Patent Application
20130305396
Provided herein is technology relating to transgenic rodents containing human endosialin and their development and use. The rodents and derived organs, cells and biological fluids are useful in the elucidation of endosialin pathways, for example in development and in normal and disease states, as well as the development of endosialin specific compounds that may block or stimulate endosialin expression/function for therapeutic benefit. The rodents may be used to determine pathways and gene elements that can regulate endosialin function during development, homeostasis, or disease for developing agents that can positively or negatively regulate human endosialin function(s).
Angiogenesis is a regulated process involving the formation of new blood vessels. It plays an essential role in normal growth, embryonic development, wound healing, and other physiological processes (Yancopoulos et. al. (2000) Nature 407(6801):242-8). Moreover, de novo angiogenesis is involved in several disease states including cancer, where the formation of new “embryonic-like” blood vessels (referred to as neovascularization herein) appear that differ from normal vasculature with regards to structure and function (Hanahan and Weinberg, (2000) Cell 100(1):57-70; Peters et. al. (2005) Nat. Med. 11:261-262). These vessels have been found to include cells such as endothelial cells, pericytes and fibroblasts. A number of in vivo and in vitro studies have demonstrated biological differences between normal and disease-associated vasculature as determined using various model systems of angiogenesis offering the ability to develop novel anti-angiogenic compounds that can selectively inhibit vessel formation of the embryonic-type, tumor-associated endothelia for therapy of neovascular disease (Dhanabal et. al. (2005) AntiCancer Agents 5:115-130). Neovascular diseases include cancer, opthalmologic indications, inflammation and infectious disease (Conejo-Garcia, et. al. (2005) Blood 105:679-681; Das et. al. (2003) Prog. Retin. Eye Res. 22:721-748; Paleolog et. al. (1999) Angiogenesis 2:295-307; Wilkinson-Berka, et. al. (2004) Cur. Pharm. Des. 10:3331-3348). In addition, diseases exist whereby enhanced angiogenesis may improve the outcome of the disease (Galiano, et. al. (2004) Am. J. Path. 164:1935-1947; Wang et. al. (2004) Stroke 35:1732-1737). In light of these opportunities for therapy, an intense search for potential targets that can specifically inhibit or stimulate disease-associated neovascularization, vessel growth and/or suppression, and function is ongoing. In an attempt to identify such targets, strategies have been designed to identify cell surface antigens of tumor stroma as well as isolate specific proteins or RNA that are expressed in neovascular endothelial and endothelial associated cells (Rettig et. al. (1992) Proc Natl. Acad Sci USA 89(22):10832-6; St. Croix, et. al. (2000) Science 289: 1197-1202). These strategies have identified a cell surface protein that appears to be specifically expressed in pericytes and other tumor stromal cells, called endosialin, CD248 or tumor endothelial marker-1 (TEM1). Studies employing antibodies that can bind to endosialin have identified a subset of cells that express this antigen in endothelial cell cultures as well as a subset of cells in normal tissue of patients. Immunohistochemistry (IHC) studies of malignant tissues have revealed good expression of endosialin in a number of neovascular associated cells in malignant tissues. Expression of endosialin in cell lines derived from embryonic-like endothelial cultures, such as but not limited to HUVEC (Human Umbilical Vein Endothelial Cells) or HMVEC-(Neonatal Dermal Microvascular Endothelial Cells) and primary human pericytes, has also been observed. Endosialin-expressing cells appear to be fibroblastic-like in morphology. Recent studies have found that endosialin is at least expressed in pericytes associated with tumor vasculature (Tomkowicz et al. (2010) Cancer Biol. Ther. 9:1-8).
In 1992, Rettig et al. described monoclonal antibodies that recognize antigens on vessels within various cancer types (Rettig et. al. (1992) Proc Natl. Acad Sci USA 89(22):10832-6). One of these was designated FB5, which recognizes a ˜100 kDa protein on the surface of a neuroblastoma cell line, LA1-5s. FB5 is a murine, IgG1, antibody that binds to endosialin and has been shown to recognize cells associated with tumor vasculature and stromal cells associated with a variety of different cancer types. Structural evaluation classified endosialin as a C-type lectin-like protein, composed of five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats). The protein also contains a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. The protein appears to be a glycoprotein. Carbohydrate analysis shows that the endosialin core protein has an abundance of sialylated, O-linked oligosaccharides, with similarities to sialomucin-like molecules.
U.S. Pat. No. 5,342,757 describes an antibody that binds to a ˜100 kDa protein (endosialin). The antibody was named FB5. Subsequent work combined the complementarity determining regions (CDR) of the mouse FB5 onto a human IgG1 backbone to create a humanized antibody that is able to also bind to vessels within malignant tissues and a subset of cells in HMVEC cultures.
U.S. Pat. No. 7,615,372 describes an antibody that binds to endosialin and can elicit immune effector activity and internalize.
Neovascularization is associated with a number of disease states. In cancer it is believed that neovascularization is important to supply tumors with blood and nutrients. In non-oncology diseases such as retinopathy and macular degeneration, uncontrolled neovascularization causes loss of sight (Wilkinson-Berka, (2004) Curr Pharm Des. 10(27):3331-48; Das and McGuire, (2003) Prog Retin Eye Res. 22(6):721-48). Moreover, several reports have identified a role of neovascularization in inflammatory disease (Paleolog and Miotla, (1998) Angiogenesis 2(4):295-307). Methods to better define the embryonic-like endothelial and precursor cells as well as methods to study cells that associate with these endothelial cells involved with the aforementioned disease states will lead to the development of novel drugs to treat these diseases. Conversely, neovascularization is associated with wound healing (Galiano et. al. (2004) Am J. Pathol. 164(6):1935-47). Many of these complex processes are difficult to study in vitro, therefore an in vivo model that can test the effects of agents that can affect the human endosialin protein will enable the development, refinement and validation for understanding the role of this pathway in humans and potentially lead to the identification of agents that can be used for therapeutic benefit including compounds to treat cancer, inflammatory disease as well as those that can enhance wound treatment associated with trauma, burns and infection.
In vivo models are valuable for studying complex, multicellular processes. The degree of homology between human and endosialin gene products of other species varies, with rodent species sharing less than 80% homology (Opavsky et al., J Biol. Chem. 2001 Oct. 19; 276(42):38795-807; Carson-Walter et al., Cancer Res. 2001 Sep. 15; 61(18):6649-55). Therefore animal models that express the human endosialin gene product would be beneficial for identifying and developing agents that can bind to human endosialin mRNA or protein to study its biological roles in angiogenesis and neovascular disease as well as other biological processes such as inflammation, ophthalmologic disease, wound healing and animal development. The use of animal models expressing human endosialin has not been described previously. Moreover the development of human endosialin expressing rodents whereby the human gene is substituted for the rodent homolog within the natural rodent genome and where the human gene expression behaves identically as the endogenous rodent offers an important use for these animals in the study of human endosialin biology in normal tissues as well as in various disease states and in the development of agents that can bind to the human gene product in vivo.
