WO2010060212A1

WO2010060212A1 - Single-domain antibody targeted formulations with superparamagnetic nanoparticles

Info

Publication number: WO2010060212A1
Application number: PCT/CA2009/001716
Authority: WO
Inventors: Abedelnasser Abulrob; Teodar Veres; Umar Iqbal; Danica Stanimirovic; Boguslaw Tomanek
Original assignee: National Research Council Of Canada
Priority date: 2008-11-26
Filing date: 2009-11-25
Publication date: 2010-06-03

Abstract

A nanoconstruct includes a superparamagnetic nanoparticle, a single domain antibody (sdAb) specific for a selected receptor on mammalian cells, and a near-infrared fluorophore. The sdAb may specifically bind to IGFBP7 or a receptor expressed by tumor endothelial cells. The nanoconstruct may be used in magnetic resonance imaging methods, optical imaging methods or both.

Description

SINGLE-DOMAIN ANTIBODY TARGETED FORMULATIONS WITH SUPERPARAMAGNETIC NANOPARTICLES

Field of the Invention

The present invention relates to single-domain antibody (sdAb) targeted formulations with superparamagnetic nanoparticles.

Background of the Invention

Magnetic resonance imaging (MRI) is a non-invasive and powerful medical diagnostic technique that offers high-resolution anatomical information about the human body, and is frequently used for the non-invasive detection of a variety of diseases. MRI creates images of a body using the principles of nuclear magnetic resonance. To enhance imaging capabilities, gadolinium (Gd-DTPA) is often used as a contrast agent. These contrast agents are nonspecific however as they rely on blood vessel density only. While molecular resonance images provide good anatomical information about disease localization and spread, it is still necessary to obtain biopsy of diseased tissue and perform ex vivo molecular analyses such as histopathology and immunochemistry, to obtain information about molecular characteristics of the disease, such as the expression of certain receptors that could be targeted by drugs or transporters that may cause resistance to certain drugs.

The ability to perform these molecular analyses non-invasively by in vivo imaging using, for example, MRI at the time of diagnosis and during disease treatment, would greatly improve treatment efficacy by obtaining early molecular information on disease, adjusting treatment to fit 'personal' characteristics of disease, and selecting appropriate patient populations for clinical trials.

Magnetic Resonance (MR) molecular imaging is a new imaging technique that is not currently used routinely in clinical applications because of the lack of appropriate contrast agents that are targeted to recognize specific molecular targets. These contrast agents need to have strong magnetic properties to provide measurable information on a specific molecular target. Target characteristics are also important, including selectivity of the target for diseased tissues and the high expression/density of the target, to enable sufficient signal changes that can be detected with MRI.

Standard MRI contrast agents are based on Gd, which changes Tl relaxation time; it is used as a non-specific contrast agent and its accumulation is based on tissue vasculature. A promising alternative to Gd as a contrast agent are paramagnetic nanoparticles that exhibit unique nanoscale properties of superparamagnetism and has the potential to be utilized as an excellent, high contrast probes for MRI. The paramagnetic nanoparticles induce strong magnetic field distortions around the particles and decrease T2 and T2* relaxation times. This effect results in marked, focal signal change in T2 and T2* weighted MR images. Some of these nanoparticles are currently available for clinical applications. Iron oxide nanoparticles are commercially available under the brand name Sinerem® in Europe (Laboratoire Guerbet, Aulnay sous Bois, France), and Combidex® in the U.S. (Advanced Magnetics, Cambridge, MA), but are not yet FDA approved. These particles can be used for lymph node staging and localization of pathological lymph nodes using a non-invasive technique called MR lymphangiography (MRL) because they are selectively taken up by the reticulo-endothelial system (RES) and macrophages. Iron oxide-contrast agents can also be used to assess inflammatory processes. Upon systemic application, circulating small (SPIO) and ultrasmall particles of iron oxide (USPIO) are preferentially phagocytosed by monocytes before clearance within the reticuloendothelial system of the liver, spleen and lymph nodes.

Currently, most iron oxide nanoparticles rely on passive targeting and accumulation in the RES and sites of inflammation, and are not targeted to any specific antigen or molecular biomarker. The contrast abilities of existing iron oxide nanoparticles have to be further improved for applications in molecular MRI. Currently available iron oxide nanoparticles are not 'stealth' in the human body, and are cleared by the RES. Therefore, there is a need in the art for a targeted contrast agent useful for molecular MRI which mitigates the shortcomings in the prior art. There is also a need for the development of multimodal contrast agents/probes that could be applied for different imaging modalities, for example optical and MRI. These probes could be used for in vivo imaging in animal and humans, as well as imaging cells and tissues ex vivo.

Summary of the Invention

The present invention comprises superparamagnetic nanoparticle formulations functionalized with fluorescent probes to form multimodal nanoparticles, as well as single domain antibodies (sdAb) useful for tissue and tumor targeting. In one embodiment, these nanoparticle formulations may be used for both optical imaging and MRI. Multimodal imaging may result in greater accessibility of diagnostic tools to clinicians and patients.

The present invention provides a nanoconstruct:

(a) a superparamagnetic nanoparticle;

(b) a single domain antibody (sdAb) specific for a selected receptor on mammalian cells; and

(c) a near-infrared fluorophore.

The nanoparticle may be coated, and/or may be functionalized with polyethylene glycol. The superparamagnetic nanoparticle may comprise iron oxide, iron cobalt (FeCo), any other particle exhibiting paramagnetic properties, or a combination thereof.

The single domain antibody may be linked to the superparamagnetic nanoparticle core/shell by a functional group, such as a carboxylate, a sulfonate, a phosphate, an amine, or any combination thereof. In one embodiment, the single domain antibody selectively binds a receptor expressed by tumor endothelial cells. In another embodiment, the sdAb may selectively bind Insulin-like Growth Factor Binding Protein 7 (IGFBP7). In one specific example, the single domain antibody may comprise complementarity determining region (CDR) sequences RTSRRYAM or RTFSRLAM (CDRl ; SEQ ID NOs: 1 and 2), GISRSGDGTHYAYSV (CDR2; SEQ ID NO:3), and AAART AFYYYGNDYNY (CDR3;

SEQ ID NO:4). Alternatively, the sdAb may comprise the sequence:

AIAIAVALAGFATVAQAQVKLEESGGGLVQAGGSLRLSCAASGRTSRRYAMGWF RQAPGKEREFVAGISRSGDGTHYAYSVKGRFTISRDNAANTVELQMNSLKPEDT AVYFCAAARTAFYYYGNDYNYWGQGTQVTVSS (SEQ ID NO:5), or a sequence substantially identical thereto. Alternatively, the sdAb may comprise the sequence:

AIAIAVALAGFATVAQAQVKLEESGGGSVQPGGSLRLSCAASGRTFSRL AMGWFRQAPGKERELVAGISRSGDGTHYAYSVKGRFTISRDNAANTV ELQMNSLKPEDTAVYFCAAARTAFYYYGNDYNYWGQGTQVTVSS

(SEQ ID NO:6), or a sequence substantially identical thereto.

The present invention also provides a method for in vivo imaging of cells or tissues in a mammal, the method comprising the steps of: (a) administering to the mammal a composition comprising the nanoconstruct of the present invention as described herein, wherein the single domain antibody (sdAb) is specific for a selected receptor;

(b) waiting a time sufficient to allow the sdAb to bind to the selected receptor; and

(c) imaging the cells or tissues with a non-invasive imaging technique whose resolution is enhanced by the presence of the nanoconstruct on or within the cells.

