Patent Application
20130064762
Cancer is a heterogeneous collection of diseases. Within a given cancer type, each individual tumor is defined by a distinct set of mutations resulting in different molecular profiles that could elicit an immune response. A neoplastic cell may have many mutations throughout its DNA (Lobe et al., 2003). Further, a particular tumor continues to mutate over time, which causes an evolving and diversifying phenotype. This variability complicates diagnosis and therapy.
It has long been thought that the immune system suppresses developing tumors (Ehrlich 1909). This is consistent with the observation that immunosuppressed transplant recipients have higher rates of non-viral associated tumors than the general population (Birkeland 1995; Penn 1995; Penn 1996; Pam 1995). Transformation leads to tumorigenesis when mutations in the cells allow them to escape the effects of the immune system (Dunn et al., 2002). Although the immune system cannot kill the tumor, it may still recognize the transitioning tissue. Autoantibodies can be generated in response to molecules that are associated with tumors. Various groups have identified a number of tumor-associated autoantibodies in the hopes of utilizing them as biomarkers, prognostic factors, and indicators of tumor recurrence. Autoantibodies to NY-ESO-1 have been identified in the sera of patients with esophageal, lung, liver, breast, thyroid, prostate, and colorectal cancers (Akcakanat et al., 2004; Chapman et al., 2008; Fosså et al., 2004; Korangy et al., 2004; Maio et al., 2003; Nakamura et al., 2006; Stockert et al., 1998; Türeci et al., 2006). The sensitivity and specificity of tumor detection in the sera is increased by testing for the presence of a panel of antibodies, rather than a single antibody (Kobold et al., 2010); however, this is still not sufficient for diagnosis in many tumor types. For example, probing for a single autoantibody in the serum gives a positive result in 10-20% of patients with hepatocellular carcinoma. The detection increases to 66% with a panel of ten autoantibodies (Zang and Tan 2010). If a panel is more useful than a single antibody, the entire collection of autoantibodies might be even more effective in detecting tumors.
There is a need for methodology that improves early detection of neoplasia, as well as detection of metastases, unrecognized foci and transformed cells at surgical margins. Such methodology would optimally be able to continue to track tumors even as they change as a consequence of mutagenesis. Early detection increases the possibility of treating a tumor before the development of metastasis, improving patient survival (Lobe et al, 2003). The present invention utilizes autoantibodies to reliably identify pre-neoplastic and neoplastic tissue with high sensitivity and distinguish these from normal tissue. Genetic instability in neoplasias make them a moving target for detection and therapeutics. Numerous therapeutics that are initially efficacious against neoplasia fail as the tumor mutates and changes. The use of the broad spectra of antibodies generated against a neoplasia ensures that individual, or even bulk changes in the tumor, will not allow the neoplasia to escape detection and treatment. Additionally, the immune system has the neoplasia under continuous surveillance and thus able to detect and respond even to large scale changes. This invention can be used for diagnosis and therapy throughout tumorigenesis.
This invention is based, at least in part, on the discovery of a method for identifying pre-neoplastic or neoplastic cells or tissue of a mammal by utilizing autoantibodies that detect the pre-neoplastic or neoplastic cells, tissue, or associated antigen. In related embodiments, the invention involves methods of killing pre-neoplastic or neoplastic cells or tissue by either binding toxins to autoantibodies that detect the pre-neoplastic or neoplastic cells or tissue or introducing toxin-conjugated molecules that can in turn recognize the autoantibodies already bound to the pre-neoplastic or neoplastic tissue.
One aspect the invention provides a method of identifying pre-neoplastic or neoplastic tissue of a mammal comprising detecting autoantibodies complexed to antigen at the tissue, wherein an increase in the amount of autoantibodies complexed to antigen at the tissue, as compared to that at a control tissue, is indicative that the tissue is pre-neoplastic or neoplastic.
In certain embodiments, the tissue is in the body of the mammal. The mammal may be human or an animal. The tissue may be in the body of the mammal or explanted from the mammal. The detection may be done intra-operatively or prior to surgery. If the tissue is an explant, the explant may be fresh, frozen or fixed.
In certain embodiments, detection occurs by detecting an increase in the amount of autoantibodies complexed to antigen at the tissue as compared to that of a control sample.
In certain embodiments, the autoantibodies are labeled with a label. In certain embodiments, the label is an optical reporter, a positron-emission tomography reporter, a magnetic resonance imaging reporter, or a biochemical marker.
In certain embodiments, the autoantibodies are detected using an antibody detection reagent. In certain embodiments, the antibody detection reagent is Anti-IgG, Protein A, Protein G, an anti-IgG, Fab(2) fragment of an anti-IgG, Fab(1) of an anti-IgG and a humanized mouse anti-human IgG, peptides that bind to the Fc region of antibodies, small molecules that recognize IgG for the species of interest, and small molecules that bind the Fc region.
In certain embodiments, the autoantibodies and tissue are autologous. In other embodiments, the autoantibodies and tissue are heterologous but are from one or more individuals of the same species.
In certain embodiments, the pre-neoplastic or neoplastic tissue is in the liver. In certain embodiments, the neoplastic tissue is a fibrolamellar hepatocellular carcinoma. In other embodiments, pre-neoplastic or neoplastic tissue is liver, skin, breast, or prostate tissue and/or cancer.
Another aspect of the invention provides a method of identifying pre-neoplastic or neoplastic tissue of a mammal comprising: (i) providing labeled autologous autoantibodies, (ii) contacting the mammal's tissue with the labeled autologous autoantibodies, and (iii) detecting labeled autologous autoantibodies complexed to an antigen at the tissue, wherein an increase in the amount of labeled autoantibodies complexed to antigen at the tissue is indicative that the tissue is pre-neoplastic or neoplastic.
In certain embodiments, the tissue is in the body of the mammal. The mammal may be human or an animal. The tissue may be in the body of the mammal or explanted from the mammal. The detection may be done intra-operatively or prior to surgery. If the tissue is an explant, the explant may be fresh, frozen or fixed.
In certain embodiments, detection occurs by detecting an increase in the amount of autoantibodies complexed to antigen at the tissue as compared to that of a control sample.
In certain embodiments, the autoantibodies are labeled with a label. In certain embodiments, the label is an optical reporter, a positron-emission tomography reporter, a magnetic resonance imaging reporter, or a biochemical marker. In certain embodiments, the label is a reporter that can be detected optically selected such as a positron emission tomography reporter, CT reporter, X-ray reporter, magnetic resonance imaging reporter, luminescence reporter, RAMAN spectroscopy reporter, surface enhanced Raman spectroscopy (SERS) reporter, second harmonic generation reporter, or biochemical detection reporter.
In certain embodiments, the autoantibodies and tissue are autologous. In other embodiments, the autoantibodies and tissue are heterologous but are from one or more individuals of the same species.
In certain embodiments, the pre-neoplastic or neoplastic tissue is in the liver. In certain embodiments, the neoplastic tissue is a fibrolamellar hepatocellular carcinoma. In other embodiments, pre-neoplastic or neoplastic tissue is liver, skin, breast, or prostate tissue and/or cancer.
Another aspect of the invention provides a method of identifying pre-neoplastic or neoplastic tissue of a mammal comprising: (i) contacting the tissue with the labeled autoantibodies, and (ii) detecting autoantibodies complexed to antigen at the tissue, wherein an increase in the amount of autoantibodies complexed to antigen at the tissue is indicative that the tissue is pre-neoplastic or neoplastic.
In certain embodiments, the tissue is in the body of the mammal. The mammal may be human or an animal. The tissue may be in the body of the mammal or explanted from the mammal. The detection may be done intra-operatively or prior to surgery. If the tissue is an explant, the explant may be fresh, frozen or fixed.
In certain embodiments, detection occurs by detecting an increase in the amount of autoantibodies complexed to antigen at the tissue as compared to that of a control sample.
In certain embodiments, the autoantibodies are labeled with a label. In certain embodiments, the label is an optical reporter, a positron-emission tomography reporter, a magnetic resonance imaging reporter, or a biochemical marker. In certain embodiments, the antibody is labeled with a reporter that can be detected optically such as a positron emission tomography reporter, CT reporter, X-ray reporter, magnetic resonance imaging reporter, luminescence reporter, RAMAN spectroscopy reporter, surface enhanced Raman spectroscopy (SERS) reporter, second harmonic generation reporter, or biochemical detection reporter.
In certain embodiments, the autoantibodies and tissue are autologous. In other embodiments, the autoantibodies and tissue are heterologous but are from one or more individuals of the same species.
In certain embodiments, the pre-neoplastic or neoplastic tissue is in the liver. In certain embodiments, the neoplastic tissue is a fibrolamellar hepatocellular carcinoma. In other embodiments, pre-neoplastic or neoplastic tissue is liver, skin, breast, or prostate tissue and/or cancer.
In yet another aspect, the invention provides a method of identifying pre-neoplastic or neoplastic tissue of a human comprising: (i) contacting the tissue with a labeled probe (e.g., anti-human IgG, antigen-binding fragments of anti-human IgG antibodies, Protein A, or Protein G, peptides that bind the Fc region, small molecules that bind the Fc region, or small molecules that bind human IgG and (ii) detecting the labeled probe complexed to antigen at the tissue, wherein an increase in the amount of labeled probe complexed to antigen at the tissue is indicative that the tissue is pre-neoplastic or neoplastic.
In certain embodiments, the tissue is in the body of the mammal. The mammal may be human or an animal. The tissue may be in the body of the mammal or explanted from the mammal. The detection may be done intra-operatively or prior to surgery. If the tissue is an explant, the explant may be fresh, frozen or fixed.