Provided herein are rodents that express a nucleotide sequence encoding human endosialin (e.g., SEQ ID NOs: 3 and 4). In preferred embodiments, the rodent is a mouse. Murine endosialin nucleotide and amino acid sequences are provided in SEQ ID NOs: 1 and 2, respectively. Preferably, the nucleotide sequence encoding human endosialin is integrated into the native or endogenous endosialin gene locus. More preferably, the endogenous endosialin gene product is functionally disrupted or is substantially reduced or null in the transgenic rodent. Preferably, the transgenic rodent comprises a nucleotide sequence encoding human endosialin that is under the control of the rodent's endogenous gene expression regulatory sequences. In some embodiments, the construct containing the human endosialin-encoding nucleotide sequence also contains a reporter gene or a selectable marker (e.g., a positive selection marker and/or a negative selection marker). The nucleotide sequence encoding human endosialin is preferably expressed in a similar development and disease response pattern as that of the native endosialin gene product in parental or wild type rodents. This feature makes these rodents useful for studying the effects of test agents to positively or negatively affect endosialin biology for therapeutic use. Use of human endosialin expressing rodents lacking native endosialin gene product (HUE rodents) is proposed as a strategy for developing agents that can positively or negatively affect the endosialin pathway and also serve as a screening tool to identify those agents that may be useful as human therapies.
Rodents expressing a nucleotide sequence encoding human endosialin from the endogenous endosialin locus whereby the human endosialin sequence is expressed similarly as the rodent endosialin sequence during various physiological processes are provided.
Provided are the progeny of the transgenic rodents expressing human endosialin.
Provided are cells isolated from the transgenic rodents expressing human endosialin. These cells can be isolated from normal tissue, malignant tissue, inflamed tissue or diseased eye.
Also provided are methods of using the human endosialin expressing rodents, referred to as HUE rodents, to study the biology of the human endosialin and to develop agents that can affect endosialin biology as it relates to primary endothelial cell and pericyte populations, malignant tissues or normal tissues as well as other diseases.
In some embodiments, the mice of the invention have a nucleotide sequence encoding human endosialin knocked into the mouse endosialin locus on the proximal region of mouse chromosome 19.
In some embodiments, the mice consist of a C57BL/6 strain, whereby studies of human endosialin can be conducted in these mice or mice in which the human endosialin locus is crossed to other mouse strains as commonly done by methods known to those skilled in the art.
Provided is a method of studying the role of human endosialin in normal development comprising measuring the level of expression of the human endosialin in the organs, tissues or cells of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent. In preferred embodiments, the level of expression of the human endosialin is measured at a particular developmental stage of the rodent.
Also provided is a method of studying the role of human endosialin function in normal development comprising localizing the expression of the human endosialin in the organs, tissues or cells of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent. In preferred embodiments, the nucleotide sequence comprises a reporter gene or selectable marker.
Provided is a method of studying the role of human endosialin in supporting or preventing a disease phenotype comprising measuring the level of expression of the human endosialin in cells of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent wherein the rodent exhibits the disease phenotype. In preferred embodiments the phenotype is cancer. In some preferred embodiments the phenotype is inflammatory disease. In further preferred embodiments the phenotype is eye disease. In yet further preferred embodiments the phenotype is reduced wound healing.
Provided is a method of studying the role of human endosialin in supporting or preventing a dysplastic phenotype comprising grafting a tumor onto a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent and measuring the level of expression of the human endosialin in cells of the rodent.
Also provided is a method of studying the role of human endosialin in supporting or preventing a dysplastic phenotype comprising grafting a tumor onto a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent and localizing the expression of the human endosialin in cells of the rodent. In preferred embodiments, the nucleotide sequence comprises a reporter gene or selectable marker.
Provided is a method of studying the role of human endosialin in supporting or preventing a dysplastic phenotype comprising grafting a tumor onto the progeny of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent and an immunocompromised rodent and measuring the level of expression of the human endosialin in cells of the progeny. In preferred embodiments, the nucleotide sequence comprises a reporter gene or selectable marker.
Also provided is a method of screening test pharmacological agents to identify a targeting agent for human endosialin comprising administering a test pharmacological agent to a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, measuring human endosialin activity in the rodent and comparing the human endosialin activity to a control. An increase or decrease in human endosialin activity relative to the control can be indicative of a targeting agent for endosialin. Also provided is a method of validating an agent for human endosialin comprising administering the agent to a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, measuring human endosialin activity in the rodent and comparing the human endosialin activity to a control. An increase or decrease in human endosialin activity relative to the control can validate the agent for endosialin. In preferred embodiments of the methods described herein, the targeting agent is an antibody or antigen-binding fragment thereof. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.). In some preferred embodiments, the agent can stimulate human endosialin activity. In further preferred embodiments, the agent can suppress human endosialin activity. In yet further preferred embodiments, the targeting agent is an endosialin binding protein. In some embodiments, the binding protein can stimulate human endosialin activity. In some embodiments, the binding protein can suppress human endosialin activity. In some embodiments, the binding protein is bound to an agent that stimulates or suppresses human endosialin activity. In some preferred embodiments, the agent is a nucleic acid that can complementary bind to human endosialin mRNA. In some preferred embodiments, the agent is a small chemical molecule. In some embodiments, the test agent is bound to a detectable label. Exemplary detectable labels include but are not limited to chemiluminescent compounds (e.g., an acridinium ester compound), a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, or an enzyme.
Further provided is a method of screening test pharmacological agents to identify a targeting agent for human endosialin comprising contacting a test pharmacological agent to a cell of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent measuring human endosialin activity in the cell and comparing the human endosialin activity to a control. An increase or decrease in human endosialin activity relative to the control can be indicative of a targeting agent for endosialin. Also provided is a method of validating an agent for human endosialin comprising contacting said agent to a cell of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of said rodent, measuring human endosialin activity in the cell and comparing the human endosialin activity to a control. An increase or decrease in human endosialin activity relative to the control can validate the agent for endosialin. In preferred embodiments of the methods described herein, the targeting agent is an antibody or antigen-binding fragment thereof. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.). In some preferred embodiments, the agent can stimulate human endosialin activity. In further preferred embodiments, the agent can suppress human endosialin activity. In yet further preferred embodiments, the targeting agent is an endosialin binding protein. In some embodiments, the binding protein can stimulate human endosialin activity. In some embodiments, the binding protein can suppress human endosialin activity. In some preferred embodiments, the agent is a nucleic acid that can complementary bind to human endosialin mRNA. In some preferred embodiments, the agent is a small chemical molecule. In some embodiments, the test agent is bound to a detectable label. Exemplary detectable labels include but are not limited to chemiluminescent compounds (e.g., an acridinium ester compound), a phosphorescent compound, a fluorescent compound, a radiolabel, biotin, or an enzyme.
