and without exposure to free anti-HER2 antibody (competitor).

[0095] Methods: In this study, 100 μg of imaging agent was incubated with 1 x 10 cells from several high-expressing HER2-positive (IHC3+) cell lines (SKBR3, BT474, HCC1954) with varying degrees of HER2 expression, as well as a moderate-expressing (IHC2+) cell line (ZR75), and one low- or non-expressing line (MCF7) for 24 hours. These studies included a competition group in which all cells were exposed to free anti-HER2 antibody (100-fold excess compared to Herceptin on NP) and then incubated with imaging agent. Binding was assessed by detection of SPMR.

[0096] Results: These results also showed that the imaging agent and SPMR signals could be competed out by free antibody (Figure 7).

[0097] Conclusion: This imaging agent is highly specific and selective for binding to HER2-expressing cells.

[0098] Additional cell selectivity and specificity testing Study Objective: Additional cell binding studies were performed to assess the effect of tumor cell titration on the level of imaging agent binding.

[0099] Methods: HER2-highly expressing cell lines (SKBR3, BT474, and HCC1954) and HER2-nonexpressing cell line (MDA-MB231) were titrated and incubated with 100 μg of imaging agent for 24 hours. The cell numbers assessed were 2.5 × 106, 5.0 × 106, and 10 × 106 cells. Binding was assessed by detection of SPMR signal, which was confirmed by measuring iron content using ICP-MS.

[0100] Results: Titration of HER2-high and HER2-nonexpressing cell lines using imaging agents demonstrated a clear and direct correlation between cell number and SPMR signal (Figure 8, left panel). These results were further confirmed by the corresponding iron content using ICP-MS (Figure 8, right panel).

[0101] Conclusion: The SPMR signal is directly related to the number of cells labeled by the imaging agent, which in turn is directly related to the amount of imaging agent present in the sample.

[0102] Nonspecific interactions of imaging agents with lymphocytes and peripheral blood mononuclear cells in vitro. Study Objective: Because the intended use of this imaging agent is to detect HER2-positive tumor cells in lymph nodes, a study was performed to determine whether there was nonspecific binding of the imaging agent to lymphocytes and PBMCs that would cause an interfering SPMR signal.

[0103] Methods: Human cadaveric lymph nodes were purchased from a commercial vendor and lymph node cells were isolated. After isolation, 10 × 106 isolated lymphocytes were incubated with 100 μg of imaging agent for 24 hours. The imaging agent was also incubated with 1 × 106 HER2-positive cells (BT474) and a coculture of both cell types (1 × 106 BT474 and 10 × 106 lymphocytes). The imaging agent was further incubated with 10 × 106 PBMCs. Binding was assessed by detection of SPMR signal.

[0104] Results: Lymphocytes incubated with the imaging agent produced an SPMR signal, indicating some nonspecific interaction with lymphocytes. HER2-expressing cells incubated with the imaging agent also produced an SPMR signal that was approximately three times higher than the nonspecific signal from lymphocyte cultures (Figure 9, left panel). PBMCs incubated with the imaging agent did not produce an SPMR signal, indicating no interaction between the imaging agent and PBMCs (Figure 9, right panel).

[0105] Conclusions: Taken together, these results indicate that this imaging agent is unlikely to generate significant false-positive signals due to nonspecific interactions with non-tumor cells in lymph nodes and blood.

[0106] Tumor detection and specificity of imaging agents in in vivo xenograft tumor models in mice Study objective: To determine whether this imaging agent can target HER2+ tumors in vivo and generate a signal sufficient for SPMR detection.

[0107] Methods: Female athymic nude mice were subcutaneously implanted with 3 × 10 HER2-positive (BT474) cells in the flank region of their bodies. After 6–10 weeks, palpable tumors ranging in size from 0.125 cm3 to 1 cm3 developed. 400 μg of imaging agent was delivered using several different administration routes, including intraperitoneal, peritumoral, and intravenous injection into the tail vein. PEGylated NPs (same composition as the imaging agent but without anti-HER2 antibody) served as a control vehicle. An in vivo competition study was also performed by pre-injecting 1 mg of free anti-HER2 antibody via the tail vein 24 h prior to delivery of the imaging agent. Mice were euthanized 24 h after dosing. Tumors and other major organs were excised, and ex vivo SPMR measurements were performed on the excised tissues in addition to measuring iron content using ICP-MS.

[0108] Results: BT474 tumor cells exposed to the imaging agent produced a higher SPMR signal compared to tumor cells exposed to the control vehicle (Figure 10). Furthermore, pre-injected free antibody competed out the imaging agent, resulting in little or no SPMR signal.

[0109] Uptake of the imaging agent into the xenograft tumors was further confirmed by measuring iron content using ICP-MS. Quantitative analysis of the SPMR signal and ICP-MS suggested that, on average, approximately 2–5 μg of imaging agent was present in the tumors, compared with levels in the control vehicle that were below the ICP-MS detection limit.