In certain embodiments, detection occurs by detecting an increase in the amount of autoantibodies complexed to antigen at the tissue as compared to that of a control sample.
In certain embodiments, the autoantibodies and tissue are autologous. In other embodiments, the autoantibodies and tissue are heterologous but are from one or more individuals of the same species.
In certain embodiments, the pre-neoplastic or neoplastic tissue is in the liver. In certain embodiments, the neoplastic tissue is a fibrolamellar hepatocellular carcinoma. In other embodiments, pre-neoplastic or neoplastic tissue is liver, skin, breast, or prostate tissue and/or cancer.
In yet another aspect, the invention provides a method of diagnosing prostate cancer in a subject comprising detecting autoantibodies complexed to antigen in the prostate secretions of the subject, wherein an increase in the amount of autoantibodies complexed to antigen in the secretions of the subject is indicative of prostate cancer.
In certain embodiments, the detection of autoantibodies complexed to the antigen comprises detecting an increase in the amount of autoantibodies complexed to antigen in the prostate secretion as compared to that of a control sample.
In certain embodiments, the mammal is a human or an animal. In certain embodiments, detection occurs intra-operatively. In certain embodiments, detection occurs prior to surgery.
In certain embodiments, the secretion is ejaculate. In certain embodiments, the secretion is urine.
In certain embodiments, the autoantibodies are labeled with a label. In certain embodiments, the label is an optical reporter, a positron-emission tomography reporter, a magnetic resonance imaging reporter, or a biochemical marker. In certain embodiments, the labeled antibody is labeled with a reporter that can be detected optically such as a positron emission tomography reporter, CT reporter, X-ray reporter, magnetic resonance imaging reporter, luminescence reporter, RAMAN spectroscopy reporter, surface enhanced Raman spectroscopy (SERS) reporter, second harmonic generation reporter, or biochemical detection reporter.
In certain embodiments, the autoantibodies are detected using an antibody detection reagent. In certain embodiments, the antibody detection reagent is Anti-IgG, Protein A, Protein G, an anti-human IgG, Fab(2) fragment of an anti-human IgG, Fab(1) of an anti-human IgG and a humanized mouse anti-human IgG, peptides that bind to the Fc region of human antibodies, and small molecules that bind IgGs.
In certain embodiments, the autoantibodies and sample are autologous. In other embodiments, the autoantibodies and sample are heterologous but are from one or more individuals of the same species.
In yet another aspect, the invention provides a method of killing pre-neoplastic or neoplastic tissue of a mammal comprising: (i) isolating autoantibodies from the mammal, (ii) complexing a toxin to the autoantibodies, and (iii) contacting the tissue with the toxin-autoantibodies complex, wherein said toxin-autoantibodies complex kill the pre-neoplastic or neoplastic tissue.
In certain embodiments, the toxin is paclitaxel, adriamycin, beta-emitters, or ricin.
In certain embodiments, the mammal is a human or an animal. In certain embodiments, the autoantibodies and tissue are autologous. In other embodiments, the autoantibodies and tissue are heterologous but are from one or more individuals of the same species.
In certain embodiments, the pre-neoplastic or neoplastic tissue is in the liver. In certain embodiments, the neoplastic tissue is a fibrolamellar hepatocellular carcinoma. In other embodiments, pre-neoplastic or neoplastic tissue is liver, skin, breast, or prostate tissue and/or cancer.
In yet another aspect, the invention provides a method of killing pre-neoplastic or neoplastic tissue of a mammal comprising: (i) complexing a toxin to molecules that recognize autoantibodies and (ii) contacting the tissue with the toxin-molecule conjugate, wherein said toxin-molecule-autoantibody complex kill the pre-neoplastic or neoplastic tissue.
In certain embodiments, the molecules are antibody recognition molecules such as anti-human IgG, antigen-binding fragments of anti-human IgG antibodies, Protein A, or Protein G, peptides that bind IgGs, and small molecules that bind IgGs.
In certain embodiments, the toxin is paclitaxel, adriamycin, beta-emitters, or ricin.
In certain embodiments, the mammal is a human or an animal. In certain embodiments, the autoantibodies and tissue are autologous. In other embodiments, the autoantibodies and tissue are heterologous but are from one or more individuals of the same species.
In certain embodiments, the pre-neoplastic or neoplastic tissue is in the liver. In certain embodiments, the neoplastic tissue is a fibrolamellar hepatocellular carcinoma. In other embodiments, pre-neoplastic or neoplastic tissue is liver, skin, breast, or prostate tissue and/or cancer.
This invention generally relates to a method of identifying pre-neoplastic or neoplastic tissue of a mammal. In one embodiment, autoantibodies, either from the same individual or a member of the same species, are directly labeled. The labeled antibodies are then introduced into the body and the pre-neoplastic or neoplastic tissue is detected by directly detecting the labeled autoantibodies.
In another embodiment, the autoantibodies are detected indirectly by detecting autoantibodies already bound to the pre-neoplastic or neoplastic tissue. In this embodiment, the autoantibodies are detected by a detection agent.
This invention also relates to diagnosing prostrate cancer in a subject by detecting autoantibodies found in prostate secretions of the subject.
This invention also relates to a method of killing tumor cells. In an embodiment, the tumor cells are killed by directly conjugating a subject's own autoantibodies, or the autoantibodies of another subject of the same species, to a toxin and reintroducing the toxin-conjugated autoantibodies into the subject. In this embodiment, the toxin-conjugated autoantibodies bind to the neoplastic cells to kill them. In another embodiment, the toxin is conjugated to molecules which, in turn, can recognize the autoantibodies already bound to the tumor. In one embodiment the subject is a human. In another embodiment the subject is an animal.
Long-term survival in patients with various types of cancer could be greatly improved by 1) earlier detection of neoplasia; 2) greater sensitivity and specificity for detecting small foci; 3) greater sensitivity for detecting altered cells at the margins; 4) the ability to continue to detect and treat tumors even as they change. As described and exemplified herein, the instant invention relates to the discovery that polyclonal spectrum of autoantibodies bound to tissue can be used diagnostically as a means to detect microscopic foci of tumor, and distinguish transformed from normal tissue. Analysis revealed a significantly higher concentration of autoantibodies in four transgenic mouse models and two xenograft mouse models of cancer than cognate wild-type. Specifically, there was greater autoantibody binding within proliferating and transitioning tissue in all six mouse models. The increasing binding of the autoantibodies was detectable from in vitro slices of tumor and healthy tissue, from in situ tissue in a living animal during surgery and in a living animal in whole body imaging. These results indicate that the spectrum of autoantibodies bound in tissue can detect tumors at early stages of transformation. Further, they continue to track transformed cells even as the tumor undergoes mutations. This invention utilizes agents that bind autoantibodies for cancer detection and therapeutics.
As exemplified below, autoantibodies bind pre-neoplastic and neoplastic tissue in transgenic and xenograft mouse models of cancer. These autoantibodies distinguished the abnormal tissue from normal tissue within the same animal and from WT controls. Thus, the heterogeneous collection of an individual's antibodies bound to tissue is useful for detecting and targeting pre-neoplastic or neoplastic lesions. This sensitivity is the consequence of two features of the autoantibodies. First, they are diverse, recognizing a wide collection of antigens. Second, they are continuously responding to mutations in the tumor over time. Probes that recognize IgG can detect this changing spectra of antibodies. This offers the potential for improved patient outcomes by earlier detection of neoplasia, unrecognized foci and transformed cells at the margins of resection, even at the level of single cells. Additionally, the ability to target autoantibodies, which are continually tracking changes of tumors, offers therapeutic potential.
As exemplified below, the results show that early transitioning cells, are distinguishable from surrounding tissue, even before there is frank neoplasia. There were numerous regions of alb-myc livers that were histopathologically “normal”, but had high concentrations of autoantibodies. These regions also bound anti-CD34, a marker of fenestrated, capillarized endothelial cells that is not found on normal liver sinusoidal cells (Scoazec and Feldmann, 1991). This suggests that autoantibodies are recognizing transitioning areas not detectable by histopathology. In the MMTV-neu model, autoantibodies bound to atypical hyperplastic glands were observed, discriminating between this pre-neoplastic tissue and surrounding normal tissue. Thus, the autoantibodies can recognize early foci of transformation.
In all models tested, the autoantibodies bound the tumor microenvironment. The tumor has been described as an “ecosystem”: neoplastic cells combined with a milieu that aids in their growth (Bissell and Radisky, 2001). The microenvironment is known to be made up of cells, including fibroblasts, immune cells, and endothelial cells, that are activated or recruited by the nearby tumor cells to aid in the sustainment and growth of the tumor itself. The communication between this reactive microenvironment and the cancer cells affects the phenotype of the tumor (Mueller and Fusenig, 2004). Disrupting this microenvironment has a detrimental effect on tumors (Mueller and Fusenig, 2004; Roskelley and Bissell, 2002). The tumor microenvironment contains a variety of immune cells including neutrophils, dendritic cells, macrophages, and lymphocytes (Coussens and Werb, 2002).
The results of the instant invention show that autoantibodies are also present in the tumor microenvironment. In the alb-myc model, antibodies bound to the hepatocytes, but also to the liver sinusoidal endothelial cells. Alterations in these cells have been reported in early stages of liver tumorigenesis (Frachon et al., 2001). In the MMTV-neu model, antibody bound to the mammary adipocytes, endothelial cells and connective tissue stroma. Stroma adjacent to mammary tumor undergoes both phenotypic and epigenetic transformations (Fiegl et al., 2006; Hu et al., 2005; Trimboli et al., 2009). Mammary endothelial cells have been reported to be involved with tumor survival, proliferation, and invasiveness (Franses et al., 2011). Furthermore, mammary cancer-associated fibroblasts have been shown to promote tumor growth. (Orimo et al., 2005; Tyan et al., 2011) Similar results were observed in the prostate models, with antibody bound throughout the microenvironment, an altered area supporting the growth of the surrounding prostate tumor (Chung et al., 2005; Dakhova et al., 2009). Autoantibodies were also observed in the secretions of the prostate.