Provided is a method of screening test pharmacological agents to identify a targeting agent for human endosialin comprising administering a test pharmacological agent to a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, measuring human PDGF receptor pathway activity in endosialin expressing cells of the rodent and comparing the human PDGF receptor pathway activity to a control. In some preferred embodiments, the agent stimulates PDGF receptor pathway in endosialin expressing cells. In some preferred embodiments, the agent suppresses PDGF receptor pathway in endosialin expressing cells. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Provided is a method of screening test pharmacological agents to identify a targeting agent for human endosialin comprising contacting a test pharmacological agent to a cell of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, measuring human PDGF receptor pathway activity in the cell and comparing the human PDGF receptor pathway activity to a control. In some preferred embodiments, the agent stimulates PDGF receptor pathway in endosialin expressing cells. In some preferred embodiments, the agent suppresses PDGF receptor pathway in endosialin expressing cells. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Also provided is a method for screening for test agents that can suppress disease comprising administering a test agent to a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent wherein the rodent exhibits the disease, measuring presence of the disease in the transgenic rodent and comparing presence of the disease in the transgenic rodent to a control. A decrease in the disease relative to the control can be indicative of an agent that can suppress the disease. Also provided is a method for validating an agent that can suppress disease comprising administering the agent to a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent wherein the rodent exhibits the disease, measuring presence of the disease in the transgenic rodent, and comparing presence of the disease in the transgenic rodent to a control. A decrease in the disease relative to the control can validate an agent that can suppress the disease. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.). In preferred embodiments, the disease is cancer. In other preferred embodiments the disease is inflammatory disease. In further preferred embodiments the disease is eye disease. In yet further preferred embodiments the disease is reduced wound healing.
Further provided is a method for screening for test agents that can suppress disease comprising contacting a test agent to a cell of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent wherein the rodent exhibits the disease, measuring presence of the disease in the cell and comparing presence of the disease in the cell to a control. A decrease in the disease relative to the control can be indicative of an agent that can suppress the disease. Also provided is a method for validating an agent that can suppress disease comprising contacting the agent to a cell of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent wherein the cell exhibits the disease, measuring presence of the disease in the cell, and comparing presence of the disease in the cell to a control. A decrease in the disease relative to the control can validate an agent that can suppress the disease. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.). In preferred embodiments, the disease is cancer. In other preferred embodiments the disease is inflammatory disease. In further preferred embodiments the disease is eye disease. In yet further preferred embodiments the disease is reduced wound healing. Also provided is optionally a tumor can be grafted onto the transgenic rodent. Also provided is optionally the transgenic rodent has metastases.
Provided is a method for screening for test agents that can suppress tumor growth comprising grafting a tumor onto a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, administering a test agent to the transgenic rodent, measuring tumor growth in the transgenic rodent and comparing tumor growth in the transgenic rodent to a control. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Provided is a method for screening for test agents that can suppress tumor growth comprising grafting a tumor onto the progeny of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent and an immunocompromised rodent, administering a test agent to the transgenic rodent, measuring tumor growth in the transgenic rodent and comparing tumor growth in the transgenic rodent to a control. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Also provided is a method for screening for test agents that can suppress tumor growth comprising grafting a tumor onto a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, administering a test agent to the transgenic rodent, localizing tumor cells in the transgenic rodent and comparing the localization of tumor cells in the transgenic rodent to a control. In preferred embodiments, the nucleotide sequence comprises a reporter gene. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Provided is a method for screening for test agents that can suppress tumor growth comprising grafting a tumor onto the progeny of a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent and an immunocompromised rodent, administering a test agent to the transgenic progeny rodent, localizing tumor cells in the transgenic progeny rodent and comparing tumor cell localization in the transgenic progeny rodent to a control. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Also provided is a method for determining the optimal dosage of an agent, for example, an agent identified by either of the previous screening methods, in treating the disease comprising administering the agent to the transgenic rodent at different dosages, measuring the presence of the disease in the transgenic rodent and determining at which dosage the agent suppresses the disease in the transgenic rodent. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Provided is a method for determining the optimal dosage of an anti-endosialin antibody or antigen-binding fragment thereof in treating a disease comprising administering the antibody or antigen-binding fragment thereof at different dosages to a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, measuring the presence of the disease in the transgenic rodent and determining at which dosage the agent suppresses the disease in the transgenic rodent. In preferred embodiments, the disease is cancer, inflammatory disease, eye disease or reduced wound healing.
Provided is a vector comprising the human endosialin open reading frame cloned in between genomic regions endogenous to the rodent endosialin locus.
Also provided is a cell isolated from a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent. In preferred embodiments the cell is isolated from normal tissue. In other preferred embodiments the cell is isolated from malignant tissue. In further preferred embodiments the cell is isolated from inflamed tissue. In yet further preferred embodiments the cell is isolated from diseased eye.
Provided is a method for screening for gene expression with a cell isolated from a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, comprising measuring an expression profile in the cell. In some embodiments, gene expression is measured before, simultaneously with, and/or after administration of a test agent. In preferred embodiments the expression profile is measured directly after isolation of the cell. In some preferred embodiments the expression profile is measured after culturing the cell in the presence of stimulants or nutrients. In further preferred embodiments the expression profile is measured after treatment with a test agent. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.). In some preferred embodiments the cell is analyzed for RNA profiles. In further preferred embodiments the cell is analyzed for cDNA profiles. In yet further preferred embodiments the cell is analyzed for protein profiles.
Provided is a method for screening test agents that inhibit the growth and/or differentiation of endosialin expressing cells comprising administering a test agent to the cell isolated from a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, measuring the growth and/or differentiation of the cell and comparing the growth and/or differentiation to a control untreated cell, wherein the test agent inhibits the growth and/or differentiation of the cell if the level of growth and/or differentiation of the cell is lower than the level of growth and/or differentiation of the control untreated cell. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Also provided is a method for screening for test agents that stimulate the growth and/or differentiation of endosialin expressing cells comprising administering a test agent to the cell isolated from a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, measuring the growth and/or differentiation of the cell and comparing the growth and/or differentiation to a control untreated cell, wherein the test agent stimulates the growth and/or differentiation of the cell if the level of growth and/or differentiation of the cell is higher than the level of growth and/or differentiation of the control untreated cell. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Provided is a method for screening for test agents that inhibit normal biological activity of endosialin expressing cells comprising administering a test agent to the cell isolated from a transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, measuring the biological activity of the cell and comparing the biological activity to a control untreated cell, wherein the test agent inhibits the biological activity of the cell if the level of biological activity of the cell is lower than the level of biological of the control untreated cell. In some embodiments the biological activity of the cell comprises the binding of endosialin to collagen Col I, Col IV or fibronectin. In some embodiments the biological activity of the cell comprises cell adhesion to extracellular matrices. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
In preferred embodiments of the previous methods, the agent is an antibody. In further preferred embodiments the antibody is a monoclonal antibody. In some preferred embodiments the antibody is a polyclonal antibody. In some preferred embodiments the antibody is an antigen-binding fragment of an antibody. In further preferred embodiments the antibody contains 1-6 CDRs that can bind to human endosialin.