[0110] Conclusion: This imaging agent can bind to tumor cells in vivo and produce a specific signal that can be measured with an MRX instrument.

[0111] Enhanced tumor detection and specificity of imaging agents in a murine in vivo bilateral flank xenograft tumor model Study Objective: A bilateral flank tumor model was used as a follow-up study to further demonstrate that imaging agents can bind to HER2-positive tumor cells but not to HER2-negative tumors in vivo and can generate specific signals via various delivery routes.

[0112] Methods: Bilateral flank tumor models were generated by implanting 3 × 106 BT474 (HER2-positive) and 1 × 106 MCF7 (low or no HER2 expression, MCF7 cells grow faster than BT474) cancer cells into each flank of female athymic nude mice. 400 μg of imaging agent was delivered using several different administration routes, including intraperitoneal, peritumoral, and intravenous injection into the tail vein. PEGylated NPs (same composition as the imaging agent but without anti-HER2 antibody) served as a control vehicle. Mice were euthanized 24 hours after dosing. Tumors and other major organs were excised, and ex vivo SPMR measurements were performed on excised tissues, in addition to Prussian blue and anti-Herceptin staining to identify the location of the imaging agent within the tumor.

[0113] Results: All administration routes tested resulted in significantly higher SPMR signals in BT474 tumors compared to MCF7 tumors (Figure 11, left panel). The presence of the imaging agent was primarily located in the outer portion of the tumor, as indicated by Prussian blue staining (blue staining) and anti-Herceptin staining (brown staining) (Figure 11, right panel).

[0114] Conclusions: These results demonstrate that the specificity of the imaging agent for targeting HER2-positive tumor cells is maintained in vivo and is detectable by SPMR measurements. Clearance of the imaging agent is primarily via the liver and spleen, as expected from the scientific literature for NPs in a similar size range.

[0115] Dispersion of imaging agents to lymph nodes in mice Study Objective: A mouse-based study was designed and performed to evaluate the time required for lymph node drainage of an imaging agent injected into the distal mammary pad.

[0116] Methods: 433 μg of imaging agent was injected into the right nipple / areola of the fourth abdominal mammary fat pad of nine female athymic naive nude mice. Axillary and inguinal lymph nodes were excised 24, 48, and 72 hours after injection (three mice per time point). Visual inspection of the color of the excised lymph nodes was performed 24 hours after injection to confirm the presence of the imaging agent in the lymph nodes, and Prussian blue staining was performed to identify the location of the imaging agent within the lymph nodes. The excised lymph nodes were measured ex vivo using an MRX instrument. To confirm the MRX results, iron content in each lymph node was also measured using ICP-MS (Figure 12).

[0117] Results: The target ALN appeared significantly darker brown / black compared to the inguinal lymph nodes from the ipsilateral side of the body and the ALN from the contralateral side (Figure 13). The presence of the imaging agent in the ALN was confirmed by Prussian blue staining, which showed that the imaging agent was primarily located in the sinuses of lymphatic tissue (indicating adequate drainage) (Figure 14).

[0118] ICP-MS further confirmed the presence of the imaging agent in the ALN by detecting elemental iron: from the same injection in mice, an average of 6 μg of Fe was detected by ICP-MS in the ALN and 1 μg in the inguinal lymph nodes.

[0119] After 72 hours, the majority of the imaging agent had drained from the lymph nodes, particularly in the inguinal lymph nodes closest to the injection site (Figure 12).

[0120] Conclusion: The imaging agent is more efficiently cleared from lymph nodes at 72 hours, thus minimizing the possibility of nonspecific signals in MRI / MRX measurements.

[0121] MRI imaging using imaging agents - Cohort 1 This imaging agent was used in six patients in an in vivo MRI study as follows.

[0122] Human participant selection was based on the following criteria: participants were diagnosed with HER-2-positive primary breast cancer and were scheduled to undergo core biopsy or pathological confirmation following preoperative clinical evaluation of axillary lymph node disease. Participants with suspicious lymph nodes identified by routine axillary evaluation were enrolled in the study. Exclusion criteria included known inflammatory breast cancer and a history of surgical axillary procedures, including sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND), or radiation therapy on the same side of the primary breast cancer. Participants with possible lymph node metastasis and scheduled for either sentinel lymph node biopsy (SLNB) and / or axillary lymph node dissection (ALND) (Arm 1), or scheduled for biopsy of suspicious lymph nodes (Arm 2), or participants in whom biopsy of suspicious lymph nodes had already been performed (Arm 3), were selected for enrollment, as determined by the investigator.

[0123] On study day 1, all eligible participants received a single dose of 22.5 mg of Fe equivalent drug substance in the subareolar stromal tissue or an area near the primary tumor. A baseline MRI assessment was performed within 3 days prior to administration of the imaging agent (the imaging agent was a biofunctional magnetic nanoparticle solution described herein). A second MRI was performed within 18–30 hours after administration of the imaging agent and prior to neoadjuvant therapy, and a third MRI was performed within 66–78 hours. Either whole lymph node(s) or lymph node tissue from core biopsies was obtained for histopathological evaluation. These specimens were also used for ex vivo MRX measurements.