The results below demonstrate autoantibody binding is the result of a tumor-specific immune response. First, in the mouse models for cancer only anti-mouse IgG antibodies bound tissue, antibodies against IgG of other species or antibodies against other immunoglobins did not bind. Second, the areas to which they bound were independently shown to either be neoplastically transformed (as assayed by H&E) or transitioning (as assayed by binding of anti-CD34). Third, the antibody binding was specific to the organs generating the tumor. For example, when comparing MMTV-neu to WT mice, the mammary gland is the only organ that has a higher autoantibody binding. Moreover, in the xenograft model, the antibody response was specific to where the tumor was implanted. There were two exceptions to this observation. In the alb-myc model, autoantibodies were found bound to organs other than the liver. However, these organs also express the albumin promoter, which was used to drive the expression of the myc oncogene. Additionally, in the xenograft model, one mouse had autoantibody within the liver, spleen, and lung. On H&E, these organs were found to contain spontaneous AMH/leukemia. These results seen in a spontaneous hematopoietic cancer are consistent with the genetically engineered or xenograft solid tumor model results. During whole animal imaging of MMTV-neu mice, antibody binding was found in one mouse extraneous to the breast and, upon autopsy, this was found to be a spontaneous tumor.
As used herein, the singular forms “a,” “an,” and “the” include plural references unless the content clearly dictates otherwise.
The term “about,” as used here, refers to +/−10% of a value.
An “antigen” refers to a molecule containing one or more epitopes (either linear, conformational or both) that elicit an immunological response. The term may be used interchangeably with the term “immunogen.” An “epitope” is that portion of given species (e.g., an antigenic molecule or antigenic complex) that determines its immunological specificity. An epitope is within the scope of the present definition of antigen. Commonly, an epitope is a polypeptide or polysaccharide or a folded domain in a naturally occurring antigen. In artificial antigens it can be a low molecular weight substance such as an arsanilic acid derivative. Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will typically include at least about 7-9 amino acids, and a helper T-cell epitope will typically include at least about 12-20 amino acids. The term “antigen” denotes both subunit antigens, i.e., antigens which are separate and discrete from a whole organism or cell with which the antigen is associated in nature, or tumor cells, such as tumor antigens.
The term “adjuvant” or “immunological adjuvant” refers to any substance that assists or modifies the action of an antigen in the immune system. Adjuvants can potentiate humoral and/or cellular immunity.
The term “antibody” refers to an immunoglobulin or antigen-binding fragment thereof, and encompasses any such polypeptide comprising an antigen-binding fragment of an antibody. The term includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific, humanized, human, single-chain, single-domain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The term “antibody” also includes antigen-binding fragments of an antibody. Examples of antigen-binding fragments include, but are not limited to, Fab fragments (consisting of the VL, VH, CL and CH1 domains); Fd fragments (consisting of the VH and CH1 domains); Fv fragments (referring to a dimer of one heavy and one light chain variable domain in tight, non-covalent association); dAb fragments (consisting of a VH domain); single domain fragments (VH domain, VL domain, VHH domain, or VNAR domain); isolated CDR regions; (Fab′)2 fragments, bivalent fragments (comprising two Fab fragments linked by a disulphide bridge at the hinge region), scFv (referring to a fusion of the VL and VH domains, linked together with a short linker), and other antibody fragments that retain antigen-binding function.
The term “autoantibody” as used herein refers to an antibody that recognizes the cells, tissues, native proteins, or molecules of the organism in which it was formed. Autoantibodies can be from an organism or individual that is heterologous to the tissue in which the detection or killing is occurring, but are from one or more individuals of the same species. The production of autoantibodies in cancer is the result of an autoimmune response directed to molecules that are overexpressed, mutated, or aberrantly regulated or localized as a result of cellular transformation of the tissue to a cancerous, neoplastic, pre-cancerous or pre-neoplastic phenotype. These molecules include, but are not limited to proteins, lipids, glycolipids, and nucleic acids.
The term “neoplastic tissue,” “neoplastic cells,” or “neoplasms” as used herein refers to an abnormal mass of tissue or a proliferation of cells. The growth of neoplastic cells exceeds that of normal tissue around it and it is not coordinated with that of the normal tissue around it. Neoplasms may be benign, pre-malignant (e.g., carcinoma in situ) or malignant (e.g., cancer). This tissue can originate from any cell type or tissue found in a mammal, including, but not limited to hepatic, skin, breast, prostate, neural, optic, intestinal, cardiac, vasculature, lymph, spleen, renal, bladder, lung, muscle, connective, tissue, pancreatic, pituitary, endocrine, reproductive organs, bone, and blood. More particularly, the neoplastic tissue is from the liver, skin, breast, prostate, and lymph. More particularly, the neoplastic tissue is from the liver. Even more specifically, the neoplastic tissue is a fibrolamellar hepatocellular carcinoma.
The term “pre-neoplastic tissue” as used herein refers to tissue preceding the formation of a benign or malignant neoplasm. This tissue can originate from any tissue found in a mammal, including, but not limited to liver, skin, breast, prostate, neural, optic, intestinal, cardiac, vasculature, lymph, spleen, renal, bladder, lung, muscle, connective, tissue, pancreatic, pituitary, endocrine, reproductive organs, bone, and blood. More particularly, the neoplastic tissue is from the liver, skin, breast, prostate, and lymph. More particularly, the neoplastic tissue is from the liver. Even more specifically, the neoplastic tissue is a fibrolamellar hepatocellular carcinoma.
The term “hyperplasia” as used herein refers to an increase in number of cells/proliferation of cells. It may result in the gross enlargement of an organ. Hyperplasia is a common pre-neoplastic response to a stimulus.
The term “fragment” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of polypeptides, a fragment may be defined by a contiguous portion of the amino acid sequence of that protein and may be at least 3-5 amino acids, at least 6-10 amino acids, at least 11-15 amino acids, at least 16-24 amino acids, at least 25-30 amino acids, and at least 30-45 amino acids. In the case of polynucleotide, a fragment is defined by a contiguous portion of the nucleic acid sequence of that polynucleotide and may be at least 9-15 nucleotides, at least 15-30 nucleotides, at least 31-45 nucleotides, at least 46-74 nucleotides, at least 75-90 nucleotides, and at least 90-130 nucleotides. In some embodiments, fragments of biomolecules are immunogenic fragments.
The term “intra-operatively” as used herein refers to occurring, carried out, or encountered in the course of surgery.
The term “autologous” as used herein refers to a situation in which the donor and recipient are the same individual.
The term “heterologous” as used herein refers to a situation in which the donor and recipient are from different individuals. The different individuals can be of the same species or different species.
The term “control sample” as used herein refers to a background signal or a signal associated with a normal tissue (i.e., a tissue that is not pre-neoplastic or neoplastic). The control sample can be autologous, from the same animal, or heterologous, from another animal. The control sample can be from the same species or different species.
The term “secretion” as used herein refers to a substance, such as saliva, fluid, mucus, tears, bile, or a hormone, that is secreted from a cell or gland.
The term “complexing” or “complex” as used herein refers to the binding or association of an antibody or autoantibody to an antigen, cell, or tissue. It can also mean the binding or association of a molecule, such as a reporter or toxin, to an autoantibody or the binding or association of a molecule, such as a reporter or toxin, to a different molecule that can recognizes an autoantibody.
While various groups have identified a number of tumor-associated autoantibodies in the hopes of utilizing them as biomarkers, prognostic factors, and indicators of tumor recurrence, they have only looked in the sera of patients. One embodiment of the present invention, however, focuses on autoantibodies as they are associated with the pre-neoplastic or neoplastic tissue itself. By allowing for identification and imaging of the tissue itself, this invention allows for the detection of both pre-neoplastic and neoplastic tissue, not just the presence of the autoantibody within the sera. Thus, this invention leads to improved staging of disease. Earlier diagnosis of tumor recurrence, the ability to detect small foci that can not be detected optically as well as detect altered cells at the margins of a resection. This detection can occur in a biopsy sample, a secretion sample, in situ (such as intra-operatively or intravital staining), or a whole body scan.
Pre-neoplastic and neoplastic tissue can be detected on biopsy samples of tissue or secretion samples removed for examination prior to surgical intervention. Types of biopsies used in relation to this invention include, but are not limited to needle biopsy, CT-guided biopsy, ultrasound-guided biopsy, liver biopsy, bone biopsy, bone marrow biopsy, liver biopsy, kidney biopsy, aspiration biopsy, prostate biopsy, skin biopsy. Secretion samples can be collected by ejaculation, urination, voiding, swabbing, collecting, scraping, or needle aspiration of the secretion.
The instant invention provides a new strategy for identifying pre-neoplastic or neoplastic tissue. In particular, autoantibodies can be used to distinguish transitioned tissue from normal tissue intra-operatively in two ways. First, abnormal tissue can be detected in freshly excised or frozen sections of tissue slides. Frozen sections are commonly used for real-time analysis of margins of surgical specimens. Further resection or enhanced treatment regimens are often employed based on these results. However, the sensitivity of frozen sections recognizing positive surgical margins is inconsistent. For example, the sensitivity of frozen section analysis for positive surgical margins during radical prostatectomy for prostate cancer is 42% (Tsuboi et al., 2005). Supplementing or supplanting current pathological frozen section analysis with the present invention increases this sensitivity leading to a more precise intra-operative test.