In preferred embodiments of the previous methods, the agent is a protein. In further preferred embodiments the protein is a full length protein. In some preferred embodiments the protein is a peptide. In some preferred embodiments the protein is a mimetic.
In preferred embodiments of the previous methods, the agent is a chemical. In further preferred embodiments the chemical stimulates endosialin gene expression. In some preferred embodiments the chemical suppresses endosialin gene expression. In some preferred embodiments the chemical suppresses endosialin protein function. In further preferred embodiments the chemical stimulates endosialin protein function.
In preferred embodiments of the previous three methods, the agent is an antisense oligonucleotide. In further preferred embodiments the agent is an RNAi molecule.
Provided is a method for testing agents for detecting human endosialin expressing cells in vivo comprising administering the agent to the transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, and measuring the level of detection of human endosialin expressing cells in the transgenic rodent by the agent and comparing the level of detection of human endosialin expressing cells in the transgenic rodent by the agent to the level of detection of human endosialin expressing cells in the transgenic rodent by a control. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Provided is a method for testing agents that modulate human endosialin expression in cells in vivo comprising measuring the expression of human endosialin in cells of the transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, administering the agent to the transgenic rodent, measuring the expression of human endosialin in cells of the transgenic rodent after administration of the agent and comparing the level of expression of human endosialin in cells of the transgenic rodent after administration of the agent to the level of expression of human endosialin in cells of the transgenic rodent before administration of the agent. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Also provided is a method for screening test agents that inhibit tumor growth comprising grafting a tumor onto the transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, administering a test agent or a control agent to the transgenic rodent, measuring tumor growth in the transgenic rodent; and comparing tumor growth in the transgenic rodent administered with the test agent to tumor growth in the transgenic rodent administered with a control agent wherein the test agent is selected as an agent that inhibits tumor growth if the tumor growth in the transgenic rodent administered with the test agent is decreased with respect to tumor growth in the transgenic rodent administered with the control agent. In preferred embodiments the tumor is syngeneic to the transgenic rodent. In some preferred embodiments the tumor is allogeneic or xenogeneic to the transgenic rodent. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Also provided is a method for screening test agents that inhibit tumor metastasis comprising introducing tumor cells into the transgenic rodent comprising a nucleotide sequence encoding human endosialin integrated into the genome of the rodent, administering a test agent or a control agent to the transgenic rodent, measuring tumor metastasis in the transgenic rodent and comparing tumor metastasis in the transgenic rodent administered with the test agent to tumor metastasis in the transgenic rodent administered with a control agent, wherein the test agent is selected as an agent that inhibits tumor metastasis if the tumor metastasis in the transgenic rodent administered with the test agent is decreased with respect to tumor metastasis in the transgenic rodent administered with the control agent. In preferred embodiments tumor metastasis is measured by quantifying in vivo bioluminescence of lung met cells employing the lung met model. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
The invention also provides a method of using the rodents described herein to identify agents that can positively or negatively affect the expression and/or protein function of human endosialin within these rodents.
The invention further provides a method of using the HUE rodents to optimize the use of the agents for maximizing their exposure to stimulate or suppress a given human endosialin biological activity within these rodents.
The reference works, patents, patent applications, and scientific literature, including accession numbers to GenBank database sequences that are referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.
Standard reference works setting forth the general principles of recombinant DNA technology known to those of skill in the art include Ausubel et al. C
Provided herein are methods for generating a human endosialin expressing rodents, preferably a mouse, whereby the endogenous endosialin gene is made non-functional. The human endosialin rodent, referred to as a HUE rodent, can be used to study the function of human endosialin in animal models as well as to develop agents that can affect human endosialin biological functions. The methods can be used to study the effect of the agents on normal endosialin physiology, or the effect of agents in disease models involving the endosialin pathway.
Without wishing to be bound by any particular theory of operation, it is believed that human endosialin positive cells (HEPCs) are precursors for cells that participate in the formation of vessels associated with neovascularization and neovascular disease such as cancer, inflammation and opthamalogic diseases. Therefore, HUE rodents may be useful in studying the effects of agents that can bind human endosialin protein or mRNA to study the role of endosialin in neovascular associated diseases. Thus, HUE rodents serve as ideal animal models to identify and develop human endosialin targeting agents for treating neovascular disease such as cancer and non-malignant pathologies. Conversely, HUE rodents serve as ideal animals to identify human endosialin targeting agents for promoting wound healing. HUE rodents are also useful for screening for active pharmacologic agents that can bind to human endosialin mRNA or protein including antibodies, endosialin binding proteins, vaccines, nucleic acid-based agents including antisense oligodeoxynucleotides and/or inhibitory micro RNAs, and viral-based gene therapies.
As used herein, the term “agent” or “targeting agent” refers to any molecule that can be used to inhibit or stimulate the activity of human endosialin, including but not limited to small chemical entities, RNAi, antisense oligonucleotides, agonists, natural ligands, antibodies, antigen-binding fragments of antibodies, endosialin binding proteins, vaccines and endosialin expressing whole cell vaccine. The term “targeting agent” or “agent” includes any molecule that binds to human endosialin that is bound to a molecule that stimulates or inhibits the activity of human endosialin.
As used herein, the term “endosialin-positive cell” (EPC) or “endosialin-expressing cell” refers to a cell that expresses endosialin. In some cases, the EPC is isolated from primary tissues or endothelial cultures, for example from HUE rodents described herein, using antibodies or binding proteins that can bind endosialin or endosialin-expressing progeny of such cells. EPCs may have the ability to form vessels.
As used herein, the term “inhibition of growth” means a decrease in the number of cells, in culture, by about 5%, preferably 10%, more preferably 20%, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 100%. In vivo inhibition of cell growth may be measured by assays known in the art. For example, “inhibition of growth” may be used to refer to the instance in which the tumor growth in a HUE rodent contacted with a test agent is reduced relative to the tumor growth in the absence of the test agent.
As used herein, the term “stimulation of growth” means an increase in the number of cells, in culture, by about 5%, preferably 10%, more preferably 20%, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 100%. In vivo stimulation of cell growth may be measured by assays known in the art. For example, “stimulation of growth” may be used to refer to the instance in which the tumor growth in a HUE rodent contacted with a test agent is increased relative to the tumor growth in the absence of the test agent.
The term “analysis” refers to analyzing RNA, protein or cellular profiles from HUE rodents. Analysis can be performed using any method used by those skilled in the art such as but not limited to differential expression methods such as microarray, cDNA libraries, SAGE, subtraction, or protein arrays.