[0124] The study consisted of screening, baseline, imaging, and follow-up periods. Participants attended a screening visit between days -28 and -1 to determine study eligibility. Each participant received dosing after obtaining a baseline MRI scan during the baseline period or on day 1 of the imaging period. Post-dosing MRI scans and pathology specimens were obtained during the imaging period. Data acquisition and evaluation included safety assessments and testing; image acquisition, storage, transfer, and central image review; lymph node sampling; ex vivo MRX measurements; participant clinical care pathology; study specimen transport; study specimen pathology review and central pathology review.

[0125] Postdose safety and imaging (including MRI and biopsy) assessments were performed at Visit 2 (Day 1) and Visits 3-5 (18-78 hours after dosing). Two postdose MRI imaging scans were performed, one at approximately 24 hours and one at approximately 72 hours. Additional safety assessments were performed at Visit 6 (Day 7 ± 2 days) and the end-of-study visit (Day 28 ± 3 days).

[0126] Evaluation of Results. While not intending to be bound by any particular theory of operation, MRI contrast agents are indirectly detected by their ability to perturb water proton relaxation and alter MRI signal intensity. The imaging agent induces changes in the T1 (spin-lattice or longitudinal relaxation) and T2 (spin-spin or transverse relaxation) or T2* (dephased spin-spin) properties of the local tissue being imaged, resulting in image contrast. The core is designed for high magnetic relaxivity, which alters T2 contrast. In normal lymph nodes, the imaging agent is taken up by resident macrophages, resulting in a relatively uniform T2 hypointense (dark) contrast. If tumor cells have metastasized to the node, they replace the macrophages (in whole or in part), and as a result, the uniform hypointensity normally seen in the area of ​​the node where the tumor cells are present is absent. However, because the imaging agent contains molecularly targeted nanoparticles (different from particles described in the literature), specific binding between targets in the tumor and ligands in the imaging agent according to embodiments herein was found to result in heterogeneous hypointensity where the nanoparticles bound to tumor cells in the infiltrated lymph nodes. Comparing pre- and post-administration MRI images, it was found that non-targeted particles did not show any change in intensity in tumor-infiltrated lymph nodes, whereas the solutions described herein showed changes in signal intensity in both tumor-infiltrated and normal lymph nodes in pre- and post-administration MRI images. However, the amount of change in signal intensity in tumor-infiltrated lymph nodes was clearly different and distinct from the change in normal lymph nodes, making it possible to distinguish tumor-infiltrated lymph nodes from normal lymph nodes on post-administration MRI images.

[0127] MRI measurements were performed using a 1.5T or 3T clinical scanner. During the MRI examination, participants were positioned supine on a spine coil with the center of the body offset, with the breast diagnosed with cancer positioned close to the center of the magnet. A high-density surface array coil was placed over the axillary region of the breast to be imaged. All image acquisition was performed axially. T1, T2, and T2* imaging sequences were used.

[0128] The primary effect of the imaging agent was expected to be on T2 and T2* relaxation, and therefore on T2 / T2*-weighted sequences. Additionally, contrast enhancement was expected for T1-weighted images. All sequences except the T2* sequence were acquired with explicit breath-hold instructions given to participants. The approximate imaging time for the above MR imaging sequences was approximately 20 minutes. To ensure consistency, the imaging scanner and protocol were required to be similar for pre- and post-drug imaging. All images were sent to a central radiology laboratory via either secure file transfer or secure transportation of CDs containing the imaging data.

[0129] This central imaging laboratory reviewed MRI scans in a cohort of six participants. Lymph nodes were assessed by both traditional radiological indices, such as size and morphology, on pre-dose images, and discriminatory factors, such as changes in contrast intensity between pre- and post-dose MRI scans and homogeneity versus heterogeneity of hypointense patterns on post-dose images. Radiologists used these image features to score lymph nodes as "suspicious," "normal," or "indeterminate" on both pre- and post-dose scans.

[0130] Tissues from lymph nodes imaged with imaging agents were collected as formalin-fixed specimens. Whenever possible, MRX measurements were performed before processing the tissue for pathology.

[0131] MRX Results. To determine whether MRX signal was detectable in participants' lymph nodes and to inform future clinical device parameters, MRX measurements were performed ex vivo using preclinical equipment in the MRX laboratory. In one participant's samples (three lymph nodes sliced ​​into nine specimens), significant MRX signal (3–10 times the LOQ) was measured in eight of the nine specimens (LOQ approximately 2.5 μg iron). Core biopsy specimens did not yield measurable MRX signal. Core biopsies represent 2%–5% of the entire lymph node, an insufficient size to inform MRX sensitivity for clinical in vivo use.