The selected receptor may be IGFBP7. The imaging technique used may be magnetic resonance imaging, optical imaging, or a combination thereof. The method as described may allows for imaging of one or more tumors, metastases, vascularized malignant cell clusters, or individual malignant cells selected from the group consisting of brain cancer, colon cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, endometrial cancer, oral cancer, liver cancer, and renal cancer or any other cancer. In another aspect, the invention may comprise a method for detecting glioblastoma in a patient, comprising:

(a) contacting a tissue of interest with a nanoconstruct as described herein, wherein the sdAb is specific for IGFBP7, and may comprise the specific antibodies described herein; and

(b) measuring the level of binding of the nanoconstruct, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.

In yet another aspect, the invention may comprise a method of imaging angiogenesis in a mammal, comprising: (a) introducing into the mammal a nanoconstruct as described herein, wherein the sdAb is specific for tumor endothelial cells;

(b) permitting the nanoconstructs to bind to angiogenic tumor vessels; and

(c) imaging the angiogenic tumor vessels using magnetic resonance imaging.

In yet another aspect, the invention may comprise a method for detecting a tissue expressing IGFBP7, comprising:

(a) contacting a tissue of interest with a nanoconstruct as described herein, wherein the sdAb is specific for the IGFBP7, and may comprise the specific antibodies described herein; and

(b) measuring the level of binding of the nanoconstruct, wherein an elevated level of binding, relative to normal tissue is indicative of the presence of a tumor expressing

IGFBP7.

In one embodiment, the step of measuring is performed by magnetic resonance imaging or by fluorescence imaging.

In yet another aspect, the invention may comprise a method for determining the location of glioblastoma brain tumor cells in a patient pre-operatively, intra-operatively, and/or postoperatively, comprising administering a composition comprising a nanoconstruct as described herein, wherein the sdAb is specific for the IGFBP7, and may comprise the specific antibodies described herein and a pharmaceutically acceptable carrier to the patient, wherein the composition is administered in an amount sufficient to image glioblastoma cells in vivo; and

(a) pre-operatively measuring the level of binding of nanoconstruct by magnetic resonance imaging to determine the location of glioblastoma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of glioblastoma cells;

(b) intra-operatively measuring the level of binding of the nanoconstruct by fluorescence imaging to determine the location of residual glioblastoma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of residual glioblastoma cells;

(c) post-operatively measuring the level of binding of the nanoconstruct by magnetic resonance imaging to determine the location of glioblastoma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of tumor cells; or

(d) any combination of (a), (b) or (c) above.

In yet another aspect, the invention may comprise a method for in vitro detection or quantification of biological or chemical molecule in a sample, comprising the steps of:

(a) contacting the sample with a nanoconstruct as described herein, so as to form a complex between the molecule and the nanoconstruct; and (b) detecting or quantifying said formed complex.

In one embodiment, the step of detecting or quantifying may be performed by magnetic resonance imaging, fluorescence imaging, or a combination thereof.

The superparamagnetic nanoparticle formulations described herein, targeted using a single- domain antibody against IGFBP7, have been evaluated in orthotopic brain tumor models in nude mice. The accumulation of the nanoconstruct formulation in the brain tumor region was demonstrated by optical in vivo imaging. In vivo MRI imaging of nude mice bearing brain tumors and injected with iron oxide nanoparticles targeted with the single-domain antibody against IGFBP7 showed enhanced contrast in the tumor region in contrast to non-targeted iron-oxide nanoparticles. The presence/accumulation of nanoparticles in the brain tumors was further demonstrated by (immuno)histochemistry in brain sections.

Brief Description of the Drawings

In the drawings, like elements are assigned like reference numerals. The drawings are not necessarily to scale, with the emphasis instead placed upon the principles of the present invention. Additionally, each of the embodiments depicted are but one of a number of possible arrangements utilizing the fundamental concepts of the present invention. The drawings are briefly described as follows:

Figure IA is a schematic representation of an iron oxide core-silica shell nanoparticle functionalized with sdAb and PEG molecules. Figures IB, 1C, and ID show electron microscopy micrographs of iron oxide core / silica shell nanoparticles of different sizes - 40 nm, 70nm, and 80 nm, respectively.

Figure 2 shows the chemical reaction for the fluorescamine assay, used to detect and quantify the number of primary amine groups on the surface of iron-oxide nanoparticles. A fluorescamine standard curve is also shown.

Figure 3 shows a table with data estimating the number of primary amine groups in different batches of synthesized iron oxide nanoparticles using fluorescamine assay described in Figure 2.

Figure 4 shows the effect of PEGylation and sdAb bioconjugation on the number of amine groups and the protein content, respectively, in iron oxide nanoparticles. Reduction in the number of amine groups indicates the attachement of PEG molecules; increase in protein content indicates a successful conjugation of sdAb to nanoparticles. Figure 5 shows pharmacokinetics of various iron-based nanoparticles (Fe₃O₄ 10 nm, Fe₃O₄ 20 nm, and FeCo 6 nm).

Figure 6 shows biodistribution of silica-coated iron-oxide nanoparticle labeled with the near- infrared probe, Cy5.5, in normal CDl mice.

Figure 7 shows ex vivo imaging of organs 4 hours post intravenous injection of silica-coated iron oxide nanoparticles.

Figure 8 shows biodistribution of IGFBP7 single domain antibody-targeted and non-targeted silica-coated iron oxide nanoparticles in nude mice bearing brain tumors.

Figure 9 shows biodistribution of IGFBP7 single domain antibody-targeted and non-targeted silica-coated iron oxide nanoparticles in nude mice bearing brain tumors (fluorescence lifetime-gated whole body dorsal scan).

Figure 10 is a graphical representation of the accumulation of IGFBP7 single domain antibody-targeted and silica-coated non-targeted iron oxide nanoparticles in the brain tumor region.

Figure 11 shows ex vivo brain imaging of the brains of nude mice from figure 8 and figure 9 bearing glioblastoma tumor (8 h and 72 hr post-injection examples, respectively).

Figure 12 shows head optical imaging of nude mice bearing brain tumors injected with IGFBP7 single domain antibody- targeted or non-targeted iron oxide nanoparticles

Figure 13 shows biodistribution of IGFBP7 single domain antibody-targeted and non-targeted superparamagnetic iron oxide nanoparticles over time in nude mice bearing brain tumors

Figure 14 shows depth-concentration analysis (A) and volumetric analysis (B) of brain tumor region in IGFBP7 single domain antibody-targeted and non-targeted superparamagnetic iron oxide nanoparticles over time in nude mice bearing brain tumors. Figure 15 shows ex vivo brain imaging of the brains of nude mice bearing glioblastoma tumors (72h post-injection) Results for depth-concentration analysis (A) and volumetric analysis (B) of brain tumor region are shown.

Figure 16 shows ex vivo optical imaging of organs of nude mice bearing glioblastoma tumors 72 h post intravenous injection of IGFBP7 targeted and non-targeted superparamagnetic iron oxide nanoparticles. Results for depth-concentration analysis (A) and volumetric analysis (B) of brain tumor region are shown.

Figure 17 shows fluorescence microscopy images of brain tumor sections to detect Cy5.5, CD31 (to visualize brain vessels), DAPI (to visualize nuclei) 72 h after the injection of non- targeted superparamagnetic iron oxide nanoparticles. Nanoparticles (Cy5.5) were detected only in the tumor core but not in the tumor periphery.