The term “biological effect” refers to the inhibition or stimulation of a condition. A biological effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a biological effect can refer to one or more of the following: (a) an increase or decrease in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of growth of tumor cells in vivo (c) promotion of cell death; (d) inhibition of degeneration; (e) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (f) enhancing the function of a population of cells. Pharmacologic agents and derivatives thereof described herein effectuate the biological effect alone or in combination with conjugates or additional components of the compositions of the invention.
As used herein, the term “about” refers to an approximation of a stated value within an acceptable range. Preferably the range is +/−5% of the stated value.
As used herein, the term “normal tissue” refers to non-diseased tissue from mammalian embryos, fetal or adult sources.
As used herein, the term “diseased tissue” refers to tissue from mammalian embryos, fetal or adult sources that are dysplastic. Tissues can be from but not limited to malignant sources; disease of the eye; or disease from tissues with infection and/or inflammation.
As used herein, the term “endosialin ligand” refers to any protein or biochemical that can bind to cell surface endosialin.
As used herein, the term “targeting construct” or “targeting vector” refers to a vector having a recombinant DNA fragment containing the human endosialin open reading frame cloned in the sense orientation. According to the methods described herein, the nucleotide sequence encoding human endosialin integrates into a genomic fragment containing the native rodent endosialin gene, preferably functionally disrupting the rodent endosialin gene. In some embodiments, the targeting vector may contain a selection marker (e.g., a neomycin selection marker) for facilitating screening of recombinant cells.
As used herein, the term “analytical mice” or “analytical rodents” refers to HUE mice or rodents that can be used to identify agents such as antibodies, antigen-binding fragments thereof or binding proteins that can recognize endosialin expression on the cell surface or inhibit tumor growth or metastasis; agents such as antisense deoxynucleotides or inhibitory RNAs that can bind to human endosialin message in the cell in which these agents can be tested for the ability to affect endosialin pathway as it relates to disease and potentially be used to discover, optimize and/or validate the use of an agent for therapeutic use or diagnosis.
The term “functionally disrupted,” as used herein, refers to a gene that has a mutation that prevents the normal function of the gene, e.g., prevents expression of a gene of interest product or prevents expression of normal amounts a gene of interest product. The mutation causing the functional disruption can be an insertion, deletion or point mutation(s) which alters the amino acid sequence of the endogenous gene product encoded therein. The mutation causing the functional disruption may also be a replacement of the endogenous gene with a transgene (i.e, a “knock-in”).
The term “substantially reduced or null” is intended to mean that essentially undetectable amounts of normal endogenous gene product is produced in cells of the animal. This type of mutation is also referred to in the art as a “null mutation” and an animal carrying such a null mutation is also referred to as a “knockout animal.” This type of mutation can also be referred to as a “knock-in” when the endogenous gene is replaced with a transgene.
The term “transgenic rodent”, as used herein, refers to a rodent having cells that contain a transgene, wherein the transgene was introduced into the rodent or an ancestor of the rodent at a prenatal, e.g., an embryonic, stage. A “transgene” is a DNA which is integrated into the genome of a cell from which a transgenic rodent develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic rodent. A type of transgenic rodent is the “knock-in,” where the endogenous gene is replaced with a transgene.
Human endosialin rodents are rodents engineered to express a human endosialin gene from the rodent's endogenous endosialin gene locus. The endogenous endosialin gene of HUE rodents is preferably functionally disrupted or substantially reduced or null. The HUE rodent therefore can be used to screen, develop and study agents that can have a direct effect on human endosialin in suppressing or promoting biological pathways associated with development and/or disease. These rodents provide unique models to study the function of human endosialin in light of the fact that rodent endosialin is functionally disrupted or substantially reduced or null by the introduction of the human gene into the functional region of the gene encoding for the rodent endosialin. This feature avoids any potential redundancy that can occur if rodent endosialin is present which may be able to support a disease state or development process. Traditional transgenic mice that contain a human endosialin gene and an intact rodent endosialin gene may diminish the ability to clearly identify agents that can bind human endosialin to elucidate the agent's pharmacologic activity on suppression or activation of human endosialin gene function in supporting a disease state.
In preferred embodiments the rodent is a mouse. Preferred mice of the invention are those in which various mouse strains are used for disease models. These strains include but are not limited to DBA2, C57BL/6, and SJ123 strains. The mice are rendered deficient for endogenous mouse endosialin but have a functional human endosialin gene product.
Preferred transgenic rodents described herein are made by homologous recombination of a targeted native or endogenous allele encoding endosialin with a transgene comprising a sequence encoding human endosialin flanked by flanking sequences which effect the homologous recombination of the transgene with the native allele, wherein the expression of the human endosialin is under the control of native gene expression regulatory sequences of the native allele. In preferred embodiments, at least a portion of the native endosialin allele is replaced with the human endosialin sequence.
HUE rodents are engineered to express human endosialin and lack endogenous endosialin gene product. In some preferred embodiments, human complementary cDNA encoding the entire endosialin open reading frame (ORF) is cloned into a rodent genomic fragment containing the rodent endosialin gene locus. The targeting construct may contain a selectable marker (e.g., a neomycin resistance gene) to aid in selection of cells that uptake the construct. The resultant construct is then linearized and introduced in a rodent embryonic stem cell (ESC) line, and screened via polymerase chain reaction (PCR) for the integration of the construct into the rodent endosialin locus by using standard methods known to those skilled in the art of generating gene knock out or transgenic embryonic stem (ES) cells. Clones found to have the targeting construct integrated into the endogenous rodent endosialin gene locus are expanded and further validated by southern blot. A clone is used to inject into rodent blastocysts and resultant rodent pups are screened using methods known to those skilled in the art, including genomic PCR or southern blotting using DNA extracted from tail clippings to identify those rodents containing the targeting construct. These chimeric rodents are then grown and crossed to non-transgenic rodents to generate heterozygotes and ultimately homozygous rodents containing null rodent endosialin and functional human endosialin expression.
Also provided are isolated genetic knock-in cells. In some embodiments, the isolated knock-in cell is a genetic knock-in primary cell obtained from a knock-in rodent as described above. In some embodiments, the isolated knock-in cell is a clone of, is genetically identical to, or, preferably is progeny of a genetic knock-in primary cell obtained from a knock-in rodent as described above.
The antibodies and binding proteins of the invention specifically bind endosialin on the cell surface within or derived from HUE rodents. In some embodiments, the antibodies and proteins bind to cells expressing human endosialin within normal tissue. In other embodiments, the antibodies and proteins bind to cells in diseased tissue. In yet other embodiments, the antibodies and proteins bind to cells in primary cultures derived from HUE rodents. In some embodiments, the antibody or binding protein is bound to a molecule that inhibits or stimulates human endosialin or to a detectable label.