Second, autoantibodies can detect abnormal tissue in situ during surgical resection of tumor. Therefore, autoantibodies can be applied to intraoperative imaging to assess for the presence of neoplasia on the cellular level. The use of near-infrared fluorescent probes to detect autoantibodies can be combined with the recent development of a real-time intraoperative imaging device (Liu et al., 2011) and other inter-operative technologies (Liu et al., 2011; Ye et al., 2011). The presence of tagged autoantibodies within transitioned tissue will also allow for a more accurate resection of all diseased tissue.
The detection can occur by one of two methods. The first method comprises the labeling of the autoantibodies, wherein the labeled autoantibodies are used to detect the pre-neoplastic or neoplastic tissue by introducing the labeled autoantibodies into the subject. The second method comprises the detection of autoantibodies indirectly by introducing an autoantibody detection agent.
In one embodiment the labeled autoantibodies or the autoantibodies detected by the reagent are bound to the pre-neoplastic or neoplastic tissue. In another embodiment, the labeled autoantibodies or the autoantibodies detected by the reagent are bound to individual pre-neoplastic or neoplastic cells. In yet another embodiment, the labeled autoantibodies or the autoantibodies detected by the reagent are bound to antigens on the pre-neoplastic or neoplastic cells. In another embodiment, the antigen is an antigen known to be expressed by a cancerous cell.
In one embodiment, the pre-neoplastic or neoplastic tissue and cells are derived from bone, blood, hepatic, skin, breast, prostate, neural, optic, intestinal, cardiac, vasculature, lymph, spleen, renal, bladder, lung, muscle, connective, pancreatic, pituitary, endocrine, or reproductive organ tissue. In one embodiment, the neoplastic tissue or neoplastic cells are cancerous. In yet another embodiment, the type of cancer is bone, blood, or hepatic, skin, breast, prostate, neural, optic, intestinal, cardiac, vasculature, lymph, spleen, renal, bladder, lung, muscle, connective, pancreatic, pituitary, endocrine, or reproductive organ tissue cancers. In another embodiment, the pre-neoplastic or neoplastic tissue is liver, breast, skin, or prostate tissue. In another embodiment, the neoplastic tissue is liver, breast, skin, or prostate cancer. In another embodiment, the cancer is fibrolamellar hepatocellular carcinoma. In another embodiment, the cancer is leukemia, adrenocarcinoma, astocytomeas, basal cell carcinoma, osteosarcoma, glioma, chordoma, retinoblastoma, squalors cancer, or T-cell lymphoma.
a. Detection of Labeled Autoantibodies
In one embodiment, autoantibodies are directly labeled. In another embodiment the autoantibodies are isolated before being labeled. In yet another embodiment the autoantibodies are isolated and purified before being labeled. The labeled antibodies are then introduced or introduced or reintroduced back into the subject and the pre-neoplastic or neoplastic tissue is detected by measuring an increase in the amount of autoantibodies complexed to the tissue, cell, or antigen. In one embodiment, the detection of autoantibodies complexed to an antigen comprises detecting an increase in the amount of autoantibodies complexed to antigen at the tissue as compared to that of a control sample.
In one embodiment, an increase in signal above control or background of at least 2% indicates the detection of a pre-neoplastic tissue. In another embodiment, an increase in signal above control or background of at 5% indicates the detection of a pre-neoplastic tissue. In another embodiment, an increase in signal above control or background of at least 10% indicates the detection of a pre-neoplastic tissue. In another embodiment, an increase in signal above control or background of at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 250% indicates the detection of a pre-neoplastic tissue.
In one embodiment, an increase in signal above control or background of at least 2% indicates the detection of a neoplastic tissue. In another embodiment, an increase in signal above control or background of at 5% indicates the detection of a neoplastic tissue. In another embodiment, an increase in signal above control or background of at least 10% indicates the detection of a neoplastic tissue. In another embodiment, an increase in signal above control or background of at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 250% indicates the detection of a neoplastic tissue.
In one embodiment, the autoantibodies are taken from the subject being tested for the pre-neoplastic or neoplastic tissue (i.e., the autoantibodies and the tissue are autologous). In this embodiment, the labeled autoantibodies are reintroduced back into the subject and the pre-neoplastic or neoplastic tissue is detected by measuring an increase in the amount of autoantibodies complexed to the tissue, cell, or antigen.
In another embodiment, the autoantibodies and tissue are heterologous but from the same species. In yet another embodiment, the autoantibodies and tissue are heterologous but from different species. In this embodiment, the labeled autoantibodies are introduced into the subject and the pre-neoplastic or neoplastic tissue is detected by measuring an increase in the amount of autoantibodies complexed to the tissue, cell, or antigen. In yet another embodiment, the heterologous autoantibodies are pooled from multiple individuals.
Methods for isolating autoantibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982. A number of companies make kits, along with instructions on their implementation: www.piercenet.com/browse.cfm?fldID=ACC3CB7A-1097-403A-BAC1-E6F8AAD12044.
Labeling or tagging of the autoantibodies of the present invention is done according to methods of antibody generally known in the art. Methods for labeling antibodies are well known in the art. Examples of labeling substances include, but are not limited to, an optical reporter [such as bioluminescence (e.g. luciferase), near-infrared and visible fluorescent labels, fluorophores/fluorochromes (e.g., Hydroxycoumarin, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-Phycoerythrin, PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, Fluor X, Fluorescein, Indocyanine Green (ICG), BODIPY-FL, TRITC, X-Rhodamine, Lissamine Rhodamikne B, Texas Red, fluorescent proteins (e.g. CFP, GFP, YPF, mCherry, mPlum, mStrawberry, and RFP and photoconvertable fluorescent proteins), Cy dyes (e.g., Cyanine, Cy3, Cy5, Cy5.5, Cy7), Alexa Fluor dyes, Atto Dyes, Dylight, IRIS Dyes, Seta dyes, SeTau dyes, SRfluor dyes and Square dyes)], a positron-emission tomography reporter [such as carbon, iodine, nitrogen, oxygen, fluoride, and zirconium isotopes (e.g., carbon-11, nitrogen-14, oxygen-15, fluorine-18, iodine-124, zirconium-89, and copper-64), fludeoxyglucose, and 11C-metomidate], a magnetic resonance imaging reporter [such as iron oxide agents (e.g., Cliavist, Combidex, Endorem, Feridex, Resovist, Sinerem), boron, magnesium chelators, and gadolinium agents (e.g., Omniscan, Multihance, Magnevist, ProHance, Vasovist, Eovist and OptiMARK, gadocoletic acid gadodenterate, gadomelitol, and gadopenamide)], and a biochemical marker [such as proteins (e.g., biotin, avidin, neutravidine, photobiotin, strep-tag, streptavidin) and enzymes (e.g., horseradish peroxidase, alkali phosphatase)].
The labeled autoantibodies can be detected by light/optical microscopy with a fluorescence microscope (e.g., confocal microscopy, multiphoton microscopy, whole animal fluorescence imaging and stimulated emission depletion microscopy), by positron emission tomography (PET) scans, magnetic resonance imaging (MRI) scans, or biochemical assays (e.g., ELISA, radioimmunoassay, magnetic immunoassays, and reverse phase protein lysate microarray).
b. Detecting Autoantibodies Using an Antibody Detection Reagent
Detecting an autoantibody using an antibody detection reagent is well known in the art. Such reagents for antibody detection include, but are not limited to an antibody made to IgG (anti-IgG), Protein A, Protein G, Fab(2) fragment of an anti-IgG, Fab(1) of an anti-IgG, peptides that bind to the Fc region, and small molecules that bind antibodies. In one embodiment, the anti-IgG is an anti-human IgG or a humanized mouse anti-human IgG In one embodiment the anti-IgG includes the affinity ligand secretory component (Brandtzaeg, 1983), peptides that are designed based on hydropathy (Fassina et al., 1992), fragments of the placental alkaline phosphatase that can bind the Fc region (Makiya and Stigbrand, 1992), multimeric peptides (Verdoliva et al., 1995), one of the peptides from the E. coli surface exposed EiB proteins than bind IgG (Sandt and Hill, 2001), variations of the Fc receptor proteins that can bind IgG (Akilesh et al., 2007; Fridman, 1991; Fridman et al., 1984), anyone of a number of protein G or protein A mimetic (PAM) identified from peptide libraries that bind IgG (Fassina, 2000; Fassina et al., 1996; Fassina et al., 1998), or identified from combinatorial chemical synthesis (Fassina et al., 2001; Kabir, 2002), or combinatorial libraries (Nielsen et al., 2010), phage display libraries (Sakamoto et al., 2009), or rationally designed non-peptidyl mimetics of Protein A (Li et al., 1998), hexamer peptide affinity resins that bind the Fc region of IgG (Yang et al., 2005, 2009a) or the specific hexamer HWRGWV (Yang et al., 2010), use of the all-D amino acid peptide ligands (D'Agostino et al., 2008; Verdoliva et al., 2002), synthetic ligands including cyclic peptides (Verdoliva et al., 2005), a synthetic triazine scaffold substituted with 3-mainopheno and 4-amino-1-maphthol (Teng et al., 2000) or through other combinatorial chemical syntheses to make IgG binding ligands (Teng et al., 1999), affinity ligands that mimic Protein L (Roque et al., 2005), trisubstituted purine derivatives as protein A mimetics (Zacharie et al., 2010; Zacharie et al., 2009), or dendrimeric peptides (Moiani et al., 2009).