[0132] Histopathology was evaluated using hematoxylin and eosin (H&E), HER-2, and Prussian blue (iron) staining. Five participants had specimens available for pathology staining. Four participants showed Prussian blue staining of lymph nodes, confirming the presence of iron particles. One participant's specimen had no detectable levels of iron. In this participant, post-administration MR images showed no evidence of imaging agent, suggesting either lymphatic drainage issues or technical injection problems. Four participants had HER-2-positive lymph node metastases, and one participant was tumor-negative.

[0133] Final Results: Concordance between MRI and pathology was assessable at the individual participant level for four of the six participants. In three participants, post-dose MRI evaluation by a central radiologist was concordant with pathological confirmation of lymph node metastasis. Radiologists reported suspicious lymph nodes in one participant with a negative pathology (pre-dose and post-dose). Of the two participants who could not be assessed, one participant had no pathology specimen, and the other had no evidence of particle shedding in the lymph nodes.

[0134] Thus, the imaging agents disclosed herein are present in lymph nodes after administration via injection into the subareolar region. Histopathological examination of excised lymph node tissue confirms the presence of tumor cells and imaging agent within the lymph node. Comparison of pre- and post-administration MR images differentiates suspicious lymph nodes from normal lymph nodes, as evidenced by post-administration intensity patterns that differ from those expected from nonspecific uptake of the imaging agent in normal lymph nodes versus specific binding between the HER-2-targeted imaging agent and the HER-2 receptor in tumor-containing lymph nodes. These data suggest that combining standard morphological assessment (size and shape) with observable changes in MRI contrast using the nanoparticle solutions described herein may improve radiological assessment, thereby improving routine clinical evaluation and treatment options for the axilla.

[0135] MRI imaging with imaging agents - Cohort 2 This imaging agent was used in seven additional patients in in vivo MRI studies as follows.

[0136] Human participant selection was based on the following criteria: participants were diagnosed with HER-2-positive primary breast cancer and were scheduled to undergo core biopsy or pathological confirmation following preoperative clinical evaluation of axillary lymph node disease. Participants with suspicious lymph nodes identified by routine axillary evaluation were enrolled in the study. Exclusion criteria included known inflammatory breast cancer and a history of surgical axillary procedures, including sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND), or radiation therapy on the same side of the primary breast cancer. Participants with possible lymph node metastasis and scheduled for either sentinel lymph node biopsy (SLNB) and / or axillary lymph node dissection (ALND) (Arm 1), or scheduled for biopsy of suspicious lymph nodes (Arm 2), or participants in whom biopsy of suspicious lymph nodes had already been performed (Arm 3), were selected for enrollment, as determined by the investigator.

[0137] On study day 1, all eligible participants received a single dose of 22.5 mg of Fe equivalent active ingredient in the subareolar stromal tissue or an area near the primary tumor. A baseline MRI assessment was performed within 3 days prior to administration of the imaging agent (the biofunctional magnetic nanoparticle solution described herein). A second MRI was performed at an additional time point of 18 to 78 hours after administration of the imaging agent and before neoadjuvant therapy. Prior to imaging, an MR-compatible clip insert was introduced under ultrasound guidance. Lymph node tissue from either whole lymph node(s) or core biopsies of clipped lymph nodes was obtained for histopathological evaluation. Specimens from patients 7 and 8 were also used for ex vivo MRX measurements.

[0138] The study consisted of screening, baseline, imaging, and follow-up periods. Participants attended a screening visit between days -28 and -1 to determine study eligibility. Each participant received medication during the baseline period or on day 1 of the imaging period after the baseline MRI scan. For each patient, a post-dose MRI scan and pathology specimen were obtained during the imaging period. Pathology specimens were obtained from clipped lymph nodes under ultrasound guidance. Data acquisition and evaluation included safety assessments and testing; image acquisition, storage, transfer, and central image review; lymph node sampling; ex vivo MRX measurements; participant clinical care pathology; study specimen transport; study specimen pathology review and central pathology review.

[0139] Postdose safety and imaging (including MRI and biopsy) assessments were performed at Visit 2 (Day 1) and Visit 3 (18-72 hours after dosing). One postdose MRI imaging scan was performed within the 18-72 hour time frame after administration of the imaging agent. Additional safety assessments were performed at Visit 4 (Day 7 ± 2 days), the end-of-study visit (Day 28 ± 3 days), and a follow-up telemetry visit (Day 90 ± 14 days).

