WO2015057920A1 - Sr-b1 companion diagnostic test - Google Patents

Abstract

A diagnostic test to detect the presence and/or quantitative amount of SR-B1 receptor on normal and diseased tissues.

Description

SR-B1 COMPANION DIAGNOSTIC TEST

FIELD OF THE INVENTION

[0001] The present invention relates to clinical diagnostic tests used to detect cellular moieties. In particular, the presence invention describes a clinical diagnostic test used to detect the absolute quantity, or the relative abundance of cellular receptors, cell surface molecules and/or cellular moieties that can then be targeted for therapeutic intervention using a targeted drug delivery vehicle directed at those same receptors, cell surface molecules, or moieties.

BACKGROUND OF THE INVENTION

[0002] Normal, diseased or cancerous cells can express a variety of different cellular receptors and unique molecules on their surfaces. These cellular receptors perform various functions for the cells and may be used to modulate the uptake and excretion of different signaling, nutrient and building-block molecules from the cell. The presence, absolute quantity or relative abundance of unique cellular receptors or cell surface molecules can be used to identify and target specific cell populations or tissues for therapeutic intervention. Antibodies, proteins, peptides or other ligands can bind specifically to these cellular receptors and can then be used to measure the abundance of a particular receptor or molecule in patient tissue samples.

[0003] Clinical diagnostic tests are used to guide medical diagnosis and treatment decisions for many types of human diseases. Recently, the term "companion diagnostic" has been used to describe a type of clinical test done on a patient (or patient tissue sample) that is then used to guide the selection and use of a specific type of therapeutic treatment or drug. Companion diagnostics tests measure a specific characteristic of the patient, or a characteristic of a patient's tissues, cells, or other patient sample that is relevant to the selection or dosing of a specific therapeutic treatment. Thus, these types of tests are specifically paired with a particular treatment or drug, thereby acquiring the distinction as a "companion" test.

[0004] A nanoparticle may be chemically modified or engineered by integrating an antibody, protein, peptide, or other targeting ligand onto the particle's surface. These engineered nanoparticles are increasingly being used to deliver therapeutic drugs for treatment of a variety of diseases. For example, in some instances a therapeutic nanoparticle is engineered to include a ligand on the particle's surface, wherein a diseased cell comprises a receptor or cell surface molecule to which the ligand binds. Such approaches have been used to develop various currently available therapies.

[0005] Engineered nanoparticles may also be used to pre- screen a patient for a disease or to determine the most effective form of treatment. Prescreening may be accomplished by providing a companion diagnostic test that includes a plurality of engineered nanoparticles, each nanoparticle being engineered to include a target ligand for a receptor or cell surface molecule of a diseased cell. A patient's blood or other tissue sample may be used with the companion diagnostic test to determine an effective or optimal treatment for the patient.

[0006] The process for developing companion diagnostic tests requires rigorous research to provide effective engineered nanoparticles for a desired indication. Thus, while companion diagnostic tests are currently available for various known indications, challenges still exist for other indications. Thus, there is a need in the art to provide companion diagnostic tests for additional indications of interest. Such a companion diagnostic test is disclosed herein.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention describes the use of diagnostic tests to detect the presence and/or quantitative amount of specific cell surface receptors or cell surface moieties. In particular, the present invention provides a companion diagnostic test that quantifies the levels of SR-Bl receptor in cancerous tissue samples. The results of this diagnostic test are then used to select patients for drug therapy using a drug delivery vehicle that specifically targets the SR-Bl receptor or other cell surface moiety in order to deliver attached or encapsulated drug to the diseased cells.

[0008] Some implementations of the present invention include a companion diagnostic test comprising an engineered nanoparticle having an SR-Bl ligand expressed or presented on the surface of the nanoparticle. Where a diseased tissue expresses the SR-Bl receptor, the engineered nanoparticles bind to the SR-Bl receptor and provide a detectable signal. In some instances, the test comprises one or more agents or reagents that provide a detectable signal to the user. For example, in some instances the test comprises radioactive, colorimetric, fluorescence, or other tracer molecules to allow the detection and quantification of the target receptor. The level or intensity of the detectable signal provides the user with information that may be used to identify the type of diseased cell or tissue, select a form of treatment, and/or select a therapeutic agent as part of a therapy to treat the diseased cells.

[0009] The companion diagnostic test may be administered using a sample of diseased tissue taken from a patient. In some instances, a companion test utilizes tissue samples taken from the patient as part of a biopsy. In other instances, a companion test utilizes tissue samples taken as part of a surgical intervention or excision. The companion test may also be performed on blood samples taken from a patient.