Figure 18 shows fluorescence microscopy images of brain tumor sections to detect Cy5.5, CD31 (to visualize brain vessels), DAPI (to visualize nuclei) 72 h after the injection of IGFBP7 sdAb-targeted superparamagnetic iron oxide nanoparticles. Nanoparticles (Cy5.5) were detected in both tumor core and periphery, associated with brain vessels.

Figure 19 shows fluorescence microscopy images of contralateral brain sections to detect Cy5.5, CD31 (to visualize brain vessels), DAPI (visualize nuclei) 72 h after the injection of IGFBP7 sdAb-targeted superparamagnetic iron oxide nanoparticles. Nanoparticles (Cy5.5) could not be detected in the contralateral (normal ) brain.

Figure 20 shows graphically the effect of silica-coated iron oxide nanoparticles and iron cobalt nanoparticles compared to ferridex on T2 relaxation measured by 9.4T MRI.

Figure 21 A shows in vivo MRI imaging of nude mouse bearing brain tumor and injected with Non-targeted pegylated superparamagnetic Fe₃O₄ nanoparticles. Quantification of MRI imaging is shown in the graph of Figure 2 IB. Figure 22 A shows in vivo MRI imaging of nude mice bearing brain tumor and injected with IGFBP7 sdAb pegylated-targeted superparamagnetic Fe₃O₄ nanoparticles. Quantification of MRI imaging is shown in the graph of Figure 22B.

Figure 23 shows quantification of in vivo MRI imaging of nude mice bearing brain tumor and injected with IGFBP7 sdAb pegylated-targeted superparamagnetic Fe₃O₄ nanoparticles (n=5).

Detailed Description of Preferred Embodiments

The present invention relates to single-domain antibody (sdAb) targeted formulations with superparamagnetic nanoparticles for imaging purposes. In particular, the invention relates to functionalized superparamagnetic nanoparticles. When describing the present invention, all terms not defined herein have their common art-recognized meanings. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention. The following description is intended to cover all alternatives, modifications and equivalents that are included in the spirit and scope of the invention, as defined in the appended claims.

One embodiment of the present invention provides a nanoconstruct comprising:

(a) superparamagnetic nanoparticle;

(b) a single domain antibody (sdAb) specific for a selected receptor on mammalian cells; and (c) a near-infrared fluorophore.

As used herein, the term "nanoparticle" (also referred to herein as "NP") means a particle having at least one dimension that is less than about 200 nm.

The nanoparticle is a nanoparticle that has paramagnetic properties, thus is detectable with

MRI. The NPs can be divided into two subgroups: ultra small superparamagnetic NPs (USPIO) with hydrodynamic size smaller than 50nm (including coating) and superparamagnetic (SPIO), that are larger than 50nm. SPIOs are characterized by a large magnetic moment in the presence of a static external magnetic field, thus visible with MRI. SPIOS exhibit a behavior similar to paramagnetism at temperatures below the Curie or the Neel temperature. This is a small length-scale phenomenon, where the energy required to change the direction of the magnetic moment of the particle is comparable to the ambient thermal energy. At this point, the rate at which the particles will randomly reverse direction becomes significant. Normally, coupling forces in ferromagnetic materials cause the magnetic moments of neighboring atoms to align, resulting in very large internal magnetic fields. This is what distinguishes ferromagnetic materials from paramagnetic materials. At temperatures above the Curie temperature (or the Neel temperature for antiferromagnetic materials), the thermal energy is sufficient to overcome the coupling forces, causing the atomic magnetic moments to fluctuate randomly. Because there is no longer any magnetic order, the internal magnetic field no longer exists and the material exhibits paramagnetic behavior. If the material is non-homogeneous, one can observe a mixture of ferromagnetic and paramagnetic clusters of atoms at the same temperature, the superparamagnetic stage. Superparamagnetic nanoparticles are described in CM. Sorensen (2001), the contents of which are incorporated herein by reference where permitted.

The superparamagnetic nanoparticles may be any suitable superparamagnetic material, including, but not limited to FeO, Fe₂O₃, Fe₃O₄, FeCo, FePt and nanoparticle clusters containing any combination of these nanoparticles. The formation of such superparamagnetic nanoparticles is well-known by those skilled in the art and need not be further described herein (see for example Hyeon, 2003, which is incorporated herein by reference where permitted). In a specific, non-limiting example, the superparamagnetic nanoparticles are comprised of Fe₃O₄.

Superparamagnetic nanoparticles (SPNs) may be used as high contrast probes for MRI. Without wishing to be bound by theory, the superparamagnetic properties of these nanoparticles induce strong magnetic field distortions around the particles and decrease T2 and T2* relaxation times. This effect results in marked, focal signal change in T2 and T2* weighted MR images in the regions of NPs accumulation. Without wishing to be bound by theory, the presence of FeCo in an iron oxide nanoparticle may produce further T2 shortening in MRI phantom measurements.

The SPNs may be further encapsulated in a shell or coating. Without wishing to be bound by theory, the shell may enable attachment of biomolecules and may reduce toxicity; these coated superparamagnetic nanoparticles are preferably biocompatible, allowing their use in clinical diagnosis. Thus, the SPN may comprise a core + shell architecture familiar to the skilled artisan. The shell or coating may be of any suitable material known in the art; for example, and not wishing to be limiting in any manner, the shell may comprise silica, one or more than one biocompatible polymer, one or more than one lipid (Meincke et al, 2008; LaConte et al, 2007; Wijaya et al, 2007), one or more than one lipidic polymer, gold (Wang et al, 2005, GoIe et al, 2008), silver, or a combination thereof. Those skilled in the art would be familiar with methods for deposition of these types of coatings onto nanoparticles (see for example Arruebo et al, 2007; Lu et al, 2007).

In a specific, non-limiting example, the coating is a silica coating. Methods for preparing a silica shell are also well-known to those of skill in the art (see for example, Lu et al, 2002; KeIl et al, 2008). For example, and without wishing to be limiting in any manner, the thickness of the silica coating may be applied in a controlled manner over the SPN core. The thickness of the silica coating, once complete, may be about 5 nm and 40 nm, or any value there between; for example, the silica coating may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nm thick. In a specific, non-limiting example, the thickness of the silica coating may be about 15 nm.

The superparamagnetic nanoparticle as just described may also be functionalized with polyethylene glycol (PEG). Without wishing to be bound by theory, the PEGylation of the nanoparticles may enable them to escape from the reticuloendothelial system and to improve their plasma stability and plasma half-life. Any suitable size PEG may be used for attachment (conjugation) to the superparamagnetic nanoparticle / shell. Without wishing to be limiting, the PEG may be in the range of about 1000 to 5000 Da; for example, the PEG may be about 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 Da, or any size therebetween. In a specific, non-limiting example, the PEG may be about 2000 Da. Those of skill in the art will be familiar with methods for functionalizing SPNs with PEG (Veiseh et al, 2009).

The SPNs may be between about 1 and 200 nm in diameter, or any size therebetween; for example, the diameter of the superparamagnetic nanoparticles may be about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 nm, or any diameter therebetween. In a specific, non-limiting example, the superparamagnetic nanoparticles may be about 5, 15, or 20 nm in diameter. The nanoparticles in a given volume may all be the same size, or may be of different sizes.