In one embodiment the reagents for detection of autoantibodies are selected from the collection of molecules that bind with high affinity to the autoantibodies in a one-to-one manner.
In one embodiment the reagents for detection of autoantibodies are selected from the collection of molecules that bind with relatively lower affinity to the autoantibodies. In this embodiment, multiple copies of the reagents for detection will be conjugated together. Thus, the lower affinity will be compensated by an increased avidity of binding to the autoantibodies.
In the case of whole animal imaging, the labeled antibody detection agent will be injected into the circulation and allowed to distribute, and then a whole body scan will be done later, such as 6 to 24 hours later. The time period for distribution will vary depending upon the antibody detection agent; for example, some equilibrate in 30 minutes some in 6 hours. In one embodiment of intraoperative imaging, the antibody detection kit is made of one component, directly targeted to the autoantibodies, that is injected into the circulation and allowed to distribute prior to surgery. In another embodiment of intraoperative imaging, the antibody detection kit is injected during surgery directly into the circulation perfusing the diseased organ. In another embodiment of intraoperative imaging the antibody detection kit will be made of two components: An agent targeted to the antibody conjugated to one fluorophore and a chemically similar agent that targets antibodies of a different species labeled with a second fluorophore. The two will be mixed together in a buffer, poured over the region of interest and then rinsed with just the buffer.
The reagents for detection of autoantibodies will be conjugated to labels or reporters that can be detected. Examples of labeling substances include, but are not limited to, an optical reporter [such as bioluminescence (e.g. luciferase), infrared, near-infrared and visible fluorescent labels, fluorophores/fluorochromes (e.g., Hydroxycoumarin, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-Phycoerythrin, PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, Fluor X, Fluorescein, Indocyanine Green (ICG), BODIPY-FL, TRITC, X-Rhodamine, Lissamine Rhodamikne B, Texas Red, fluorescent proteins (e.g. CFP, GFP, YPF, mCherry, mPlum, mStrawberry, and RFP and photoconvertable fluorescent proteins), Cy dyes (e.g., Cyanine, Cy3, Cy5, Cy5.5, Cy7), Alexa Fluor dyes, Atto Dyes, Dylight, IRIS Dyes, Seta dyes, SeTau dyes, SRfluor dyes and Square dyes)], a positron-emission tomography reporter [such as carbon, iodine, nitrogen, oxygen, fluoride, and zirconium isotopes (e.g., carbon-11, nitrogen-14, oxygen-15, fluorine-18, iodine-124, zirconium-89, and copper-64), fludeoxyglucose, and 11C-metomidate], a magnetic resonance imaging reporter [such as iron oxide agents (e.g., Cliavist, Combidex, Endorem, Feridex, Resovist, Sinerem), boron, magnesium chelators, and gadolinium agents (e.g., Omniscan, Multihance, Magnevist, ProHance, Vasovist, Eovist and OptiMARK, gadocoletic acid gadodenterate, gadomelitol, and gadopenamide)], and a biochemical marker [such as proteins (e.g., biotin, avidin, neutravidine, photobiotin, strep-tag, streptavidin) and enzymes (e.g., horseradish peroxidase, alkali phosphatase)]. Thus, the labeled autoantibodies can be detected by light/optical microscopy with a fluorescence microscope (e.g., confocal microscopy, multiphon microscopy, whole animal fluorescence imaging microscopy, and stimulated emission depletion microscopy), by positron emission tomography (PET) scans, magnetic resonance imaging (MRI) scans, or biochemical assays (e.g., ELISA, radioimmunoassay, magnetic immunoassays, and reverse phase protein lysate microarray).
c. Detection in Frozen Sections
Tissue samples are obtained from surgically-removed tissue that has been frozen in a fixed or unfixed state and sectioned. The tissue can be sectioned by any means known in the art. For example, the tissue can be placed in a cryoprotective embedding medium (e.g., Tissue Tek O.C.T., TBS or Cryogel), and then the tissue sample can be snap frozen in isopentane cooled by liquid nitrogen. Tissue is then sectioned in a freezing microtome or cryostat.
Examples for suitable tissue fixatives include, but are not limited to, crosslinking fixatives (e.g., paraformaldehyde, formalin, and gluteraldehyde), precipitating fixatives (e.g., ethanol, methanol, acetone, and an alcohol in combination with acetic acid), oxidizing agents (e.g., osmium tetoxide, potassium dichromate, chromic acid, and potassium permanganate), mercurials (e.g., B-5 and Zenker's), picrates, HOPE Fixative, or other standard histological preservatives.
d. Detecting in Fresh Explants
Tissue samples are obtained from surgically-removed or biopsied tissue in a fixed or unfixed state.
In one embodiment the tissue will be excised and then the explant will be incubated with an anti host-IgG, conjugated to one reporter and an anti-non-host IgG conjugated to a different reporter. For example, if the sample is from a human than the explanted sample will be simultaneously incubated with a humanized-mouse anti human IgG conjugated to Cy5.5 and a humanized-mouse anti-chicken IgG conjugated to ICG (indocyanine green). The fluorescence of the ICG and the Cy5.5 will be measured simultaneously and transformed cells will be assayed for by the ratio of the fluorescence of Cy5.5:ICG. The fluorescence can be quantified either by fluorescence microscopy or by a spectrofluorimeter. For the examples show (
In another embodiment the tissue will be excised and then the explant will be incubated with labeled autoantibodies from the patient and then the fluorescence monitored by fluorescence microscopy. Fluorescence microscopy is a preferred method of assaying because it helps distinguish tumor for healthy regions.
In another embodiment the animal will be injected with an anti host-IgG, conjugated to one reporter and an anti-non-host IgG conjugated to a different reporter. For example, if the host is a human than the host will be simultaneously incubated with a humanize-mouse anti-human IgG conjugated to Cy5.5 and a humanized-mouse anti-chicken IgG conjugated to ICG (indocyanine green). The antibodies will be allowed to circulate prior to surgery. Then, during surgery the sample will be removed and then imaged by fluorescence microscopy, as shown in
In another embodiment the animal will be injected with labeled autoantibodies which will be allowed to circulate prior to surgery. Then, during surgery the sample will be removed and then imaged by fluorescence microscopy, as shown in
e. Detection Interoperative
In one embodiment the animal will be injected with autoantibodies that are conjugated to a probe that can be detected by fluorescence, PET or MRI. The antibodies will be allowed to circulate prior to imaging as in
In another embodiment the animal will be injected with an anti host-IgG, conjugated to a probe that can be detected by PET or MRI. The antibodies will be allowed to circulate prior to imaging. In a variation on this implementation the labeled anti-host IgG will be injected into a blood vessel feeding the diseased organ.
f. Detection in Whole Body Scans.
Detection of an antibody in a whole animal is well established by a variety of means including detection by PET, MRI and whole body fluorescence. These techniques have been used to track specific antibodies, such as Trastuzumab (trade name heurceptin). In one embodiment of this invention, the whole IgG from the mammal will be labeled as described above, and then the labeled autoantibodies introduced back into the mammal and then the mammal will be scanned in a whole body scan as described above (e.g. a PET imager, MRI, whole body fluorescence imager) as seen in
In one embodiment of this invention, the autoantibodies that are already bound at the tumor will detected by labeling an agent that can detect the autoantibodies as described above. These agents will be injected into the animal and then detected (by PET, MRI, whole animal imaging as described above).
The most common tests for prostate cancer in its earlier states is with regular digital prostate exams and the prostate specific antigen (PSA) blood tests. PSA is a specific type of protein whose blood level tends to increase in the presence of prostate cancer. Currently 20% of men test positive for PSA, which frequently results in a biopsy. However, most of these are false positives. A test that is more accurate than PSA (fewer false positives with no increase in false negatives) could decrease unnecessary expense and discomfort. A prostate ultrasound and biopsy are both used to evaluate the abnormal results obtained in the digital rectal exam or an elevated PSA test.
With the biopsy, transrectal ultrasound imaging is used to guide several small needles through the rectum wall into areas of the prostate where abnormalities are detected. The needles remove a tiny amount of tissue. Usually six or more biopsies are taken to test various areas of the prostate. Prostate biopsy can lead to harmful side effects such as prolonged or heavy bleeding, pain, swelling, difficulty urinating, fever, discharge from the penis, and erectile dysfunction. A prostate biopsy can also result in spreading cancer cells to other parts of the body and may also be the reason that men have a recurrence of disease many years after the prostate was removed. Additionally, it is an invasive and costly procedure. The present invention, however, is a non-invasive way to diagnose prostate cancer.
In one embodiment, diagnosing prostate cancer in a human subject comprises detecting autoantibodies complexed to antigen in the prostate secretions of the subject, wherein an increase in the amount of autoantibodies complexed to antigen in the secretions of the subject is indicative of prostate cancer. Such a test is non-invasive and does not require a biopsy. In another embodiment, the detection of autoantibodies complexed to the antigen comprises detecting an increase in the amount of autoantibodies complexed to antigen at the tissue as compared to that of a control sample.
In one embodiment, the autoantibodies are labeled autoantibodies. In another embodiment, the autoantibodies are detected using an antibody detection reagent.
In one embodiment, the secretion sample is tested for autoantibodies endogenously present in the sample by using an autoantibody detection reagent. In another embodiment, the secretion sample is contacted with autoantibodies, which are in turn detected with an autoantibody detection reagent to detect pre-neoplastic or neoplastic tissue. In another embodiment, the secretion sample is contacted with autoantibodies which are in turn detected with an autoantibody detection reagent to detect pre-neoplastic or neoplastic cells. In another embodiment, the secretion sample is contacted with autoantibodies which are in turn detected with an autoantibody detection reagent to detect pre-neoplastic or neoplastic antigen associated with prostate cancer. In one embodiment, the autoantibody is autologous. In another embodiment, the autoantibody is heterologous.