[0140] Evaluation of Results: In some lymph nodes, the uniform hypointensity typically seen in completely normal lymph nodes is present in parts of the node, while the remainder of the node has heterogeneous hypointensity. Areas of uniform hypointensity are assumed (or hypothesized) to be normal lymph node tissue where nanoparticle shedding or nonspecific uptake has occurred. Areas of heterogeneous hypointensity (patchy) indicate binding of the ligand to tumor cells. Areas that do not appear changed after contrast indicate no nanoparticle shedding. Irregularly darkened areas within the lymph node, with the remainder of the node appearing uniformly hypointense, indicate a heterogeneous architecture due to a combination of nonspecific uptake in large normal areas within the lymph node, specific binding to accessible tumor cells, and areas of non-draining lymph node. It is hypothesized that the nanoparticle administration route via lymphatic drainage follows the same route as tumor infiltration, resulting in a patchy or otherwise irregularly darkened, heterogeneous architecture after administration, including nonspecific uptake by macrophages in areas of lymph nodes not infiltrated by tumors and specific binding of particles to tumors or their interfaces or contact points with tumors in areas with tumors. Such differences in particle uptake within the same lymph node indicate the presence of normal and abnormal lymph node tissue, and therefore lymph nodes suspected of tumor infiltration. Some lymph nodes show no particle uptake at all, likely due to high tumor burden causing blockage of lymphatic vessels leading to them. However, the presence of hypointensity in surrounding adjacent lymph nodes can be used to assess lymph node status at the patient level.

[0141] Final Results: Concordance between MRI and pathology was assessable at the lymph node level for four of the seven participants. In four participants, post-dose MRI evaluation by a central radiologist of clipped and biopsied lymph nodes was concordant with pathological confirmation of lymph node metastasis in core biopsy specimens from the same lymph nodes. Of the three participants who were not assessable, two participants showed no signs of particle shedding from the injection site, and one participant's images were uninterpretable due to MRI susceptibility artifact.

[0142] Figure 8 shows an example of a post-dose MRI evaluation of a lymph node exhibiting a heterogeneously hypointense (patchy) appearance. This lymph node was assessed by a radiologist as suspicious for tumor and confirmed by pathology.

[0143] Figure 9 shows an example of a post-medication MRI evaluation of a lymph node showing heterogeneous architecture (partial irregular darkening), which was assessed by a radiologist as suspicious for tumor and confirmed by pathology.

[0144] Figure 10 shows an example of a post-medication MRI evaluation of a lymph node showing both heterogeneous architecture (irregular partial darkening) and heterogeneous hypointensity (patchy). This lymph node was evaluated by a radiologist as suspicious for tumor and confirmed by conventional clinical evaluation methods as an abnormally enlarged lymph node highly suspicious for tumor, and pathology confirmed that this patient presented with 15 metastatic lymph nodes.

[0145] Figure 11 shows an example of post-dose MRI evaluation of a lymph node without contrast uptake. The lymph node appears identical in pre- and post-contrast MRI scans. However, the adjacent lymph node appears uniformly hypointense after contrast, indicating contrast uptake by this adjacent lymph node. Presumably, the tumor completely infiltrated the lymph node, preventing the contrast from penetrating the lymph node, but the contrast still drained into adjacent normal lymph nodes, resulting in a uniformly hypointense appearance. In this patient, conventional clinical evaluation revealed three enlarged lymph nodes highly suspicious for tumor, one of which was biopsied and had a positive pathology diagnosis.

[0146] Thus, the imaging agents disclosed herein are present in lymph nodes after administration by injection into the subareolar or peritumoral area. Histopathological examination of excised lymph node tissue confirms the presence of tumor cells and imaging agent within the lymph node. Comparison of pre- and post-administration MR images differentiates suspicious lymph nodes from normal lymph nodes, as evidenced by a post-administration intensity pattern that differs from that expected from nonspecific uptake of the imaging agent in normal lymph nodes versus specific binding between the HER2-targeted imaging agent and HER2 receptors in tumor-containing lymph nodes.

[0147] In a first embodiment, a method for evaluating a target tissue by magnetic resonance imaging includes: introducing a biofunctionalized magnetic nanoparticle solution into a subject, the biofunctionalized magnetic nanoparticle solution being composed of a plurality of biofunctionalized 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 biofunctionalized magnetic nanoparticle solution to bind to the target molecule, if present, wherein the target molecule is indicative of a health status of the subject; performing magnetic resonance imaging of the subject to obtain a magnetic resonance image or image file representing the target tissue of the subject; and evaluating the magnetic resonance image or image file for areas of heterogeneous hypointensity in the target tissue indicative of the presence of biofunctionalized magnetic nanoparticles bound to the target molecules, thereby identifying that the areas of heterogeneous hypointensity in the target tissue indicate the presence of the target molecule in the target tissue.

[0148] In a second embodiment, the first embodiment includes that each of the biofunctional magnetic nanoparticle structures in the nanoparticle solution has a diameter of 10 to 150 nanometers.

[0149] In the third embodiment, the first embodiment includes a biofunctional magnetic nanoparticle solution in which the diameter of each of the nanoparticle structures in the nanoparticle solution is uniform.

[0150] In a fourth embodiment, each of the first to third embodiments includes those in which the biofunctional magnetic nanoparticle solution is an aqueous solution of nanoparticles in 0.9% NaCl containing 0.05% polysorbate 20.

[0151] In a fifth embodiment, each of the first to third embodiments includes those in which the biofunctional magnetic nanoparticle solution is an isotonic solution that supports product stability and safe injection into humans.