The nanoconstructs of the present invention comprise the SPNs as described above and also comprise single-domain antibodies (sdAb) as a targeting moiety. By the term "single-domain antibody" or "sdAb", it is meant an antibody fragment comprising a single protein domain. Single domain antibodies may comprise any variable fragment, including V_L, V_H, V_HH, V_NAR, and may be naturally-occurring or produced by recombinant technologies. For example V_HS, V_LS, V_HHS, V_NARS, may be generated by techniques well known in the art (Holt, et al., 2003; Jespers, et al., 2004a; Jespers, et al., 2004b ; Tanha, et al., 2001 ; Tanha, et al., 2002; Tanha, et al., 2006 ; Revets, et al., 2005 ; Holliger, et al., 2005 ; Harmsen, et al., 2007 ; Liu, et al., 2007 ; Dooley, et al., 2003 ; Nuttall, et al., 2001 ; Nuttall, et al., 2000 ; Hoogenboom, 2005; Arbabi- Ghahroudi et al., 2009). In the recombinant DNA technology approach, libraries of sdAbs may be constructed in a variety of ways, "displayed" in a variety of formats such as phage display, yeast display, ribosome display, and subjected to selection to isolate binders to the targets of interest (panning). Examples of libraries include immune libraries derived from llama, shark or human immunized with the target antigen; non-immune/naϊve libraries derived from non-immunized llama, shark or human; or synthetic or semi-synthetic librairies such as V_H, V_L, V_HH or V_NAR libraries.

While, in principle, monoclonal antibodies could be used to target an imaging contrast agent to the antigen recognition site, these antibodies are relatively large proteins (150 kDa) and can only be attached to nanoparticles in low numbers, typically less than 25 per nanoparticle. Moreover, repetitive display of large proteins on the surface of nanoparticles can also be immunogenic, and in some instances further accelerate biological clearance. sdAbs have several properties which may make them preferable in certain circumstances over more popular antibody formats such as IgGs or scFvs as the recognition component of nanoconstruct. First, they are highly stable against proteases and chemical denaturants; under nonphysiological conditions they regain their activity following removal from denaturing conditions (Tay et al, 2007; Arbabi-Ghahroudi et al, 2005). This allows for flexibility in terms of choosing optimal conjugation chemistry conditions (Tay et al, 2007), leads to a more active end product. Second, sdAbs, which are typically about 13kDa in size, can be conjugated on the surface of nanoparticles with a much higher binding site density (> 5 fold compared to IgGs and 2-fold compared to scFvs) and do not promote nanoparticle aggregation associated with scFvs and IgGs, resulting in much more active nanoconjugates,and more robust signal amplification strategy. The latter is a key issue for molecular imaging strategies. Higher levels of imaging signal per unit level of target-probe interaction lead to higher sensitivity for any particular imaging modality. Third, sdAbs can be easily engineered to contain amino acid residues for conjugation in an active orientation or for performing a variety of conjugation chemistries (Shen et al, 2008; Shen et al, 2005).

In one embodiment, the sdAb may recognize and bind to an antigen present in tumor endothelial cells. For example, and without wishing to be limiting in any manner, the sdAb may selectively bind Insulin-like Growth Factor Binding Protein 7 (IGFBP7), which is strongly upregulated in vessels of glioblastoma tumors undergoing neovascularization. This target is less expressed in vessels of low grade gliomas. Without wishing to be limiting in any manner, the single domain antibody may be an sdAb as described in PCT/CA2009/001460 entitled "Formulations Targetting IGFBP7 for Diagnosis and Therapy of Cancer", the disclosure of which is incorporated herein by reference where permitted. In a specific, non-limiting example, the sdAb may comprise complementarity determining region (CDR) sequences RTSRRYAM [SEQ ID NO. 1] or RTFSRLAM [SEQ ID NO. 2] (CDRl), GISRSGDGTHYAYSV [SEQ ID NO. 3] (CDR2), and AAARTAFYYYGNDYNY [SEQ ID NO. 4] (CDR3). Alternatively, the single domain antibody may comprise the sequence:

AIAIAVALAGFATVAQAQVKLEESGGGLVQAGGSLRLSCAASGRTSRR YAMGWFRQAPGKEREFVAGISRSGDGTHYAYSVKGRFTISRDNAANT VELQMNSLKPEDTAVYFCAAARTAFYYYGNDYNYWGQGTQVTVSS [SEQ ID NO. 5] or a sequence substantially identical thereto. Alternatively, the sdAb may comprise the sequence:

[SEQ ID NO. 6] or a sequence substantially identical thereto.

A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant polypeptide with no substantial change in physiological, chemical, or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered "substantially identical" polypeptides. Conservative amino acid mutation may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity). In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term "basic amino acid" it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term "neutral amino acid" (also "polar amino acid"), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (GIn or Q). The term "hydrophobic amino acid" (also "non-polar amino acid") is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (He or I), phenylalanine (Phe or F), valine (VaI or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (GIy or G). "Acidic amino acid" refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (GIu or E), and aspartate (Asp or D).

Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at http://ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art.

The substantially identical sequences of the present invention may be at least 75% identical; in another example, the substantially identical sequences may be at least 70, 75, 80, 85, 90, 95, or 100% identical at the amino acid level to sequences described herein. Importantly, the substantially identical sequences retain the activity and specificity of the reference sequence.

The sdAb may be conjugated (also referred to herein as "linked" or "coupled") to the superparamagnetic nanoparticle, or to a silica shell, using any suitable method known in the art. For example, and without wishing to be limiting, the single domain antibody may be linked to the superparamagnetic nanoparticle core/shell by a functional group such as a carboxylate, a sulfonate, a phosphate, an amine, and any combination thereof.

Conjugation of sdAbs to the nanoparticle or shell may be accomplished using methods well known in the art (see for example Hermanson, 1996). Single domain antibodies have several exposed lysine (primary amine) residues, and thus one method of covalently anchoring the sdAb to the carboxylic acid-modified nanoparticle surface is through bioconjugation chemistry. Suitable coupling reagents include l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which is often used in combination with N- hydroxysuccinimide (NHS). For example, the sdAb as described above may have, or may be engineered to have, one or more lysine residues opposite or away from its antigen binding site, which is used in covalent conjugation to the nanoparticle surface. In one embodiment, the number of sdAbs conjugated to the surface of the nanoparticle is controllable and controlled.

Alternatively, the sdAb may be conjugated to the nanoparticle/shell surface through an amino acid with a carboxylic acid (i.e., GIu or Asp) on the sdAb and primary amines on the nanoparticle, or through binding of the sdAb (detecting entity) to a molecule that has binding activity towards the sdAb and is already attached to the nanoparticle. For example, this molecule could be an antibody which binds to the sdAb or to tags (C-Myc tag, His6 tag) on the sdAb such as anti-C-Myc or anti-Hisό antibodies, or through binding of a biotinylated sdAb to a biotin binder on the surface of nanoparticles. Biotin binders are well known and may include streptavidin, neutravidin, avidin, or extravidin. The sdAb could also be coupled to the nanoparticle by means of nickel -nitrilotriacetic acid chelation to a His6-tag. In another alternative, sdAbs can also be engineered to have cysteines opposite their antigen binding sites. Conjugation via a maleimide cross-linking reaction allows the directional display of single domain antibodies where all single domain antibodies are optimally positioned to bind to their antigens. Amine-terminated nanoparticle is activated with maleimide in DMF followed by an incubation of cysteine-terminated single domain antibody to achieve covalent binding through the formation of sulfide bond formation.