[0010] In some implementations, the companion diagnostic test is performed in-vivo, thereby negating the need to remove cells, blood, tissues or organs from the patient. Such in- vivo tests could be done using any type of visualization technology such as x-ray, CT scanning, PET scanning, or other technique capable of detecting a signal from the agents or reagents of the test. In some instances, a signal from the test is used to detect and/or quantify the presence and amount of target receptor or cell surface moiety of the diseased tissue.

[0011] Following detection and/or quantification of the receptor, the present invention further provides a method whereby the patient receives one or more therapeutic treatments using a drug delivery system that specifically directs a therapeutic drug to the receptor or cell surface moiety, as determined by the results of the companion diagnostic test.

DETAILED DESCRIPTION OF THE INVENTION

[0012] While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined by the claims. The present invention is not limited to the methods and materials described herein but include any methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. In the event that one or more of the incorporated literature references, patents or similar materials differ from or contradict this application, including but not limited to defined terms, term usage, described techniques or the like, this application controls.

[0013] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0015] It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

[0016] Furthermore, the following terms shall have the definitions set out below.

[0017] As used herein, the term "nanoparticle" refers broadly to an ultrafine particle sized between 1 and 100 nanometers.

[0018] As used herein, the term "SR-B1" is an abbreviation for scavenger receptor class B member 1. SB-B1 is a protein that in humans is encoded by the SCARB1 gene and functions as a receptor for high-density lipoprotein. SR-B1 is an integral membrane protein found in numerous cell types/tissues, including the liver and adrenal. It is best known for its role in facilitating the uptake of cholesteryl esters from high-density lipoproteins in the liver. This process drives the movement of cholesterol from peripheral tissues towards the liver for excretion. This movement of cholesterol is known as reverse cholesterol transport and is a protective mechanism against the development of atherosclerosis, which is the principal cause of heart disease and stroke.

[0019] As used herein, the term "scavenger receptor(s)" refers broadly to a group of receptors that recognize modified low-density lipoprotein (LDL) by oxidation and acetylation. Class B scavenger receptors are identified as oxidized LDL receptors and are typically concentrated in the caveolae plasma membrane domain.

[0020] As used herein, the term "peptide" refers broadly to a sequence of two or more amino acids joined together by peptide bonds. It should be understood that this term does not connote a specific length of a polymer of amino acids, nor is it intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. [0021] The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L- amino acid residue, as long as the desired functional is retained by the peptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. Ac refers to the free carboxy group present at the carboxy terminus of a peptide.

[0022] It should be appreciated that also within the scope of the present invention are nucleic acid sequences encoding the peptides of the present invention, which code for a polypeptide having the same amino acid sequence as the sequences disclosed herein, but which are degenerate to the nucleic acids disclosed herein. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid.

[0023] The present invention further includes disulfide peptides that have been substituted with various amino acid conjuates. For example, some disulfide peptides of the present invention include Dab (diaminobutyric acid), Dap (diaminopimelic acid), Pen (penicillamine), Sar (sarcosine), Cit (citrulline), Cav (Cavanine), 4-guan (4-guanidino), and various N-methylated amino acids. One having skill in the art will appreciate that additional substitutions may be made to achieve similar desired results, and that such substitutions are within the teaching and spirit of the present invention.

[0024] Some embodiments of the invention comprise a diagnostic test that quantifies the abundance of SR-B1 receptor in tissue taken from a patient. In some instances, the tissue is removed as part of a biopsy procedure. In other instances, the tissue is removed as part of a surgical intervention. The diagnostic test may also be used with blood cells taken from the patient as part of phlebotomy procedure.

[0025] The diagnostic tests described herein will be used to quantify the amount and the relative abundance of the cell surface receptor known as SR-B1 in cancer tissue and normal tissue samples taken from patients diagnosed with cancer. The purpose of performing this test is to use the information gained from the test to determine whether the patient is a good candidate for cancer drug treatments that direct cancer killing drugs specifically at cells that express SR-B1 receptor.

[0026] In some instances, the present invention comprises a diagnostic test that further includes a targeted nanoparticle, such as those described in United States Patent Application 13/961,296 filed August 7, 2013, which is incorporated herein by reference. For example, in one embodiment a diagnostic test is provided comprising an enzyme or molecule (ie. fluorescein) that is readily detectable at low levels. In other instances, a radioactive tracer is further incorporated in a nanoparticle.