[0152] In a sixth embodiment, each of the first to third embodiments includes those in which at least one stealth generating compound is a polyethylene glycol polymer.

[0153] In a seventh embodiment, each of the first to third embodiments includes those in which at least one stealth production compound comprises PEG having a molecular weight of 500 Da to 20,000 Da.

[0154] In an eighth embodiment, each of the first to third embodiments includes those in which the at least one stealth generating compound includes methoxypolyethylene glycol 2000 and methoxypolyethylene glycol 10000.

[0155] In a ninth embodiment, each of the first to third embodiments includes one in which the organic coating includes an organic acid including oleic acid.

[0156] In a tenth embodiment, each of the first to third embodiments includes one in which the organic acid is oleic acid.

[0157] In an eleventh embodiment, each of the first to third embodiments includes one in which the polymer coating comprises dextran.

[0158] In a twelfth embodiment, each of the first to third embodiments includes those in which the targeting ligand is an anti-HER-2 antibody or a functional binding fragment thereof.

[0159] In a thirteenth embodiment, each of the first to third embodiments includes those in which the targeting ligand is trastuzumab or a functional binding fragment thereof.

[0160] In a fourteenth embodiment, each of the first to third embodiments includes those in which the polymer coating on the outside of the organic coating is poly(maleic anhydride-alt-octadecene).

[0161] In a fifteenth embodiment, each of the first to third embodiments includes one in which performing magnetic resonance imaging uses a T1 imaging sequence.

[0162] In a sixteenth embodiment, each of the first to third embodiments includes one in which performing magnetic resonance imaging uses a T2 imaging sequence.

[0163] In a seventeenth embodiment, each of the first to third embodiments includes where the biofunctional magnetic nanoparticle solution is injected into the peritumoral region of a tumor present in a human subject.

[0164] In an eighteenth embodiment, each of the first to third embodiments includes one in which performing magnetic resonance imaging is performed using a 1.5T or 3T clinical scanner.

[0165] In a nineteenth embodiment, each of the first to third embodiments further comprises evaluating the image of the target tissue for heterogeneous architecture.

[0166] In a twentieth embodiment, each of the first to third embodiments further comprises assessing the morphology of the target tissue for the presence of a suspected tumor.

[0167] In a twenty-first embodiment, each of the first to third embodiments further comprises performing magnetic resonance imaging on the human subject 24 hours after the first imaging to reassess the target tissue.

[0168] In a twenty-second embodiment, each of the first to third embodiments includes those in which the one or more targeting ligands are capable of specifically binding to a protein or cell associated with cancer.

[0169] In the twenty-third embodiment, each of the first to third embodiments includes those in which the target molecule is the HER2 protein, or a domain or region thereof.

[0170] In a twenty-fourth embodiment, each of the first to third embodiments further comprises evaluating the image of the target tissue for heterogeneous architecture.

[0171] In a twenty-fifth embodiment, each of the first to third embodiments includes one in which the identification of heterogeneous hypointensity in the target tissue is further indicative of the malignancy of the target tissue.

[0172] In a twenty-sixth embodiment, each of the first to third embodiments includes one in which the identification of heterogeneous architecture in the target tissue is further indicative of the malignancy of the target tissue.

[0173] In a 27th embodiment, each of the first to third embodiments further includes subjecting the subject to treatment for a condition, the treatment involving administration of a drug, a biopsy, surgery, or forgoing a biopsy or surgery.

[0174] In a twenty-eighth embodiment, a method for evaluating axillary node lesions by magnetic resonance imaging is provided, the method comprising: introducing a biofunctionalized 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; evaluating an image of the at least one lymph node for heterogeneous hypointensity; and evaluating the at least one lymph node for axillary node lesions, wherein the biofunctionalized magnetic nanoparticle solution comprises nanoparticle structures, each structure comprising an iron core 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 polymer coating layer, and at least one stealth generating compound bound to 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.

[0175] In a twenty-ninth embodiment, the twenty-eighth embodiment includes wherein each of the biofunctional magnetic nanoparticle structures in the nanoparticle solution has a diameter of 10 to 150 nanometers.

[0176] In a thirtieth embodiment, the twenty-eighth embodiment includes a biofunctional magnetic nanoparticle solution in which the diameter of each of the nanoparticle structures in the nanoparticle solution is uniform.

[0177] In a thirty-first embodiment, the twenty-eighth embodiment includes the twenty-eighth embodiment in which the polymer coating on the outside of the organic coating is poly(maleic anhydride-alt-octadecene).

[0178] In a thirty-second embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the biofunctional magnetic nanoparticle solution is an aqueous solution of nanoparticles in 0.9% NaCl containing 0.05% polysorbate 20.

[0179] In a thirty-third embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the biofunctional magnetic nanoparticle solution is an isotonic solution that supports product stability and safe injection into humans.