In the case of the prostate, the secretions are believed to not be pre-neoplastic nor neoplastic. Instead, they are normally never exposed to the immune system. It is only upon neoplasia that the basal layer of cells that surround the secretions are compromised, allowing the secretions to be exposed to the immune system, that you get autoantibodies to the secretions. Thus, there are autoantibodies to the secretions not because they neoplasia but because the neoplasia has compromised the barrier between the immune system and the secretions. The immune system reacts to these secretions, as it is normally naive to these secretions.
In another embodiment, the secretion sample is contacted with labeled autoantibodies to detect pre-neoplastic or neoplastic antigen. In another embodiment, the secretion sample is contacted with labeled autoantibodies to detect pre-neoplastic or neoplastic tissue. In another embodiment, the secretion sample is contacted with labeled autoantibodies to detect pre-neoplastic or neoplastic cells. In another embodiment, the secretion sample is contacted with labeled autoantibodies to detect an antigen associated with prostate cancer. In one embodiment, the labeled autoantibody is autologous. In another embodiment, the labeled autoantibody is heterologous.
In one embodiment, the labeled autoantibodies or the autoantibodies detected by the reagent are bound to the pre-neoplastic or neoplastic tissue. In another embodiment, the labeled autoantibodies or the autoantibodies detected by the reagent are bound to individual pre-neoplastic or neoplastic cells. In yet another embodiment, the labeled autoantibodies or the autoantibodies detected by the reagent are bound to antigens on the pre-neoplastic or neoplastic cells.
In one embodiment, an increase in signal above control or background of at least 2% indicates the detection of a pre-neoplastic tissue. In another embodiment, an increase in signal above control or background of at 5% indicates the detection of a pre-neoplastic tissue. In another embodiment, an increase in signal above control or background of at least 10% indicates the detection of a pre-neoplastic tissue. In another embodiment, an increase in signal above control or background of at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 250% indicates the detection of a pre-neoplastic tissue.
In one embodiment, an increase in signal above control or background of at least 2% indicates the detection of a neoplastic tissue. In another embodiment, an increase in signal above control or background of at 5% indicates the detection of a neoplastic tissue. In another embodiment, an increase in signal above control or background of at least 10% indicates the detection of a neoplastic tissue. In another embodiment, an increase in signal above control or background of at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 250% indicates the detection of a neoplastic tissue.
The ability to specifically target the autoantibodies and their bound target in the tumor offers a potential therapeutic target for stalling, senescing, killing, or eradicating pre-neoplastic and neoplastic tissue.
In one embodiment, autoantibodies are isolated (i.e., by purification) from the subject and complexed with toxin. The toxin-autoantibody complex is then introduced to the subject, wherein said toxin-autoantibodies complex kill the pre-neoplastic or neoplastic tissue.
In one embodiment, a sample containing autoantibodies is taken from the subject and the autoantibodies are complexed with toxin. The toxin-autoantibody complex is then reintroduced back into the subject harboring the pre-neoplastic or neoplastic tissue or cells.
In one embodiment, autoantibodies are isolated from the subject and complexed with toxin. The toxin-autoantibody complex is then introduced into the general circulation of the subject, wherein said toxin-autoantibodies complex kill the pre-neoplastic or neoplastic tissue.
In one embodiment, autoantibodies are isolated from the subject and complexed with toxin. The toxin-autoantibody complex is then introduced into the blood vessels that perfuse the organ(s) containing the pre-neoplastic or neoplastic cells, wherein said toxin-autoantibodies complex kill the pre-neoplastic or neoplastic tissue.
In one embodiment, a sample containing autoantibodies is taken from the subject and the autoantibodies are complexed with toxin. The toxin-autoantibody complex is then introduced into the general circulation of the subject, wherein said toxin-autoantibodies complex kill the pre-neoplastic or neoplastic tissue.
In one embodiment, a sample containing autoantibodies is taken from the subject and the autoantibodies are complexed with toxin. The toxin-autoantibody complex is then introduced into the blood vessels that perfuse the organ(s) containing the pre-neoplastic or neoplastic cells, wherein said toxin-autoantibodies complex kill the pre-neoplastic or neoplastic tissue.
In another embodiment, the autoantibodies are heterologous but are from the same species. In yet another embodiment, the autoantibodies are heterologous but are from different species. In yet another embodiment, the heterologous autoantibodies are pooled from multiple individuals.
In one embodiment, the toxin-autoantibody complex is introduced into the subject harboring the pre-neoplastic or neoplastic tissue or cells. In another embodiment, the toxin-autoantibody complex is introduced into the circulation of the subject. In another embodiment, the toxin-autoantibody complex is introduced into the blood vessels perfusing the organ with the pre-neoplastic or neoplastic tissue or cells.
In one embodiment, toxins are complexed with molecules that recognize auto-antibodies and the toxin-molecule complex are then administered to the mammal harboring the pre-neoplastic or neoplastic tissue. In one embodiment, the molecules are IgG (anti-IgG), Protein A, Protein G, Fab(2) fragment of an anti-IgG, Fab(1) of an anti-IgG, peptides that bind to the Fc region, and small molecules that bind antibodies. In one embodiment, the anti-IgG is an anti-human IgG or a humanized mouse anti-human IgG In one embodiment the anti-IgG includes the affinity ligand secretory component (Brandtzaeg, 1983), peptides that are designed based on hydropathy (Fassina et al., 1992), fragments of the placental alkaline phosphatase that can bind the Fc region (Makiya and Stigbrand, 1992), multimeric peptides (Verdoliva et al., 1995), one of the peptides from the E. coli surface exposed EiB proteins than bind IgG (Sandt and Hill, 2001), variations of the Fc receptor proteins that can bind IgG (Akilesh et al., 2007; Fridman, 1991; Fridman et al., 1984), anyone of a number of protein G or protein A mimetic (PAM) identified from peptide libraries that bind IgG (Fassina, 2000; Fassina et al., 1996; Fassina et al., 1998), or identified from combinatorial chemical synthesis (Fassina et al., 2001; Kabir, 2002), or combinatorial libraries (Nielsen et al., 2010), phage display libraries (Sakamoto et al., 2009), or rationally designed non-peptidyl mimetics of Protein A (Li et al., 1998), hexamer peptide affinity resins that bind the Fc region of IgG (Yang et al., 2005, 2009a) or the specific hexamer HWRGWV (Yang et al., 2010), use of the all-D amino acid peptide ligands (D'Agostino et al., 2008; Verdoliva et al., 2002), synthetic ligands including cyclic peptides (Verdoliva et al., 2005), a syntheic triazine scaffold substituted with 3-mainopheno and 4-amino-1-maphthol (Teng et al., 2000) or through other combinatorial chemical syntheses to make IgG binding ligands (Teng et al., 1999), affinity ligands that mimic Protein L (Roque et al., 2005), trisubstituted purine derivatives as protein A mimetics (Zacharie et al., 2010; Zacharie et al., 2009), or dendrimeric peptides (Moiani et al., 2009).
Complexing of the toxin to the autoantibody or molecule is performed by general methods of binding the autoantibody or molecules to a toxin. (Yoo et al., 2000)
In one embodiment, the toxin is paclitaxel, adriamycin, beta-emitters, or ricin.
In one embodiment, the toxin complexed with the autoantibody or molecule includes, but is not limited to, alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, camomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE™), Aventis, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In one embodiment the autoantibody, or the agent that recognizes the autoantibody is conjugated via a “slow-release” linkage to a toxin, such as Paclitaxel. The timing of the agent would be adjusted to maximize the extent to which antibody/agent—Paclitaxel which as not bound to the neoplasia has been cleared from the body and the binding of the antibody/agent—Paclitaxel to the neoplasma has been maximized prior to release of the toxin from the antibody (Grube et al., 2003; Jackson et al., 2000; O'Brien et al., 2003; Tanabe et al., 2003; Yang et al., 2009b).
Dosage can be by a single dose schedule or a multiple dose schedule. In a multiple dose schedule the various doses may be given by the same or different routes, e.g., intravenous, blood transfusion, or injection into the artery feeding the organ with the neoplasia. Multiple doses will typically be administered daily, every other day, three times a week, twice a week, or at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).
In another aspect of the invention, the toxin-antibody or the toxin-molecule conjugates of the invention is a pharmaceutical composition suitable for administration to a mammal, preferably a human. To administer the toxin-antibody or the toxin-molecule conjugate composition to humans or animals, it is preferable to formulate the molecules in a composition comprising one or more pharmaceutically acceptable carriers. The phrase “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce allergic, or other adverse reactions when administered using routes well-known in the art. “Pharmaceutically acceptable carriers” include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
Examples of pharmaceutically acceptable carriers or additives include water, a pharmaceutical acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like. Additives used are chosen from, but not limited to, the above or combinations thereof, as appropriate, depending on the dosage form of the present invention.
Dosage can be by a single dose schedule or a multiple dose schedule. In a multiple dose schedule the various doses may be given by the same or different routes, e.g., intravenous, blood transfusion, or injection into the artery feeding the organ with the neoplasia. Multiple doses will typically be administered daily, every other day, three times a week, twice a week, or at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).
Efficacy of therapy can be assessed using any suitable method. Ways of checking efficacy of treatment involves monitoring levels of autoantibodies at the tissue or secretion level as discussed above.