[0027] In some instances, the test is administered through a binding assay between the SR-Bl targeting molecules and tissue samples taken from the patient. In some embodiments, normal and diseased or diseased tissue is taken from a patient as control and test samples, respectively. The tissue samples are prepared and then exposed to the SR-Bl targeting molecules for a desired period of time.

[0028] Following the binding assay, the tissues are rinsed and the abundance of SR-

Bl receptor present in the diseased and normal tissue samples is then measured. In some instances, the SR-Bl targeting molecule is directly detectable by scintillation counting, fluorescence, colorometric visualization or another direct method of detection. For example, in some embodiments the SR-Bl targeting molecule radio-labeled or includes a fluorescence tag. In other instances, the SR-Bl targeting molecule is further bound by a secondary ligand that is capable of being detected by a known diagnostic assay or procedure.

[0029] The SR-B 1 targeting molecules may include any protein, peptide, antibody or other molecule that specifically binds to SR-Bl receptor, and that can be quantified, may be used as a diagnostic ligand for this test. The sample tissues are exposed to the diagnostic ligand in a binding assay, as described above. Typically, the diagnostic protein, peptide, antibody or other SR-B 1 receptor binding moiety will be linked to an enzyme or molecule (ie. fluorescein) that can be readily detected at very low levels. Radioactive tracers may also be linked to the diagnostic protein, peptide, antibody or other moiety and can also serve as sensitive markers for quantitation of SR-Bl abundance.

[0030] As a non-limiting example of a diagnostic test in accordance with the present invention, blood or tissue samples (cancerous and non-cancerous) may be obtained from a patient and washed or incubated with a solution containing an SR-Bl binding moiety or ligand (for example, a peptide that binds to the SR-B 1 binding region or an SR-B 1 targeting antibody). After the wash or incubation period, the unbound ligand is washed away with buffer, and then the amount of bound protein, peptide, antibody or other SR-Bl binding moiety is quantified to indicate absolute or relative amount of SR-Bl expression on the tissue to determine the tissue's appropriateness for treatment or the tissue's response to treatment. In some instances, a binding affinity between the SR-Bl ligand and the tissue may provide information concerning the tumor type or stage. For example, SR-Bl receptor may be expressed at a high level in various stages of many types of cancers, including but not limited to breast, ovarian, colorectal, prostate, pancreatic, bone, liver, lung, etc. A decision to treat a given patient using SR-Bl targeting drug nanoparticles may be based on the results of the diagnostic test described here and on other factors. A decision to treat may be based on the absolute or average number of SR-Bl receptors present on a patients cancer cells, or may be based on a ratio of SR-Bl receptors present on a patient's cancer cells compared to those present on that patient's normal cells. Thus, a decision to test a patient's cancer tissue for SR-Bl abundance may be based on the type of cancer identified in a given patient, or on the site of the cancer seen in a given patient.

[0031] The ratio of SR-Bl receptor in the normal and diseased tissue samples serve as an indicator of the likelihood that these disease targeting drug nanoparticles will be effective in treating the patient's disease. For example, where the results of the binding assay indicate an increased expression of SR-Bl receptor on the diseased tissue as compared to the normal tissue, these results may indicate that the diseased tissue will have a greater likelihood of successful treatment by therapeutic agents that are carried in SR-Bl targeting nanoparticle micelles.

[0032] In some embodiments, the number of detected SR-Bl receptors on the tissue sample provides the user with an indication the success for certain types of therapies. For example, in some instances the detection of SR-Bl receptors above a pre-determined threshold indicates that the diseased tissue may be receptive to treatment using a therapeutic agent that is engineered to bind SR-Bl receptors. In some instances, this minimum threshold of detected SR-Bl receptor per Z micrograms of tumor tissue is from approximately XX/ppm to approximately YY/ppm. For some embodiments, a decision to treat a given patient with SR-Bl targeting drug nanoparticles is based on the relative abundance (ratio) of SR-Bl expressed on that patient's cancer cells compared to his/her normal (non-malignant) cells. In some instances, a preferred relative abundance or ratio of diseased tissue to normal tissue SR- Bl levels is from approximately 2: 1 to 100,000: 1, from approximately 100: 1 to 25,000: 1, from approximately 1000: 1 to 10,000: 1, and in a preferred embodiment a ratio of approximately 5000: 1. In some embodiments, a cancer patient may be selected for drug treatment using a drug containing nanoparticle that specifically targets the SR-Bl receptor if the ratio of SR-Bl receptor on that patient's cancer tissue compared to their normal tissue is 2: 1 or greater.