[0180] In a thirty-fourth embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which at least one stealth generating compound is a polyethylene glycol polymer.

[0181] In a thirty-fifth embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the at least one stealth generating compound comprises a polyethylene glycol (PEG) polymer, including PEG having a molecular weight of 500 Da to 20,000 Da.

[0182] In a thirty-sixth embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the at least one stealth generating compound includes methoxypolyethylene glycol 2000 and methoxypolyethylene glycol 10000.

[0183] In a thirty-seventh embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the organic coating includes an organic acid.

[0184] In a thirty-eighth embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the organic acid is oleic acid.

[0185] In a thirty-ninth embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the polymer coating comprises dextran.

[0186] In a fortieth embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the targeting ligand is an anti-HER-2 antibody or functional binding fragment thereof.

[0187] In a forty-first embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the targeting ligand is trastuzumab or a functional binding fragment thereof.

[0188] In a forty-second embodiment, each of the twenty-eight to thirty-first embodiments includes one in which one lymph node is evaluated.

[0189] In a forty-third embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which more than one lymph node is evaluated.

[0190] In a forty-fourth embodiment, each of the twenty-eighth to thirty-first embodiments includes one in which performing magnetic resonance imaging uses a T1 imaging sequence.

[0191] In a forty-fifth embodiment, each of the twenty-eighth to thirty-first embodiments includes one in which performing magnetic resonance imaging uses a T2 imaging sequence.

[0192] In a forty-sixth embodiment, each of the twenty-eighth to thirty-first embodiments includes the human subject holding their breath while magnetic resonance imaging of the axillary region is performed.

[0193] In a forty-seventh embodiment, each of the twenty-eighth to thirty-first embodiments includes one in which the biofunctional magnetic nanoparticle solution is injected into the peritumoral region of a tumor present in a human subject.

[0194] In a forty-eighth embodiment, each of the twenty-eighth to thirty-first embodiments includes one in which performing magnetic resonance imaging is performed using a 1.5T or 3T clinical scanner.

[0195] In a forty-ninth embodiment, each of the twenty-eighth to thirty-first embodiments further comprises performing a biopsy on the lymph node if assessed to be diseased.

[0196] In a fiftieth embodiment, each of the twenty-eighth to thirty-first embodiments further comprises evaluating the image of at least one lymph node for heterogeneous architecture.

[0197] In a fifty-first embodiment, each of the twenty-eighth to thirty-first embodiments further comprises evaluating the morphology of at least one lymph node for the presence of a suspected tumor.

[0198] In a 52nd embodiment, each of the 28th to 31st embodiments further comprises performing magnetic resonance imaging on the human subject 24 hours after the first imaging to reassess at least one axillary lymph node for axillary nodal involvement.

[0199] In a fifty-third embodiment, each of the twenty-eighth to thirty-first embodiments includes those in which the identification of heterogeneous hypointensity in at least one lymph node is further indicative of lymph node malignancy.

[0200] In a 54th embodiment, each of the 28th to 31st embodiments and the 52nd embodiment includes those in which the identification of heterogeneous hypointensity in the target tissue is further indicative of lymph node malignancy.

[0201] In a 55th embodiment, there is provided an agent 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 biofunctionalized 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; evaluating images of the at least one lymph node for heterogeneous hypointensity; and evaluating the at least one lymph node for axillary node lesions, wherein the biofunctionalized magnetic nanoparticle solution comprises nanoparticle structures, each structure comprising an iron core 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 polymer coating layer, and at least one stealth generating compound bound to 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.

[0202] In a fifty-sixth embodiment, the fifty-fifth embodiment includes one in which the identification of heterogeneous hypointensity in the target tissue or lymph node is further indicative of malignancy of the target tissue.

[0203] In a 57th embodiment, each of the 55th and 56th embodiments includes evaluating an image of at least one lymph node for heterogeneous architecture.

[0204] In a fifty-eighth embodiment, the fifty-seventh embodiment includes one in which the identification of heterogeneous architecture in the target tissue or lymph node is further indicative of the malignancy of the target tissue.

[0205] Other features and advantages of the invention will become apparent from the following detailed description and claims.

[0206] The above examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Many variations on those described above are possible. Since modifications and variations on the above examples will be apparent to those skilled in the art, it is intended that this invention be limited only by the scope of the appended claims.

[0207] References 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

[0208] 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.

[0209] H.S.Cody III,N.Houssami,Axillary management in breast cancer:What’s new for 2012? The Breast 21,2012,411e415412

[0210] 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

[0211] 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

[0212] 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.

[0213] 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

[0214] NCCN Guidelines for Breast Cancer 2022,Version 4.