Another way of assessing efficacy or therapy is to screen patient sera or secretions in a suitable immuno-assay (e.g., immunoblot, ELISA, microarray) for immunological reactivity to the antigen used to immunize the subject. A positive reaction between the antigen and the patient sample indicates that the patient has mounted an immune response to the antigen in question. This method may also be used to identify immunodominant antigens and/or epitopes within pathogens or antigens. Efficacy can also be determined in vivo using appropriate animal models of infection by the pathogen of interest, for example, by challenging the animal model with the pathogen of interest.
Efficacy of treatment can also be determine by the complete spectra of tools currently used to follow the efficacy of treatment including whole body scans (CAT Scan, x-ray, PET scans, MRI) and blood tests.
The compositions described herein can be administered in combination with one or more additional therapeutic agents. The additional therapeutic agents may include, but are not limited to antibiotics or antibacterial agents, antiemetic agents, antifungal agents, anti-inflammatory agents, antiviral agents, immunomodulatory agents, cytokines, antidepressants, hormones, alkylating agents, antimetabolites, antitumour antibiotics, antimitotic agents, topoisomerase inhibitors, cytostatic agents, anti-invasion agents, antiangiogenic agents, inhibitors of growth factor function inhibitors of viral replication, viral enzyme inhibitors, anticancer agents, α-interferons, β-interferons, ribavirin, hormones, and other toll-like receptor modulators, immunoglobulins (Igs), and antibodies modulating Ig function (such as anti-IgE (omalizumab)).
In one embodiment, the kit comprises a container with reagents for detecting an autoantibody and instructions for using and measuring the reagent and thus autoantibody to detect or diagnose a pre-neoplastic or neoplastic tissue.
In another embodiment, the kit comprises a container for enriching a subject's IgGs from its serum and then conjugating a label for detecting to the IgG. A subject's blood or serum is introduced into the container and the resulting labeled IgG is then reinjected back into the subject for detection. In one embodiment the subject is human. In another embodiment the subject is an animal.
Kits that utilize reagents for detection of the autoantibodies can be supplied for the present invention. The molecules that detect autoantibodies are typically provided in lyophilized form, either alone or in conjunction with buffers, stabilizers, inert proteins, or the like, in accordance with well-known manufacturing procedures. In one embodiment the agents that detect antibodies are conjugated to labels for detecting where the autoantibodies are bound in either histological sections, in fresh explants, during interoperative procedures or in whole subject scanning. In another embodiment the agents that detect antibodies are conjugated to toxins for killing the pre-neoplasia or neoplasia. In another embodiment the agents that detect autoantibodies are conjugated both to labels for detection and simultaneously conjugated to toxins.
Kits that utilize heterologous autoantibodies, from the same species but not from the subject, can be supplied for the present invention. The autoantibodies are typically provided in lyophilized form, either alone or in conjunction with buffers, stabilizers, inert proteins, or the like, in accordance with well-known manufacturing procedures. In one embodiment the heterologous autoantibodies are conjugated to labels for detection in either histological sections, in fresh explants, during interoperative procedures or in whole subject scanning. In another embodiment the heterologous autoantibodies are conjugated to toxins for killing the pre-neoplasia or neoplasia. In another embodiment the heterologous autoantibodies are conjugated both to labels for detection and simultaneously conjugated to toxins.
Kits that can utilize a subject's own autoantibodies can be supplied for the present invention. The kit comprises a container for enriching a subject's IgGs from its serum. In one embodiment the kit contains reagents for conjugated the subjects IgGs to labels for detection in either histological sections, in fresh explants, during interoperative procedures or in whole subject scanning. In another embodiment the kit contains reagents for conjugating toxins to the subjects IgGs for killing the pre-neoplasia or neoplasia. In another embodiment the kit contains reagents for conjugating both toxins and labels for detection to the subjects IgGs.
Kits can also be supplied for use with the autoantibodies of the present invention. Thus, the autoantibodies are typically provided in lyophilized form, either alone or in conjunction with buffers, stabilizers, inert proteins, or the like, in accordance with well-known manufacturing procedures. In a preferred method of the present invention, antibodies will be utilized in immunohistochemical staining procedures for use in detecting the markers in tissue samples. Tissue samples may be obtained from surgically-removed tissue, which has been frozen and sectioned. The fixed frozen section may be analyzed fixed, such as in formalin, acetone, or other standard histological preservatives, or analyzed unfixed as discussed above.
In one embodiment, the kit comprises a container containing the labeled autoantibody and instructions for using and measuring the autoantibody to detect or diagnose a pre-neoplastic or neoplastic tissue.
In another embodiment, the kit comprises a container with an autoantibody, another container contains the reagents for detecting the autoantibody, and instructions for using and measuring the autoantibody and reagent to detect or diagnose a pre-neoplastic or neoplastic tissue.
The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
1. Mouse Models:
The Alb-c-myc mouse models (Murakami et al., 1993) were a generous gift from Herman Stellar and both prostate models (Pb-_ENREF—23MYC (Ellwood-Yen et al., 2003) in FBV background and conditional Pten knockout (Pb-Cre X Ptenf/f) in C57/B6 background (Trotman et al., 2003a, b)) were a generous gift from Charles Sawyers. Alb-myc and prostate sample controls were C57BL/6J mice purchased from The Jackson Laboratory. All FVB/N-Tg(MMTVneu)202Mul/J mice, and the corresponding control FVB/NJ mice were purchased from The Jackson Laboratory. In addition six week old BALB/c mice (Jackson Labs) and CBySmn.CB17-Prkdcscid/J mice (Jackson Labs) were injected with a 4T1 cell line. One-hundred thousand cells were injected into a single mammary fat pad of each mouse, and mice were euthanized 14 days after injection. All experiments were approved by the Institutional Animal Care and Use Committee at The Rockefeller University.
2. Preparation of Tissue:
All tissue was dissected and placed in 4% paraformaldehyde for fixed tissue samples or Tissue Tek O.C.T. compound for frozen section samples. Frozen sections were then flash frozen in liquid nitrogen. A test sample and an age-matched corresponding WT sample were placed on the same slide for all experiments. In alb-myc and corresponding WT mice, at least one liver section was taken from each liver lobe.
3. Immunofluorescence:
The immunofluorescence detection of endogenous tissue was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems, Tucson Ariz.). Avidin Biotin block was then applied for 12 minutes. The tissue samples were then incubated with biotinylated secondary mouse (Vector Labs, MOM Kit BMK-2202) in 1:200 dilution (6.5 ug/mL). Detection was performed with Blocker D, Streptavidin-HRP D (Ventana Medical Systems) and followed by incubation with Tyramide-Alexa Fluor 488 (Invitrogen, cat #T20992). Negative controls were performed using biotinylated secondary rabbit IgG. (Vector Labs) IgM and IgA studies were processed as above (Invitrogen cat #M31515, M31115). Slides stained with CD34 were done in a similar fashion; however, a rat anti-mouse CD34 antibody (eBioscience, cat #14-0341) was used in 5 ug/ml concentrations. The protocol involves blocking (10% normal rabbit serum, 2% BSA) for 30 minutes, Protease 3 for 4 minutes, and a 7 hour incubation with primary antibody, followed by 16 minute incubation with biotinylated rabbit anti-rat IgG (Vector, cat #BA-4000, 1:200 dilution), Blocker D, Streptavidin-HRP (from DAB detection kit, Ventana Medical Systems), followed by incubation with Tyramide-Alexa Fluor 488 (Invitrogen, cat #T20922). Frozen section slides were dried at room temperature under the hood for 20 minutes, then baked at 56 degrees Celsius for an hour with the slide warmer, prior to immunofluorescence staining.
4. Image Analysis:
All slides were digitally scanned at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using the Zeiss Mirax Scanner with 20×/0.8NA objective and an exposure time of 5 ms with a gain of 2 for the transgenic mice and 10 ms for the xenograft mice.
5. Pathology Analysis:
Adjacent sections were given to a veterinary histopathologist in the Center of Comparative Medicine and Pathology at the Laboratory of Comparative Pathology who was blinded to all sources of tissue and all immunofluorescence results. These slides were stained with hematoxylin and eosin stain (“H&E”) and all tissue was analyzed and graded. All abnormal areas were marked and described by the degree of abnormality. In the liver histology sections, the following grading system, determined by the pathologist, was applied: 0=normal tissue, 1=areas found to have karyomegaly, cytomegaly, cystoplasmic vacuolization or other cellular changes, 2=a focus of defined cellular alteration, 3=an adenoma causing compression of adjacent parenchyma, and 4=carcinoma. The mammary tissue grading scale is as follows: 0=normal tissue, 1=hyperplastic regions without atypia/physiological hyperplasia, 2=hyperplasia with atypia, and 3=carcinoma. The prostate histology grading scale was: 0=normal tissue, grade 1=PIN, and grade 2=carcinoma.
6. EM:
Liver tissues from the alb-myc model and C57Bl/6 model of mice were fixed in 4.0% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) overnight. Sections were processed for immuno electron microscopy to recognize existing antibody as described previously (Uryu et al., 2001). Summation of tissue processing included quenching endogenous peroxidase with 0.5% hydrogen peroxide, blocking nonspecific antibody binding with 3% bovine serum albumin, applying biotinylated anti-mouse IgG, visualizing the immunocomplex with Vectastain ABC Kit (Vector Laboratories, Burlingame, Calif.) and peroxidate base reaction in the presence of 0.5% 3,3′ diaminobenzidine, and application of silver enhancement procedure to DAB immunoreactive products. Subsequently, sections were re-fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, dehydrated by a graded series of ethanol, postfixed with 1% osmium tetra-oxide and embedded in EMBed812. Ultra-thin sections were cut and examined in the electron microscope (100CX JEOL, Tokyo, Japan) with the digital imaging system (XR41-C, Advantage Microscopy Technology Corp, Danver, Mass.). All EM was done in the Electron Microscopy Resource Center at The Rockefeller University.