The number of sdAb molecules conjugated to the surface of the superparamagnetic nanoparticle may vary, based on various factors, such as the size of the nanoparticle. The conjugate of the present invention may comprise at least 1 to 100 sdAb molecules conjugated to the surface of the SPN; for example, the conjugate may carry at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 sdAb moieties linked to the superparamagnetic nanoparticle/shell. As a person of skill in the art would recognize, it may be possible to conjugate more sdAb molecules to the surface of the nanoparticle, depending on particle size, sdAb size and characteristics, and on immobilization efficiency. It is to be noted that each of the sdAb molecules linked to the nanoparticle may be the same, or may differ from one another.

In one embodiment, the nanoconstruct of the present invention may also comprise a fluorophore, such as a near-infrared fluorophore (NIRF). Any suitable near-infrared fluorophore known in the art may be used in the nanoconstructs of the present invention. For example, NIRF that can be used include, but are not limited to Cy5.5, Cy7, Cy7.5. Alexa 680, Alexa 750, ICG, IR800, or any fluorophore that emits between 650 nm and 900 nm. The fluorophore may be conjugated to the SPN via a variety of classical conjugation methods known to those skilled in the art; for example, and without wishing to be limiting in any manner, fluorophores can be maleimide or NHS activated, or activated by other methods, and subsequently attached to the nanoparticle. Without wishing to be bound by theory, the use of fluorophores with long emission in the near-infrared (NIR) region can achieve deeper tissue penetration and lower background in in vivo applications. The overall size of the nanoconstruct of the present invention may be between about 30 and 200 nm in diameter. For example, and without wishing to be limiting, the nanoconstruct may have a diameter of about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm, or any value therebetween.

An exemplary embodiment of the nanoconstruct of the present invention comprising an iron oxide core-silica shell nanoparticle functionalized with sdAb and PEG molecules is shown in the schematic of Figure IA.

Formulations and compositions comprising the nanoconstruct of the present invention and useful for diagnostic, preventative or therapeutic purposes are also provided. In addition to the nanoconstruct of the present invention, such formulations or compositions may include pharmaceutically acceptable excipients or diluents, buffers, and/or water. The formulations may be powder, suspensions, or any other suitable pharmaceutical formulation.

The present invention also provides a method for in vivo imaging of cells or tissues in a mammal, the method comprising the steps of: (a) administering to the mammal a composition comprising the nanoconstruct of the present invention as described above, wherein the single domain antibody (sdAb) is specific for a selected receptor;

(c) imaging the cells or tissues with a non-invasive imaging technique whose resolution is enhanced by the presence of the particles on or within the cells.

The selected receptor may be IGFBP7. The imaging technique used may be magnetic resonance imaging, optical imaging, or a combination thereof. The method as described may allows for imaging of one or more tumors, metastases, vascularized malignant cell clusters, or individual malignant cells selected from the group consisting of brain cancer, colon cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, endometrial cancer, oral cancer, liver cancer, and renal cancer or any other cancer. Also provided is a method for detecting glioblastoma in a patient, comprising:

(a) contacting a tissue of interest with the nanoconstruct of the present invention, wherein the sdAb is specific for the IGFBP7, and may comprise the specific antibodies described above; and (b) measuring the level of binding of the nanoconstruct, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.

The present invention further provides a method of imaging angiogenesis in a mammal, comprising:

(a) introducing into the mammal the nanoconstruct of the present invention, wherein the sdAb is specific for tumor endothelial cells;

(b) permitting the nanoconstructs to bind to angiogenic tumor vessels; and

(c) imaging the angiogenic tumor vessels using magnetic resonance imaging.

In yet another aspect, the present invention provides a method for detecting a tissue expressing IGFBP7, comprising: (a) contacting a tissue of interest with the nanoconstruct of the present invention, wherein the sdAb is specific for the IGFBP7, and may comprise the specific antibodies described above; and

(b) measuring the level of binding of the nanoconstruct, wherein an elevated level of binding, relative to normal tissue is indicative of the presence of a tumor expressing IGFBP7.

In the method as just described, the step of measuring is performed by magnetic resonance imaging or by fluorescence imaging.

The present invention also provides a method for determining the location of glioblastoma brain tumor cells in a patient pre-operatively, intra-operatively, and/or post-operatively, comprising administering a composition comprising the nanoconstruct of the present invention, wherein the sdAb is specific for the IGFBP7, and may comprise the specific antibodies described above and a pharmaceutically acceptable carrier to the patient, wherein the composition is administered in an amount sufficient to image glioblastoma cells in vivo; and

(d) a combination of (a), (b) or (c) above.

A method for in vitro detection or quantification of biological or chemical molecule in a sample is also provided by the present invention. The method comprises the steps of:

(a) contacting the sample with a nanoconstruct of the present invention, so as to form a complex between the molecule and the nanoconstruct; and (b) detecting or quantifying said complex formed.

The step of detecting or quantifying may be performed by magnetic resonance imaging, fluorescence imaging, or a combination thereof.

The superparamagnetic nanoparticles bioconjugated to sdAb can be used for molecular imaging and diagnosis of tumors. Brain tumors may be imaged or diagnosed by in vivo non- invasive imaging (MRI and optical) using sdAb against IGFBP7, rather than by invasive biopsy. Furthermore, the nanoconstruct formulation may be combined with an anti-cancer (chemotherapeutic) drug which will kill the brain tumor vessels and stop the tumor growth.

Similarly, disclosed formulations of nanoconstruct may be used for diagnosing, imaging and treatment or immunotreatment of other cancers characterized with increased angiogenesis and IGFBP7 production in brain vessels, for example colon cancer.

In one embodiment, an optical imaging contrast may be added to the formulation to create a bi-modal imaging contrast agent for dual application in optical and MRI imaging. In one embodiment, these bi-modal silica-coated superparamagnetic nanoparticles may be targeted to recognize brain tumor vasculature and are therefore applicable for diagnosis and molecular imaging of angiogenesis in brain tumors.

The following examples show the synthesis of iron oxide nanoparticles with a core size of about 5-10 rtm and coated with silica. These silica-coated iron oxide nanoparticles may then be functionalized with hydroxyl or amine groups, or both. PEG molecules may be attached to the surface of the silica-coated iron oxide nanoparticles to slow its renal clearance and uptake by reticuloendothelial system, and to optimize the blood circulation half-life. Single domain antibodies against IGFBP7 are conjugated to the silica-coated iron oxide nanoparticles while preserving antibody activity.

The sdAb targeted nanoparticles can further be functionalized with the near-infrared fluorescent probe, Cy5.5, to achieve bi-modal imaging contrast (MRI and optical) for in vivo imaging. In vivo optical imaging demonstrates that IGFBP7-targeted silica-coated iron oxide nanoparticles tagged with Cy5.5 (in contrast to non- targeted particles) can selectively detect intracranial glioblastoma tumor. Fluorescence microscopy confirms the presence of IGFBP7 targeted iron oxide nanoparticles in the brain tumor sections.

In MRI using 9.4T MRI phantoms, the functionalized iron oxide nanoparticles of the present invention show T2 shortening effect equal or better to commercially available iron oxide nanoparticles. In animal MRI studies using 9.4T system, IGFBP7 sdAb targeted iron oxide nanoparticles showed T2 shortening effect in the brain tumor region compared to normal contralateral brain.

As will be apparent to those skilled in the art, various modifications, adaptations and variations of the foregoing specific disclosure can be made without departing from the scope of the invention claimed herein.

EXAMPLES

The following examples are intended to exemplify embodiments of the present invention, and not to limit the claimed invention in any manner.