[0215] 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

[0216] 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. 1. A method for assessing a target tissue by magnetic resonance imaging, comprising: introducing a biofunctionalized magnetic nanoparticle solution into a subject, the biofunctionalized magnetic nanoparticle solution being comprised of a plurality of biofunctionalized 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; Binding the biofunctionalized magnetic nanoparticle solution to the target molecule if the target molecule is present, wherein the target molecule is indicative of a health condition of the subject; and performing magnetic resonance imaging of 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 for areas of heterogeneous hypointensity in the target tissue indicative of the presence of biofunctionalized magnetic nanoparticles bound to the target molecule, thereby identifying that the areas of heterogeneous hypointensity in the target tissue indicate the presence of the target molecule in the target tissue; The method comprising:

2. 10. The method of claim 1, wherein each of the biofunctional magnetic nanoparticle structures in the nanoparticle solution has a diameter of 10 to 150 nanometers.

3. The method of claim 1 , wherein in 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 in 0.9% NaCl containing 0.05% polysorbate 20.

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 humans.

6. The method of any one of claims 1 to 3, wherein said 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 with a molecular weight of 500 Da to 20,000 Da.

8. The method of any one of claims 1 to 3, wherein the at least one stealth-generating compound comprises methoxypolyethylene glycol 2000 and methoxypolyethylene 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. 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 polymer coating on the outside of the organic coating is poly(maleic anhydride-alt-octadecene).

15. The method of any of claims 1 to 3, wherein performing the magnetic resonance imaging uses a T1 imaging sequence.

16. The method of any of claims 1 to 3, wherein performing the magnetic resonance imaging uses a T2 imaging sequence.

17. The method according to 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 of claims 1 to 3, wherein performing the magnetic resonance imaging is performed using a 1.5T or 3T clinical scanner.

19. The method of any of claims 1 to 3, further comprising evaluating the image of the target tissue for heterogeneous architecture.

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

21. 4. The method of claim 1, further comprising performing magnetic resonance imaging on the human subject 24 hours after the first imaging to reassess the target tissue.

22. The method of any one of claims 1 to 3, wherein the one or more targeting 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 of claims 1 to 3, further comprising evaluating the image of the target tissue for heterogeneous architecture.

25. The method of any of claims 1 to 3, wherein identification of heterogeneous hypointensity in the target tissue further indicates malignancy of the target tissue.

26. The method of any one of claims 1 to 3, wherein the identification of heterogeneous architecture in the target tissue further indicates the malignancy of the target tissue.

27. 4. The method of any of claims 1-3, further comprising subjecting the subject to treatment for said condition, wherein said treatment involves administration of a drug, a biopsy, surgery, or forgoing biopsy or surgery.

28. 1. A method for assessing axillary node lesions by magnetic resonance imaging, said 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; evaluating the image of the at least one lymph node for heterogeneous hypointensity; assessing the at least one lymph node for axillary node involvement; The method, wherein the biofunctionalized magnetic nanoparticle solution comprises nanoparticle structures, each structure comprising an iron core 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 polymer coating layer, and at least one stealth generating compound bound to 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. 29. The method of claim 28, wherein each of the biofunctional magnetic nanoparticle structures in the nanoparticle solution has a diameter of 10 to 150 nanometers.

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

31. 29. The method of claim 28, wherein the polymer coating on the outside of the organic coating is poly(maleic anhydride-alt-octadecene).

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

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

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

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

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

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

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

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

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

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

44. The method of any of claims 28 to 31, wherein performing the magnetic resonance imaging uses a T1 imaging sequence.

45. The method of any of claims 28 to 31, wherein performing the magnetic resonance imaging uses a T2 imaging sequence.

46. 32. The method of any of claims 28 to 31, wherein the human subject holds their breath while magnetic resonance imaging of the axillary region is performed.

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

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

49. 32. The method of any of claims 28-31, further comprising performing a biopsy on said lymph node if assessed to be diseased.

50. 30. The method of claim 28, further comprising evaluating the image of the at least one lymph node for heterogeneous architecture.

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

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

53. The method of any of claims 28 to 31, wherein identification of heterogeneous hypointensity in said at least one lymph node further indicates malignancy of said lymph node.

54. 53. The method of claim 28, 29, 30, 31, or 52, wherein identification of heterogeneous architecture in the at least one lymph node further indicates malignancy of the lymph node.

55. 1. An agent for use in magnetic resonance imaging formulated for use in a method for assessing and / or treating a condition characterized by the presence of a protein or cell associated with cancer, said 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; evaluating the image of the at least one lymph node for heterogeneous hypointensity; assessing the at least one lymph node for axillary node involvement; The drug, wherein the biofunctionalized magnetic nanoparticle solution comprises nanoparticle structures, each structure comprising an iron core 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 polymer coating layer, and at least one stealth generating compound bound to 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. 56. The agent of claim 55, wherein identification of heterogeneous hypointensity in the target tissue or lymph node further indicates malignancy of the target tissue.

57. 57. The method of claim 55 or 56, further comprising evaluating an image of the at least one lymph node for heterogeneous architecture.

58. 58. The agent of claim 57, wherein the identification of heterogeneous architecture in the target tissue or lymph node further indicates the malignancy of the target tissue.

Images