7. Statistical Analysis:
All statistical analyses were performed using R version 2.12 using the library lme4 (www.r-project.org). All intensity values were normalized per pixel of tissue. Ratios of test sample to control sample were obtained for each slide comparing the number of pixels with intensity values of the top 40% of intensities. These were log-transformed to ensure that the distribution of ratios were approximately normal. One-way random effects model was used to generate p-values while taking into account the clustering resulting from the multiple observations contributed to the analysis by each mouse. (5 contributions in alb-myc tissue, 3 contributions in MMTV-neu, Pb-myc, and PTEN tissue). Random effects model parameters were estimated and tested by restricted maximum likelihood. All p values less than 0.05 were considered statistically significant. All ratios are presented as mean±SEM, with SEM calculated taking into account clustering, again, from the random effect models.
8. In Situ Imaging:
Balb/c mice bearing 4T1 xenograft tumors in the flank were euthanized and dissected to expose the tumor. Rat anti-mouse IgG conjugated to Cy5.5 was purchased from Jackson ImmunoResearch Laboratories (West Grove, Pa.), and goat anti-rabbit IgG was obtained from Invitrogen Corporation (Carlsbad, Calif.). Antibodies were diluted to a 0.67 nanomolar concentration in PBS, incubated with the tissue for 5 minutes, and washed with PBS. All images were acquired using the IVIS Lumina imaging system (Caliper Life Sciences, Inc).
MYC oncogene overexpression is frequently seen in patients with hepatocellular carcinoma (Shachaf et al., 2004). Therefore, the presence of autoantibodies in an alb-myc mouse model of cancer was probed.
Liver tissue samples from this tumor model (Murakami et al., 1993) and from wild-type (“WT”) mice were paraffin embedded, mounted on the same slide, and probed with fluorescently tagged horse anti-mouse IgG antibody. In the alb-myc mice, anti-mouse IgG recognized autoantibodies throughout the tissue. Fluorescence was speckled with some areas considerably brighter than others. The anti-mouse IgG associated fluorescent intensity was approximately 50-fold higher in all liver tissue from alb-myc mice relative to tissue from WT mice (1.68±0.366 log 10-fold greater, p<0.001, 12 alb-myc/WT pairs, 3-5 tissue slices per mouse) (
Similar results were observed with goat anti-mouse IgG. In contrast, there was no difference observed between alb-myc and WT tissue when probed with goat anti-rabbit IgG or chicken anti-rabbit IgG (Supplemental
Adjacent sections were stained with H&E and analyzed by a histopathologist. All alb-myc tissue was graded as either 0 (normal), 1 (areas with cellular alterations), 2 (foci of alteration), 3 (adenoma causing compression) or 4 (carcinoma). All WT liver sections were histopathologically normal and will be referred to as WT. Two alb-myc mice had liver sections with grade 4 lesions, ten had grade 3 lesions, eight had grade 2 lesions, and five had grade 1 changes. Two alb-myc mice had no abnormalities on H&E. The fluorescent intensity of anti-mouse IgG was greater in all tumor grades relative to WT (grade 4: 1.74±0.322 log-fold greater, 3: 1.67±0.386, 2: 1.82±0.403, 1: 1.71±0.553; 0: 1.68±0.458; all p<0.001). When all abnormal grades were analyzed together (grades 1-4), the mean intensity was 1.76±0.351 log-fold greater than the WT group (p<0.001) (
Regions graded 1-4 in the alb-myc tissue were also compared to grade 0 regions within the same tissue. Anti-mouse IgG localized to areas marked as abnormal on histopathology (
The variability of binding observed under the lower magnification (
Human breast tumors contain amplification of HER-2/neu in 25-30% of patients (Slamon et al., 1989). Thus, the presence of endogenous antibodies in mammary tissue from virgin and multiparous mice expressing the un-activated neu oncogene, driven by a mouse mammary tumor virus (MMTV) promoter was probed (Guy et al., 1992).
Breast tissue was paraffin embedded and probed with fluorescently labeled anti-mouse IgG; adjacent sections were stained with H&E (n=10 MMTV-neu/WT pairs, 1-2 mammary glands taken from each mouse). Regions were histopathologically graded as either 0 (normal), 1 (hyperplasia without atypia/physiological hyperplasia), 2 (hyperplasia with atypia), or 3 (carcinoma). In the eighteen MMTV-neu tissue samples, six had grade 3 lesions, four had grade 2 lesions, and the remaining samples were grade 0. In the eighteen WT samples, five had grade 2 lesions, eleven had grade 1 regions, and two samples were uniformly grade 0. In the alb-myc model where all cells expressed the activated oncogene, there was a significant difference between grade 0 in the tumor model and grade 0 in the wild-type. In this model where unactivated neu was expressed there were no obvious differences between grade 0 in the wt and tumor model. Further, in many of the WT mice many abnormalities were observed in the breast. Therefore, on each slide, all regions that were histopathologically given the same grade were analyzed as a group, whether the tissue was from a MMTV-neu mouse or a WT mouse.
Grade 3 and 2 lesions had greater antibody binding than histopathologically normal, grade 0 mammary tissue (
Mouse IgG was present in abnormal mammary tissue bound to abnormal ductal and alveolar cells (arrows in
The presence of tissue-bound autoantibodies in prostate cancer using two different mouse models was tested.
The first model expressed human myc under the prostate-specific Pb promoter (Ellwood-Yen et al., 2003). Myc is overexpressed in 30% of human prostate cancers and expression in mice leads to prostatic intraepithelial neoplasia (PIN), followed by invasive adenocarcinoma (Ellwood-Yen et al., 2003).
The second prostate tumor model was a knock-out of the PTEN tumor suppressor. This gene is deleted in 70-80% of human prostate cancers (Gray et al., 1998; Whang et al., 1998) and prostate specific deletion also results in murine prostate cancer (Trotman et al., 2003a). The previous mouse tumor models used were based on overexpression of an oncogene. In this example, a deleted gene was explored to evaluate whether tissue autoantibodies were identifying neoplasia as an inflammatory response to overexpression of a transgene.
In six prostate model samples (four Pb-myc and two PTEN knockout), two Pb-myc were grade 2 (tumor) and the remaining four were grade 1 (PIN). All WT prostates were histopathologically normal. Grade 2 lesions had 2.21±0.373 log-fold greater intensity (p<0.001) than the respective WT (
The levels of antibody binding varied throughout the tumor and PIN cells (
To test if the presence of a tumor causes an immune response that increases autoantibody binding elsewhere in the body, the presence of autoantibodies was probed in other organs, including the brain, stomach, colon, spleen, kidney, lung, liver, and mammary tissue. All organs, other than MMTV-neu mammary tissue and alb-myc liver tissue, were found to be histopathologically normal.
There was no detectable difference in autoantibody binding in the liver, colon, spleen, stomach, lung, brain, and kidney in the MMTV-neu mice compared to WT. The only tissue with greater antibody binding than normal tissue was abnormal (grade 2-3) mammary gland, which had 0.819±0.485 log-fold greater intensity than grade 0 (normal) tissue (
In the alb-myc mice, there was a greater fluorescence in mammary tissue (1.93±0.85 log-fold), brain (0.859±0.23 log-fold) kidney (0.987±0.56 log-fold) and liver (1.28±0.31 log-fold), relative to WT (all p<0.001) (
A xenograft mouse model of cancer was also tested, which gave greater control of tumor cell localization than in transgenic models. The mouse breast cancer 4T1 cell line was injected into a single mammary fat pad in 3 immunocompetent and 3 SCID mice, and mice were dissected on day 14. Assays for autoantibody binding demonstrated mouse IgG bound within the tumor and the microenvironment in immunocompetent mice; however, no detectable antibody was bound in immunosuppressed mice (
The utility of autoantibodies as a diagnostic tool in frozen sections was tested in seven pairs of abnormal and normal mammary tissue (
Mice bearing 4T1 xenografts were used to determine whether the increased antibody binding identified in tissue slices could be used to localize tumor in situ. IgG antibodies from the mice were enriched and labeled with one fluorophore and reinjected back into the mice (
Mice bearing 4T1 xenografts were used to determine if injection of the labeled autoantibodies could be used for intraoperative image guided surgery. Upon opening the abdomen, a fluorescence signal was selectively observed only for the autoantibodies, and not for the non-specific antibodies, and only at the site of the tumor (
Mice bearing 4T1 xenografts were used to determine if agents that can detect autoantibodies can be used intraoperative to guide surgery. The tumor was partially resected from the mouse's flank, and the entire flank region was incubated with a rat anti-mouse IgG conjugated to Cy5.5. To account for non-specific staining of the tissue by the conjugated antibody, a goat anti-rabbit IgG conjugated to AlexaFluor 568 was incubated simultaneously in the same region. Subtracting the anti-rabbit signal from the anti-mouse signal gave specific localization of the remnant xenograft tumors (
The presence of human auto-antibodies bound in a neoplasia was tested in a liver cancer. A biopsy of a fibrolamellar hepatocellular carcinoma from a human was probed with a fluorescently tagged anti-human IgG (
The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications, patents, and GenBank sequences cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments.
This application claims priority to U.S. provisional application Ser. No. 61/534,341, filed Sep. 13, 2011, which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61534341 | Sep 2011 | US |