Example 1 - Synthesis of Iron Oxide nanoparticles

5 nm and 15 ran Fe₃O₄ NPs were synthesized according to literature (Di Marco et al, 2007; Bonnemain, 1998; Sonvico et al, 2005), with minor modifications. To synthesize 5 nm Fe₃O₄ nanoparticles (NPs), Fe(acac)3 (2mmol), 1 ,2-hexadecanediol (10 mmol), oleic acid (6 mmol), oleylamine (6 mmol), and phenyl ether (20 mL) were mixed and magnetically stirred in a three neck 100 ml glass flask under nitrogen protection. The mixture was heated to 200 ⁰C for 30 min and then, the temperature raised up to 265°C, this temperature being maintained for another 30 min. During the heating process, the color of the reaction mixture changes from brown to black. Then the mixture was cooled to room temperature and ethanol was added in order to precipitate the product, the solid phase being obtained by centrifugation. The final product, 5 nm Fe₃O₄ NPs, may be separated and purified from unreacted residues by dissolving/precipitating cycles using a hexane/ethanol solvent pair. The final solution can be prepared by redispersing the Fe₃O₄ wet solid into hexane. The powder form, which is used in X-ray diffraction (XRD) and magnetic characterization, is obtained by drying the wet solid in vacuum. To prepare 15 nm Fe₃O₄ NPs, Fe(CO)s 0.4 ml (3.04 mmol) was rapidly injected into a mixture containing 20 ml of octyl ether and 2.5 ml of oleic acid at 100⁰C. The resulting mixture was slowly heated to 285°C for 2 h. During the heating process, the color of the reaction mixture changed from brown, to colorless and then to black. The reaction mixture was then cooled down to room temperature. Both precipitation and purification procedures are similar to those for 5 nm Fe₃O₄ NPs discussed above.

Example 2 - Synthesis of iron oxide silica core/shell nanoparticles

The silica-coated Fe₃O₄ NPs were prepared according to a microemulsion route. Briefly, 0.24 g Polyoxyethylene nonylphenyl ether (0.56 mmol, Igepal CO-520) was dispersed in cyclohexane (4.2 mL) by sonication. Then, 300 μL cyclohexane solution of Fe₃O₄ solution

(0.8 mg/mL of cyclohexane) was added. The resulting mixture was vortexed, and ammonium hydroxide (29.4%, 35 μL) was added to form a transparent, brown solution of reverse microemulsion. Next, TEOS was added, and the reaction was stirred for 24 h at room temperature. When methanol was added into the reaction solution, Fe₃O₄/SiO₂ nanoparticles were precipitated. They were collected by a magnet, washed with methanol, and redispersed in ethanol.

Example 3 - Nanoparticle Characterization.

UV- visible spectra of the NP solution were acquired with a Perkin-Elmer Lamda 950 UV-vis- NIR spectrophotometer. Transmission electron microscopy (TEM) samples were prepared by depositing from dilute hexane solution of the NPs on carbon-coated copper grids. The images were taken using Hitachi S-4700 (30 KV). X-ray diffraction (XRD) patterns of the NPs were collected on a Siemens D-500 diffractometer under Co Ka radiation. Three types of magnetic measurements were performed by using a Quantum Design PPMS model 6000 magnetometer: DC major hysteresis loops (MHL), first order reversal curves (FORC) and zero-field cooled

(ZFC) magnetization curves. MHLs were recorded between -60 kOe and +60 kOe in uniform steps of 500 Oe and a constant temperature of 300 K. FORC measurements were performed according to standard procedure. The sample is first saturated to Hsat = 2 000Oe and then demagnetized to reversal fields Hri uniformly distributed in the interval [ ] 0, - Hsat. For each reversal point, the magnetic moment m of the sample is recorded at five points namely - Hri

-2ΔH, - Hri - ΔH, - Hri , - Hri + ΔH and - Hri +2ΔH wehre ΔH stands for the field resolution and was set to 50 Oe in all the measurements. The distribution of switching fields of the particles is then obtained by the second order mixed derivative of the dependence m(Hri,H). As for the ZFC magnetization curves, the samples were cooled down in zero field from room temperature to about 5 K. A DC magnetic field of 50 Oe was then applied and the sample heated from 10 to 300 K in uniform temperature steps of about 2 K. In all the magnetic measurements, the samples were prepared by filling gelatin capsules with magnetic powders, which was subsequently sealed with Parafilm.

Example 4 - Fluorescamine Assay

Fluorescamine (278.26 g/mol) is non-fluorescent until it reacts with primary amines. The reaction is completed in seconds, as un-reacted fluorescamine is rapidly converted to a non- fluorescent product by water. In a tube: add 200 μl of methanol + 50 μl (50 μg) of lmg/ml stock solution of fluorescamine + 50 μl of amine-silica nanoparticles. Mix well for a few minutes. Then add, 200 μl, to hydrolyze un-reacted fluorescamine. Read sample using fluorescent plate reader at sensitivity 125, excitation 390nm, and emission 470nm. For quantitative fluorescamine assay, generate a primary amine standard curve using a range of glycine concentrations to determine an appropriate linear range for fluorescent detection.

Figure 2 shows the scheme for a fluorescamine assay for detection of primary amine groups, along with the fluorescamine standard curve. Figure 3 shows a table with data estimating the number of primary amine groups in different batches of synthesized iron oxide nanoparticles.

Example 5 - PEGylation, Cy5.5 and single-domain antibody conjugation to silica coated iron oxide nanoparticles with surface primary amines

Iron oxide nanoparticles with surface primary amines in PBS, pH 7.4 were reacted with IOOOX molar excess of both maleimide PEG2000-NHSester and cy5.5-NHSester simultaneously under nitrogen gas for 2 hours at room temperature with mixing. Sample was purified using a centricon 10OkDa cutoff membrane to remove unreacted PEG linker and cy5.5 dye, then re-suspended in PBS, pH 7.4. For single domain antibody (sdAb) attachment, cysteine-sdAb was reacted with the maleimide-PEG-iron oxide conjugate for 2 hours under nitrogen gas at room temperature with mixing. The unreacted cysteine-sdAb was removed by ultracentrifugation using a centricon 10OkDa cutoff membrane, and the purified sample was re-suspended in PBS, pH 7.4.

Bioconjugation of nanoparticles with single-domain antibodies using amine chemistry: in some experiments IGFBP7-sdAb was reconstituted in MES buffer (MES 0.1 M, NaCl 0.5M, pH 6) using the aforementioned Amicon columns. To produce NHS-ester functionality on the sdAb, Sulfo-NHS and EDC were added to at 180- and 70-fold molar excess respectively and reacted for 30 min at room temperature. Subsequently, EDC is removed by centrifugation using Amicon columns.

Fluorescent labeling of iron oxide nanoparticles: Nanoparticles were solubilized in nanoparticle buffer (PBS, 0.5mM EDTA, pH 7.4) such that the final concentration of iron was lmg Fe/ml. Cy5.5-NHS ester (GE Healthcare, Amersham Place, UK) was added to the nanoparticle solution at 500-fold molar excess and reacted for 1 hour at room temperature. Unreacted dye was removed using Amicon ultra centrifugal units (Millipore, MA, USA) according to manufacturer's instructions. Labeling was optimized such that each sdAb has a dye/antibody ratio of approximately two.