Preferred antibodies or antigen-binding fragments thereof and proteins suitable for use in the method of the invention, include, for example, fully human antibodies, human antibody homologs, humanized antibody homologs, chimeric antibody homologs, Fab, Fab′, F(ab′)2 and F(v) antibody fragments, single chain antibodies, and monomers or dimers of antibody heavy or light chains, endosialin ligands or mixtures thereof that can isolate the cells from tissue or culture.
The antibodies may include intact immunoglobulins of any isotype including types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). The light chains of the immunoglobulin may be kappa or lambda.
The antibody-binding fragments of antibodies include portions of intact antibodies that retain antigen-binding specificity, for example, Fab fragments, Fab′ fragments, F(ab′)2 fragments, F(v) fragments, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. Thus, antigen binding fragments, as well as full-length dimeric or trimeric polypeptides derived from the above-described antibodies are themselves useful.
The antibodies, antigen-binding fragments, or endosialin binding proteins may be used alone or as a conjugate. Conjugation of antibodies and binding proteins is well-known in the literature.
The antibodies and antigen-binding fragments thereof include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to its epitope. Examples of suitable derivatives include, but are not limited to glycosylated antibodies and fragments, acetylated antibodies and fragments, pegylated antibodies and fragments, phosphorylated antibodies and fragments, and amidated antibodies and fragments. The antibodies and derivatives thereof of the invention may themselves by derivatized by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other proteins, and the like. Further, the antibodies and derivatives thereof of the invention may contain one or more non-classical amino acids.
Polyclonal antibodies such as those derived from peripheral blood of mammalian cell hosts also may be used. Such cells may be fused with myeloma cells, for example to form hybridoma cells producing antibodies that can bind to human endosialin in vivo.
Without wishing to be bound by any particular theory of operation, it is believed that the antibodies or antigen-binding fragments thereof are particularly useful to bind endosialin expressing tissues and cells within HUE rodents due to a binding of the antibody or antigen-binding fragment thereof to an extracellular epitope. This leads to a decrease in the dissociation (Kd) of the antibody. This is an especially good feature for targeting cells for isolation from tissue or tissue culture.
U.S. Publ. No. 2008-0248034 describing antibodies that bind human endosialin is incorporated by reference herein. In some embodiments, the agent is a monoclonal antibody that binds human endosialin or an antigen binding fragment thereof. In preferred embodiments, the agent is a humanized monoclonal antibody that binds endosialin, or an antigen binding fragment thereof. Preferably, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively. Most preferably, the agent is a humanized monoclonal antibody comprising a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:5, 6, and 7, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 8, 9, and 10, respectively, or an antigen binding fragment thereof. In one embodiment, the agent is MORAb-004 (Morphotek Inc., Exton, Pa.).
Nucleic acids agents of the invention are those that can be administered via intravenous, intramuscular, aerosolized, topical or oral routes to inhibit or suppress the in vivo expression of human endosialin. Nucleic acids include antisense deoxynulceotides, siRNA and miRNA, as well as derivatives thereof. Not wanting to be limited on use, however, the development of nucleic acid agents that work in vivo is not obvious using in vitro based systems. Therefore the use of HUE rodents to screen for nucleic acid agents enables the development of the agents for potential pharmacologic use. Nucleic acid agents may be used alone or as a conjugate. Conjugation of nucleic acids is well-known in the literature.
Nucleic acid screening includes the effect of the agents in suppressing human endosialin expression in positive normal or diseased tissues whereby analysis is done measuring steady state mRNA, protein and/or endosialin expressing cells derived from exposed HUE rodents.
The analytical rodents of the invention include rodents that are prepared to exhibit a disease state such as but not limited to cancer, inflammatory or opthalmologic disease whereby human endosialin agents can be tested for altering the disease state. Conversely, analytical rodents can be used in models where defective or suboptimal endosialin activity exists such as but not limited to wound healing whereby the agents can be screened to identify those capable of stimulating a defective biological process via human endosialin activity.
The invention also encompasses the use of HUE rodents to identify agents that can be used as diagnostic probes to screen for the presence or absence of disease. Diagnostic probes include molecular entities that are used by those skilled in the art, including but not limited to monoclonal antibodies, binding proteins and labeled complementary nucleic acids to the human endosialin gene product.
Screening for antibodies, antigen-binding fragments, or endosialin-binding proteins that specifically bind to cells or tissues expressing human endosialin within HUE rodents or cells, tissues, or fluids derived therefrom may be accomplished using immunoassay techniques known in the art, e.g., immunohistochemistry (IHC) or immunocytochemistry (ICC) in tissues or cells that express endosialin. Conversely, antibodies, antigen-binding fragments, or endosialin binding proteins may be labeled with a detectable label (e.g., radioactive isotope, chromophoric fluorescent dye, chemiluminescent compound, phosphorescent compound, biotin or an enzyme) and used for detection of endosialin-expressing cells, e.g., in in vivo imaging within HUE rodents or tissues thereof. Positive binding antibodies or binding proteins identified from analytical mice can be further screened for reactivity in a cell ELISA-based assay to identify agents that can bind human endosialin expressing cells derived from tissues of HUE rodents. Clones that produce antibodies or binding proteins that are reactive to human endosialin are selected for further expansion and development.
The antibodies, derivatives and binding proteins thereof of the invention have binding affinities that include a dissociation constant (Kd) of less than 1×10−2. In some embodiments, the Kd is less than 1×10−3. In other embodiments, the Kd is less than 1×10−4. In some embodiments, the Kd is less than 1×10−5. In still other embodiments, the Kd is less than 1×10−6. In other embodiments, the Kd is less than 1×10−7. In other embodiments, the Kd is less than 1×10−8. In other embodiments, the Kd is less than 1×10−9. In other embodiments, the Kd is less than 1×10−10. In still other embodiments, the Kd is less than 1×10−11. In some embodiments, the Kd is less than 1×10−12. In other embodiments, the Kd is less than 1×10−13. In other embodiments, the Kd is less than 1×10−14. In still other embodiments, the Kd is less than 1×10−15.
The methods of the invention are suitable for use in disease and non-disease states.
The invention is suitable for screening of agents in HUE rodents that exhibit a dysplastic disorder that is marked by increased expression of human endosialin in the diseased tissue in relation to normal tissues. Malignant tissues that may be studied for anti-human endosialin activity include but are not limited to ovarian tumors, renal tumors, colorectal tumors, pancreatic tumors, prostate tumors lung tumors, fallopian tube tumors, uterine tumors, and brain tumors. Tissues affected with inflammation may also be studied which include but are not limited to arthritic tissue or tissue from inflamed airways. Eye tissue may also be studied for anti-endosialin activity in the mice. Conversely, disease states that can benefit from enhanced endosialin activity can be used to screen for active agents that enhance endosialin biological activity. These can be agents that act as endosialin ligand agonists as well as agents that modify endosialin protein structure to enhance it endogenous activity(s). Recently, it has been demonstrated that endosialin pathway crosstalks with the platelet derived growth factor (PDGF) receptor pathway to promote biological activities associated with neovascularization (Tomkowicz, B., et. al. (2010) Cancer Biol. Ther. 9:1-8). Endosialin binding agents that can promote activity of PDGF receptor pathway may offer benefit to wound healing and other related disorders.