[0033] If the detected levels are within the range, the patient may be treated with one or more appropriate anti-cancer therapies. In general, the greater the difference in SR-Bl receptor abundance between the diseased tissue and the normal tissue of the patient, the greater the selectivity and specificity will be for the therapy targeted at those receptors.

[0034] The measurement of SR-Bl receptor in patient's tissues may be done by any compatible method. For example, in some instances patient tissue is obtaining biopsy of tumor tissue. In some embodiments, immunohistochemistry is used on sections of the biopsied tissue to measure the abundance of SR-B l receptor. In other embodiments, the level of SR-Bl receptor is determined by binding a labeled protein or peptide to the biopsy sample. In these embodiments, the abundance of SR-Bl receptor is determined via peptide detection, such as by mass spectrometrometery or HPLC. Similarly, in- vivo diagnostic techniques such as CT scanning or PET scanning may be utilized using an injected or ingested marker directed at SR-Bl receptor. These methods may be desirable as non-invasive alternatives, or for situations in which it is unsafe to obtain a biopsy of the diseased tissue.

[0035] Following identification and quantification of the SR-Bl receptor for the diseased tissue, some embodiments of the present invention provide a method of treatment whereby the patient is given a therapeutic agent that is directed to the diseased tissue via the SR-Bl receptor. Various non-limiting methods for directing drug therapy at or through the SR-Bl receptor are described in U.S. Patent Application 61/682,057, which is incorporated herein in its entirety.

[0036] Some embodiments of the present invention further comprise a diagnostic test to identify cells that express SR-Bl receptor in tissue samples from patients for the purpose of introducing any kind of therapeutic or cell modulating nucleic acid into those cells via these receptors.

[0037] EXAMPLES

[0038] EXAMPLE #2: In-Vitro Diagnostic: Histochemistry

[0039] Diseased and/or normal tissue is taken from a patient either by a biopsy procedure or through a tissue excision procedure or other surgery.

[0040] A portion of the excised diseased and/or normal tissue is either immediately frozen or fixed and prepared for sectioning using any of a variety of fixation techniques consistent with the preservation of cellular morphology, cellular structures and immunological integrity of biological molecules in the tissue sample.

[0041] Fixed tissue sample is then embedded in a sectioning media (typically paraffin) and thin slide sections are cut. [0042] Frozen tissue samples are kept frozen for and thin slide sections are cut from the frozen tissue; typically slide sections taken from frozen tissue samples are next fixed in place on the slide using fixation techniques consistent with the preservation of cellular morphology, cellular structures and immunological integrity of biological molecules in the tissue sample.

[0043] Slide mounted thin sections are then treated to block the non-specific binding of diagnostic antibodies, peptides or other ligands to the tissue sections.

[0044] The tissue sections are next incubated with an antibody, peptide or ligand that binds specifically to the same cell surface receptor, cell surface molecule or moiety to be targeted by the therapeutic nanoparticle. The targeting antibody, peptide or ligand may be identical to the targeting moiety used on the nanoparticle, or may be different but have been previously shown to target (identify) the same cellular receptor, cell surface molecule or moiety that is used to target the therapeutic nanoparticle.

[0045] A "reporter" or "tracer" molecule may either have been linked directly to the antibody, peptide or ligand or that reporter molecule may be bound to the specific targeting molecule in a subsequent incubation step.

[0046] Reporter or tracer molecules include radioisotopes, fluorescein compounds, peroxidase compounds or other molecules designed to facilitate the quantitation or visualization of cellular receptors or other cellular structures that might be utilized as targets for directed nanoparticle therapies.

[0047] After the tissue sections have been allowed to bind the specific targeting moiety, and (if necessary) allowed to then bind the reporter molecule, the sections are rinsed thoroughly and the quantity of bound targeting moiety is measured.

[0048] This diagnostic test may be used to look for the simple presence of the target receptor or cell surface molecule; this test may be used to quantify the absolute number of target receptors present on a patient's cells; this test may be used to quantify the ratio of target receptors on diseased tissue compared to normal tissue.