Pharmacokinetic analysis of iron-based nanoparticles

Nanoparticles were injected via the tail vein in normal CD-I mice. Blood samples of 25 μl volume were collected by creating a small nick in the tail vein followed by collection of blood in a heparanized tube. Blood samples were collected at multiple time points at 5 min, 30 min, lhr, 1.5hr, 2h, 4h and 24 h. Samples were analyzed for labeled nanoparticles using a fluorescent plate reader with excitation 670nm and emission 690 nm and compared to a standard curve of a range of known concentrations of the labeled nanoparticles diluted in whole blood. Pharmacokinetic parameters were calculated using the WinNonlin pharmacokinetic software package (Pharsight Corporation, CA). A two-compartment, IV- Bolus model was selected for pharmacokinetic modeling, as it best represented the actual data. This model is described by the following equation: C(t) = A exp (- at) + B exp (- βt) where C{t) represents the concentration of agent in serum. A represents the zero time intercept of the alpha phase, B is the zero time intercept of the beta phase, αand β are disposition rate constants, α > β and ka is the absorption rate constant. The area under the serum concentration-time curve was calculated with the equation AUC o-∞ ⁼ D/V/Kio, D =dose given, V = apparent distribution volume and Kw = elimination rate constant. Total clearance was determined from the equation Cl/F = D/ AUC o-_∞.

To provide the example of silica-coated iron oxide nanoparticle targeting by single-domain antibodies to molecular-recognition sites in vivo, the nanoparticles were functionalized with a single-domain antibody against IGFBP7, a target selectively expressed in glioblastoma tumor vessels. sdAb (clone 4.43) against IGFBP7 was raised, purified and expressed as described in PCT Application No. PCT/CA2009/001460 and entitled Formulations of Targeting IGFBP7 or Diagnosis and Therapy of Cancer, its sequence is described herein as SEQ ID NO:5. Since the target is vascular, this formulation is applicable in imaging of intracranial glioblastoma tumors.

Figure 4 shows the effect of PEGylation and sdAb bioconjugation on the number of amine groups in iron oxide nanoparticles.

Figure 5 shows Pharmacokinetic analysis of Fe3O4 10 nm, Fe3O4 20 nm and FeCo 6 nm in mice. Table 1 also shows data supporting the graphs of Figure 5.

Table 1. Pharmacokinetics of various Iron-based Nanoparticles

[Clearance (ml/min) |θ.OOO362 |θ.OQQ582 |θ.OOQ324 |

Example 6 - Intracranial and xenograft models of U87MG deltaEGFRvIII glioblastoma in nude mice

U87MG deltaEGFRvIII is a highly malignant glioblastoma cell line derived from a human brain tumor and has been engineered to overexpress the EGFRvIII mutant receptor (kind gift from (Dr W.K. Cavenee, Ludwig Institute for Cancer Research, San Diego, CA, USA). Cells were maintained in DME medium containing 10% fetal bovine serum (FBS), penicillin/streptomycin and 200μg/ml of G418. Cells were grown at 37°C in a humidified atmosphere of 5% CO₂. Before cell implantation cells were harvested by trypsinization in EDTA/trypsin, washed in phosphate-buffered saline (PBS), and centrifuged at 20Og three times and cell density was determined. Cells were kept on ice until injection. Animal procedures were performed according to a protocol approved by Institution Animal Care Committee. Nude CD-I mice, obtained from Charles River Laboratories, Inc. (Cambridge, MA) at 4-6 weeks of age. The animals were housed in cages, in groups of 3 maintained on a 12-h light/dark schedule with a temperature of 22° C and a relative humidity of 50±5%. Food and water was available ad libitum. For intracerebral stereotactic implantation of U87MG deltaEGFRvIII, mice underwent isofluorane deep anesthesia and the scalp was swabbed with iodine and alcohol. The skin was incised and a 1 Oμl syringe was used to inoculate 5 μl of 5 x 10 U87MG deltaEGFRvIII cell suspension into the corpus striatum in the right hemisphere (3.0 mm deep; 1 mm anterior and 2 mm lateral to the bregma). The skin was sutured with three knots, followed by application of tissue glue. The animals developed solid tumors for 10 days before experiment started.

Example 7 - In vivo near-infrared fluorescence imaging

Mice were anesthetized with 1.5% isoflurane administered with a face mask. Single domain antibodies or conventional antibodies (each at 80 nmol/kg) bioconjugated to Unilamellar vesicles carrying 40% Gd-DTPA-BOA and labeled with the near-infrared fluorescent probe, Cy5.5, were administered via tail vein using a 0.5-ml insulin syringe with a 27-gauge fixed needle (vehicle, 0.9% saline; injection volume, 120 ul). Mice (n = 5-10 per group) were imaged using small animal time-domain explore Optix pre-clinical imager MX2 (Advanced Research Technologies, QC) prior to and at different time intervals (4 h, 8h and 24 h) after nanoparticles injections. In all imaging experiments, a 670-nm pulsed laser diode with a repetition frequency of 80 MHz and a time resolution of 12 ps was used for excitation. The fluorescence emission at 700 nm was collected by a highly sensitive time-correlated single photon counting system and detected through a fast photomultiplier tube offset by 3 mm for diffuse optical topography reconstruction. Each animal was positioned prone on a plate that was then placed on a heated base (36°C) in the imaging system. A two-dimensional scanning region (ROI) encompassing the whole body or the head was selected via a top-reviewing realtime digital camera. The optimal elevation of the animal was verified via a side viewing digital camera. The animal was then automatically moved into the imaging chamber where laser excitation beam controlled by galvomirrors was moved over the selected ROI. Laser power and counting time per pixel were optimized at 30 μW and 0.5 s, respectively and these values were maintained constant during the entire experiment. The raster scan interval of 1 mm was held constant during the acquisition of each frame; 1024 points were scanned for each ROI. The data were recorded as temporal point-spread functions (TPSF) and the images were reconstructed as fluorescence intensity, and fluorescence concentration maps. Following the last imaging session, mice were sacrificed by perfusion, organs were removed, placed into an imaging system and imaged ex vivo as described above.

eXplore Optix OptiView software program (Advanced Research Technologies, QC) was used to estimate fluorescence intensity; 3D reconstruction software by Advanced Research Technologies (Montreal, QC) was used for reconstruction of topography and optical sectioning.

Figure 5 shows pharmacokinetics of various iron-based nanoparticles (Fe₃O₄ 10 nm, Fe₃O₄ 20 nm, and FeCo 6 nm). Figure 6 shows biodistribution of silica-coated iron-oxide nanoparticle labeled with the near- infrared probe, Cy5.5, in normal CDl mice.

Example 8 - Fluorescence microscopy

After completion of the in vivo tumor growth, animals were perfused with heparinized saline, organs and tumor were disected and then frozen on dry ice and stored. Mouse tissues were embedded in Tissue-Tek freezing medium (Miles Laboratories, Elkhart, IN) and sectioned on a cryostat (Jung CM3000; Leica, Richmond Hill, ON, Canada) at lOμm thickness, then mounted on Superfrost Plus microscope slides (Fisher Scientific, Nepean, ON, Canada).

Slides were stored at -80⁰C until fluorescence microscopy studies. Frozen mouse brain tumor sections were thawed for a few seconds then incubated in methanol for 10 min at room temperature. Slides were rinsed with 0.2 M PBS (pH 7.3), followed by incubation with 5% goat serum in PBS for 1 hour with 0.1% triton-X 100 at room temperature. After blocking, slides were incubated with DAPI to visualize nuclei. CD31 antibody followed by Alexa 488 anti-rabbit secondary antibody was used to visualize brain vessels. Coverslips were mounted using DAKO fluorescent mounting media and were allowed to harden at 4⁰C overnight and then visualized under fluorescent microscope. Sections were then visualized under an

Olympus 1X81 inverted motorized microscope (Markham, Ontario, Canada). The software used to acquire images is In Vivo and ImagePro 6.2 to analyze and correct for background.