Endosialin binding proteins as well as anti-endosialin antibodies and antigen binding fragments thereof may be used to study pharmacologic activity in HUE rodents. These activities include maximizing therapeutic activity and/or studying effects of agents in toxicologic studies. The anti-endosialin antibodies and antigen binding fragments may be used to study dosing effects and drug concentration for maximizing anti-endosialin activity.
The invention is also suitable for screening for agents that can suppress disease using HUE rodents. These agents may include an angiogenesis inhibitor molecule that modulates an endosialin protein. The methods comprise contacting an HUE rodent bearing a tumor with a test molecule and detecting the inhibition of the tumor growth, wherein the test molecule is identified as an angiogenesis inhibitor molecule that modulates the endosialin protein or activity when the tumor growth in the mouse contacted with the test molecule is reduced relative to the tumor growth in the mouse not contacted by the test molecule.
Any suitable tumor can be injected in any suitable manner to provide a model for the testing of anti-angiogenic molecules. The rodent recipient of the tumor can be any suitable strain. The tumor can be syngeneic, allogeneic, or xenogeneic to the transgenic rodent. The recipient can be immunocompetent or immunocompromised in one or more immune-related functions, included but not limited to nu/nu, scid and beige mice. The HUE rodents can be inoculated with syngeneic tumor cells, e.g. B16 melanoma (ATCC). The effect of antibody or antigen-binding fragment thereof administration on tumor growth can be ascertained by quantizing the primary or metastatic tumor growth using conventional methods.
A variety of different test anti-angiogenic molecules may be identified using the method as provided herein. Anti-angiogenic molecules can encompass numerous chemical classes. In certain embodiments, they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Anti-angiogenic molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The anti-angiogenic molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Anti-angiogenic molecules also include biomolecules like peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Test anti-angiogenic molecules of interest also can include peptide and protein agents, such as antibodies or binding fragments or mimetics thereof, e.g., Fv, F(ab′)2 and Fab.
Test anti-angiogenic molecules also can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
A test anti-angiogenic molecule is identified as an inhibitor when it is capable of specifically inhibiting the growth of a tumor by at least 20%, often 30, 40, 50, 60, 70, 80 or 90%, and sometimes 100%. The growth inhibition can be quantified using any convenient method of measurement. For example, for primary tumor growth, perpendicular measurements are taken of the tumor mass to calculate tumor volume. Metastatic growth can be ascertained by microscopic or macroscopic analysis, as appropriate. The tumor can be syngeneic, allogeneic, or xenogeneic to the transgenic animal. The test molecules can be administered at the time of tumor inoculation, after the establishment of primate tumor growth, or after the establishment of local and/or distant metastases. Single or multiple administration of the test molecule can be given using any convenient mode of administration, including but not limited to intravenous, intraperitoneal, intratumoral, subcutaneous, and intradermal.
The following Examples are provided to illustrate the present invention, and should not be construed as limiting thereof.
HUE mice were engineered to express human endosialin and lack endogenous endosialin gene product. A human complementary cDNA encoding the entire endosialin open reading frame (ORF) was cloned into a mouse genomic fragment containing the mouse endosialin gene locus.
The homology arms and the knock-in region of the targeting vector were created by PCR using the following primers and conditions.
Capital letters represent sequences belonging to the arms. The lower case letters represent restriction endonuclease sites added for subsequent cloning steps. PCR cycling conditions were as follows: 94° C. 15 sec, 58° C. 15 sec, 68° C. 6 min, 25 cycles.
The knock-in region contained the human CD248 cDNA plasmid. These fragments were cloned in the LoxNwCD, and were confirmed by restriction digestion and end-sequencing. The final vector was obtained by standard molecular cloning. Aside from the homology arms and the knock-in human CD248 fragment, the final vector also contains loxP sequences flanking the Neo expression cassette (for positive selection of the ES cells), and a DTA expression cassette (for negative selection of the ES cells). The final vector was confirmed by both restriction digestion and end sequencing analysis. The human endosialin targeting sequence is as follows (SEQ ID NO: 11) (homology arms in italics; knock-in region in bold; LoxP sites underlined; Confirmed sequences highlighted/shaded):
5161 TACTTACAGT TCTTTTGTTT CAGTTCTTTT GGTTTGGTTT TTTTTTTTTT TTTTTTTTTT
6541 GCGTCGCCCT GCATGCTTTG AGCATAAGAC TTATGCTCAA AGGCAAGTGC TGTATAAACA
6601 GCAGTTCTCA CAGCCTGGCT GTGTCTGCAG TGCCGACCTG GGCACTGTGA GGAAGTTAGT
6661 GAAAGCACCC GGGTCCCCAC TGGCATCCAG ATTTGCTCAG GAGACTACCA AACCTTGGTC
6721 CTGTACAAAA CATATATGCA AACAGGGAGG GCTATAACAC AATAGCGGGC TGAAGAGTCC
6781 GCACACATGT ATCCCCATGT TGTGGAGGGG GACAATGGAA ACCTGGCAAG GTCACCTACA
6841 CCTATAATCT CAGTACTGCT GCACAGAGAC CTACACAAAA GGGAAGAGGA GGCAGGAAAA
6901 GAGAAGAGAA AGGGAGGGAA GGGAAGTGGA AAGGATTGAA ATAATGAAGA GGAGAAAGAA
6961 GCGTGTGCGT GCTCGTGTGT GCGTGCATGC GTGAGTGTGT ATCCTAGCAC TTGGGAAGCA