Examples of reagents that may be used for this histochemical analysis of SR-B1 presence and quantity in patient tissue samples include commercially available anti-SR-Bl receptor antibodies (LifeSpan Biosciences, Inc., SR-B1 Antibody LS-C2880, Novus Biologicals, SR- Bl Antibody NB400-113, Abeam, Anti-SR-Bl Antibody EP1556Y), and SR-B1 receptor binding peptides such as those previously described in U.S. Provisional Patent Application 61/682,057. [0049] EXAMPLE #2: In-Vitro Diagnostic: Western or Dot Blot Analysis

[0050] Western blot and dot blot analyses are well established techniques used to detect the presence and quantity of specific proteins, glycoproteins or receptors in samples of homogenized cells or tissues (Burnette, N., Anal Biochem 1981;112: 195-203).

[0051] Briefly, in this application of Western or dot blotting techniques, diseased and/or normal tissue is taken from a patient either by a biopsy procedure or through a tissue excision procedure or other surgery.

[0052] Known amounts of those tissue samples are then homogenized; typically on ice and in the presence of a cocktail of protease inhibitors.

[0053] The homogenized cell or tissue sample is then transferred to a support membrane (typically nitrocellulose) by filtration or direct application; the homogenized tissue sample may be first separated electrophoretically and then transferred to the support membrane. This transfer of the cell or tissue homogenate results in the permanent binding of cellular proteins, glycoproteins and receptors to the support membrane.

[0054] The membrane is subsequently treated with anti-SR-B l antibody that specifically binds to any SR-B1 receptor that has been bound to the support membrane. Any excess anti-SR-Bl antibody is washed away, and the amount of antibody remaining bound to the support membrane is then analyzed by colorimetric, florescent or radiologic techniques, yielding a quantitative measure of the amount of SR-B1 receptor present in the diseased and normal tissue samples.

[0055] The reagents that may be used to perform this quantitative Western or dot blot analysis are the same as those described in Example Method #1.

[0056] EXAMPLE 3: In Vivo Diagnostic

[0057] The presence and/or quantity of a specific cellular receptor or cell surface moiety to be targeted for therapeutic intervention with a drug containing nanoparticle may be assessed in-vivo using radioisotope or other labeling in conjunction with a variety of different tissue scanning technologies such as PET scanning, CAT scanning, NMR scanning and others.

[0058] The underlying principle for identifying and/or quantifying a specific cellular receptor or cell surface moiety in-vivo is the same as for in-vitro techniques, and requires the specific binding of an antibody, peptide or other ligand to the receptor of interest, then measuring or visualizing the bound antibody with the help of a reporter molecule that is radioactive itself, is "opaque" to scanning radiation, or in some other way can be measured or visualized while bound to tissue within a patient's body.

[0059] In-vivo diagnostic methods requires that the targeting antibody, peptide or other ligand be administered to the patient (typically intravenously) and then allowed to bind to the specific target tissue or cells prior to carrying out the scanning or measuring procedure.

[0060] As described for the in-vitro diagnostic technique, the targeting antibody, peptide or ligand may be identical to the targeting moiety used on the nanoparticle, or may be different but have been previously shown to target (identify) the same cellular receptor, cell surface molecule or moiety that is used to target the therapeutic nanoparticle.

Claims

1. A method for performing a clinical diagnostic measurement to assess the presence or abundance of a specific cellular receptor or cell surface moiety on a patient's cells, blood, tissues or organs that is then used to select, guide, or inform subsequent therapeutic intervention with a pharmaceutical agent that is targeted at that same cellular receptor or cell surface moiety.

2. The method of claim 1, wherein the specific cellular receptor or cell surface moiety is the Scavenger Receptor B-l (SR-B1).

3. The method of claim 1, wherein the pharmaceutical agent is a drug containing nanoparticle such as described in U.S. Provisional Patent Application 61/682,057.

4. The method of claim 1, wherein the presence or abundance of the specific cellular receptor or cell surface moiety is assessed using a radioactive tracer.

5. The method of claim 1, wherein the presence or abundance of the specific cellular receptor or cell surface moiety is assessed using a colorimetric tracer.

6. The method of claim 1, wherein the presence or abundance of the specific cellular receptor or cell surface moiety is assessed using Computerized Tomography (CT) scanning.

7. The method of claim 1, wherein the presence or abundance of the specific cellular receptor or cell surface moiety is assessed using Positron Emission Tomography (PET) scanning.

8. The method of claim 1, wherein the presence or abundance of the specific cellular receptor or cell surface moiety is assessed using an antibody directed against the specific cellular receptor or cell surface moiety. The method of claim 1, wherein the presence or abundance of the specific cellular receptor or cell surface moiety is assessed using a peptide or protein that binds to the specific cellular receptor or cell surface moiety.