Figure 17-19 shows fluorescence microscopy of brain rumor and contralateral sections to detect Cy5.5 (red) 72 h after the injection of non-targeted or IGFBP7 sdAb-targeted superparamagnetic iron oxide nanoparticles. Example 9 - MRI measurement

The T₂ relaxivity properties of iron oxide nanoparticle samples (FeCo /Au: 3.10%Co and

3.25%Fe weight (size: 10 nm); Fe₃(VSiO₂ both PEGylated and non-PEGylated, Ocean

Nanoparticles - 10 nm iron oxide nanocrystal in water with amine surface (NH2) core and 3-4 nm coating with amino -derivatized Genovis FeOdot PEG- Amine 15 nm.) were acquired.

These samples were scanned using a quadrature volume RF coil and a Bruker Biospec Avance

II MRI system with a 9.4T magnet and Paravision 4 software. T₂ maps of cross-sectional slices through the tubes were acquired using a MSME pulse sequence with 128 echo times. T₂ for each sample was calculated using a single exponential fitting of the echoes curve. (Paravision 4 software, Bruker).

Figure 20 shows graphically the effect of silica-coated iron oxide nanoparticles on T2 relaxation measured at 9.4T MRI.

Example 10: In vivo MRI measurements using IGFBP7 sdAb tarfieted superparamagnetic iron oxide nanoparticles.

The MR imaging sessions were carried out 12 days after cell inoculation. A 9.4T/21cm horizontal bore magnet (Magnex, UK) with a Biospec console (Bruker, Germany) was used. Data acquisition was gated with the respiratory cycle. A volume (3 cm diam, 2.5 cm long) radio-frequency coil was placed over the animal's head covering the ROI. T₂ images were acquired from the tumor region and from normal tissue. Axial slices were positioned within the tumor. A multislice, multiecho sequence was used with TR = 5000 ms, 16 echoes, 10 ms apart each, first echo at 10 ms, FOV = 3 ^χ 3 cm, matrix size 256 x 256 and slice thickness of 1 mm. T₂ values of the tumor tissues were measured using a single exponential fitting of the echo train from ROIs (Marevisi, NRC, Canada).

Figures 21-23 show representative in vivo MRI images and graphic plots of the T2 shortening effect of IGFBP7 sdAb targeted and non-targeted superparamagnetic nanoparticles in intracranial tumors in nude mice. References

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PCT/CA2009/001460 entitled "Formulations Targetting IGFBP7 for Diagnosis and Therapy of Cancer"

Claims

We claim:

1. A nanoconstruct comprising:

(a) a superparamagnetic nanoparticle;

(c) a near-infrared fluorophore.

2. The nanoconstruct of claim 1, wherein the superparamagnetic nanoparticle further comprises a shell or coating.

3. The nanoconstruct of claim 1 or 2, wherein the superparamagnetic nanoparticle is functionalized with polyethylene glycol.

4. The nanoconstruct of any one of claims 1 to 3, wherein the superparamagnetic nanoparticle comprises iron oxide doped with iron cobalt (FeCo).

5. The nanoconstruct of any one of claims 1 to 4, wherein the single domain antibody is linked to the superparamagnetic nanoparticle core/shell by a functional group comprising a carboxylate, a sulfonate, a phosphate, an amine, or any combination thereof.

6. The nanoconstruct of any one of claims 1 to 5, wherein the single domain antibody selectively binds Insulin-like Growth Factor Binding Protein 7 (IGFBP7).

7. The nanoconstruct of one of claims 1 to 5 wherein the single domain antibody selectively binds a receptor expressed by tumor endothelial cells.

8. The nanoconstruct of claim 6, wherein the single domain antibody comprises complementarity determining region (CDR) sequences RTSRRYAM or RTFSRLAM (CDRl), GISRSGDGTHYAYSV (CDR2), and AAARTAFYYYGNDYNY (CDR3).

9. The nanoconstruct of claim 6, wherein the single domain antibody comprises SEQ ID NO. 5 or SEQ ID NO. 6, or a sequence substantially identical thereto.

10. A method for in vivo imaging of cells or tissues in a mammal, the method comprising the steps of:

(a) administering to the mammal a composition comprising the nanoconstruct of any one of claims 1 to 5, wherein the single domain antibody (sdAb) is specific for a selected receptor;

(b) waiting a time sufficient to allow the sdAb to bind to the selected receptor; and (c) imaging the cells or tissues with a non-invasive imaging technique whose resolution is enhanced by the presence of the particles on or within the cells.

11. The method of claim 10, wherein the imaging technique is magnetic resonance imaging, optical imaging, or a combination thereof.

12. The method of claim 10 or 11, wherein the selected receptor is specifically expressed by tumor endothelial cells.

13. The method of claim 9 or 10, wherein the selected receptor is IGFBP7.

14. The method of claim 13, wherein the single domain antibody comprises complementarity determining region (CDR) sequences RTSRRYAM or RTFSRLAM (CDRl), GISRSGDGTHYAYSV (CDR2), and AAARTAFYYYGNDYNY (CDR3).

15. The method of claim 13, wherein the single domain antibody comprises SEQ ID NO. 5 or SEQ ID NO. 6, or a sequence substantially identical thereto.

16. The method of any one of claims 10 to 15, wherein one or more tumors, metastases, vascularized malignant cell clusters, or individual malignant cells are imaged, selected from the group consisting of brain cancer, colon cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, endometrial cancer, oral cancer, liver cancer, and renal cancer or any other cancer.

17. A method for detecting glioblastoma in a patient, comprising:

(a) contacting a tissue of interest with the nanoconstruct of any one of claims 1 to 8; and (b) measuring the level of binding of the nanoconstruct, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.

18. A method of imaging angiogenesis in a mammal, comprising:

(a) introducing into the mammal the nanoconstruct of claim 7;

(b) permitting the nanoconstructs to bind to angiogenic tumor vessels; and (c) imaging the angiogenic tumor vessels using magnetic resonance imaging.

19. A method for detecting a tissue expressing IGFBP7, comprising:

(a) contacting a tissue of interest with the nanoconstruct of any one of claims 6, 8 or 9; and

IGFBP7.

20. The method of claim 19, wherein the step of measuring is performed by magnetic resonance imaging

21. The method of claim 19, wherein the step of measuring is performed by fluorescence imaging.

22. A method for determining the location of glioblastoma brain tumor cells in a patient pre- operatively, intra-operatively, and/or post-operatively, comprising the step of administering a composition comprising the nanoconstruct of any one of claims 6, 8 or 9 and a pharmaceutically acceptable carrier to the patient, wherein the composition is administered in an amount sufficient to image glioblastoma cells in vivo; and (a) pre-operatively measuring the level of binding of nanoconstruct by magnetic resonance imaging to determine the location of glioblastoma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of glioblastoma cells;

(d) a combination of (a), (b) or (c) above.

23. A method for in vitro detection or quantification of biological or chemical molecule in a sample, the method comprising the steps of:

(a) contacting the sample with the nanoconstruct of any one of claims 1 to 5, so as to form a complex between the molecule and the nanoconstruct; and

(b) detecting or quantifying said complex formed.

24. The method of claim 23, wherein the step of detecting or quantifying is performed by magnetic resonance imaging, fluorescence imaging, or a combination thereof.