7021 GAGGCATTTG GATCTCTGTG AGCTCCAGAC CAGCCAGAAC TACATGGTCT AAACTACATA
7081 GTGTCTTTAA AAAGGGCTGG GGTGTATGGG GGTGGGACAT GAACGAGGAC TCAGAGAGCC
7141 TCGAACTCAC ACAGGTTAGT AGCAGAGTAT CCAGGCCAGG ACAGTGTTCC AAAGAACAAA
7201 CCTTCTCTTT CACTTTCTCA TCCATGGGTC ATGAGATTTT TAGTGCTGGG AATTCAACCC
7261 AGGACCAGAC ATCACCCTAC CACGAAGCCA CATCCCCAGC TGGCTCAGAA TCTGCGGAGA
7321 ATATAAAGGA TCGTGTGTTA ATGCCAGACT CAGAGGTGAT GGGAAGACCA GGACTTTAAG
7381 GTCATCCTGG ACCACACAGT GAGCTCCAGC TTAGCCTGGG CAACATGTAA GACTTTGCCT
7441 CAAAAAATCA AAAACGGACA GGGCGTAGTG GTGCATGCCT TTAATCCCAG CACTCGGGAG
7501 GCAGAAGCAG GCGGATTTCT GAGTTCGAAG CCATCCTGGT CTACAGAGTG AGTTCCAGGA
7561 CAGCCAAGGC TATACAGAGA AACCCTGTCT CGGAAAAATA AATAAATAAA AAACGGGCTG
7621 GAGTGATGCC TAAGCAGCGA AGAGTACTTC TTGCTCTTGC AGAGGACCCA AGTTTGGTTC
7681 CCAGCACCCA CACGGTAGTT TAAAAATACC CGTAACTCCA GTTCCAGGGA ATTTGATGCC
7741 CTCTTCTGAC TTCTGAAGGT AACAGGCACA CACACGGTAC ACACGCATAC ATGCAGGAAA
7801 AACACTCATA AACAGAAAGC AAAAATGTTT TTAAAAGTCG AAATAAAAAC CAAGAGTAGC
7861 CGGGCACGAT GGCACACTCG GGAGGCAGAG GCAGGCAGAT TTCTGAATTC AAGGCCAGCC
7921 TGGTCTACAG AGTGAGTTCC AGGACAGCCA GGGCTACACA GAGAAACCCT GTCTCAGAAA
7981 ACCAAAACAA ACAAACAAAC AAACAAAAAA CCCCAAGGGT ATAAAAGGAT TACCTGGTGT
8041 TGGGTGACTG TAGTACCTGC TACTTAGAAT CCAGGAGTTT GGAATAGCCT TGGCAACAGA
8101 GCAAACTCCA TCTCTTAAAA ATAAATAAAT AAATAAATAA AGGTAAAAAG AGAATGCAGG
8161 GGTTAATCCT TCACACAGAA TCCCAGGGCC ACCCTGGGTG CCTGGCCCAG TGTGCAAACC
8221 TCCAGAGGCC TCCAGGAAGG GCTGGAAGAG GACCCTGGGA GTAAGGAGGA CAATGTGATG
8281 CCTGTGTCAC ACTTACTATC CTGTTGTGTC CCCATGCCCA TACATGGTCT TGCACAGGTC
8341 CCCTCATAGC AGCCGTGCTC ACCCTGAGCA GAGCTCAGTC TCCACAGAGC CCAGAGTCAT
8401 GCACCCTGCC TTCCTGCAGC AGAAGCCTGG AGAGGCATTT CCTGTTAGGG GGCAGCCTCC
8461 TCCCGCTCTA AGCCCAGGTT CCCAGGGCCC CTGACTGAGC CAGGGTAAGG AGAGAGCAGG
8521 CCTTGGCCTC CTCCCCCCTC CCTAACCCCT AATGCTGGTC CTGCTGGAGC CAAAAAAACT
8581 TGGGAGTGAG GTAGGCTGAG CGAAGGGCTC ATCAAATACG TGTCTGGTTG AGGTGGTTCT
8641 CAAGGAGGTT ATCAAGTTGA TTCTCACGCA GGGCCCAGGC AGGAATGGGT GGGGCCTCAT
8701 CGTTGAGACC GTTTAGCAGG CCACATCATC CTAGCTTATC CACCTAAGGG CATCCCCAGC
8761 ATGTCAGGAG AGCTGCGTGT GTGAGGCTGG GGTTTGGCGG CCTTAGATAG GGTGTCAGAG
8821 AAGATGCTCT TGCCAGGCTT GGAAGGAGTC GTTCACATAT GTGTGATGAG AAAGGGCTGG
8881 CCCGATCAAG TCGTTGAGCT CAGAATGCAG CCAGTATACT GATACTGGTG AATCCAGATC
8941 CCGGCTTTCG GGAGTCTCTC TGGAAGACAG CTTCACCCCA GCTGCCTTAG CTGCAAACAA
9001 CGCTGAAGTT TCAGAGGGTT GACTGGGGAT GCTTCTCAGA CAGACAGGTT CCCTTTTCCC
9061 TGACTCTCAG CTCTGTAACG CCTGAGGTAG CCCGTTAGTT GTACCTCCCC CCTCCTCTGC
9121 CCTTCGCCTC TCACCCCAGA ACCCCCCCCA CCCCACTGCT TCCTGCTCCA GCAGCCCCCG
9181 GGGAGCTAGC AGGGGAGCTG GCAGCGGCCC CAGCCCACTC CTTACAAGGC GTGAGTCCGC
KpnI was used for linearizing the final vector prior to electroporation. The resultant construct was then linearized and introduced in mouse embryonic stem cell (ESC) line, and screened via polymerase chain reaction (PCR) for the integration of the construct into the mouse endosialin locus by using standard methods known to those skilled in the art of generating gene knock out or transgenic embryonic stem (ES) cells. Clones found to have the targeting construct integrated into the endogenous mouse endosialin gene locus were expanded and further validated by southern blot. Clone A was used to inject into mouse blastocysts and resultant mouse pups were screened using methods known to those skilled in the art, including genomic PCR or southern blotting using DNA extracted from tail clippings to identify those mice containing the targeting construct. These chimeric mice were then grown and crossed to C57BL/6 (also referred to as C57 mice) mice to generate heterozygotes and ultimately homozygous mice containing null murine endosialin and functional human endosialin expression.
To demonstrate the utility of HUE analytical mice to screen for agents capable of binding human endosialin, HUE mice were employed in two different cancer models. One model (named SC model) entails the grafting of syngenic tumors implanted subcutaneously into the flank of HUE and wild type C57 control mice, the latter of which has intact mouse endosialin and does not express human endosialin. The other model (Lung Met model) employed the use of C57 syngenic metastatic Lewis lung carcinoma cells, which are stably transduced to express luciferase that can be used to measure tumor volume via in vivo bioluminescence imaging (Min, J, et. al. Nat. Med. 2010 March 16(3): 286-294). These cells, when introduced into HUE or C57 control mice, have been previously shown to be capable of metastasis to the lung of the host animal. See also Jenkins et al, Clin Exp Metastasis. 2003; 20(8):733-44 (human tumor cell lines engineered to express luciferase grafted onto SCID mouse and measured for bioluminescence using bioluminescent imaging (BLI) in response to treatment) which is incorporated herein by reference in its entirety.
In the SC model, after successful implantation of tumor cells into the flank, mice were treated with the humanized anti-human endosialin (MORAb-004) antibody that does not recognize mouse endosialin or a control antibody and tested for the ability to suppress tumor growth as measured on day 17. As shown in
A second example of using HUE analytical mice is shown employing the Lung Met model. Cancer cells were introduced into HUE mice. The treatment with FB5, MORAb-004 or vehicle (phosphate buffer solution (PBS)) were initiated a day prior and continued on day 3, 5, 7, and 10. The magnitude of metastatic disease in the lung was quantified by in vivo bioluminescence imaging three times a week up to day 14. As shown in
In summary, the data described above demonstrate the utility of HUE mice when employed as analytical mice to identify agents that can target human endosialin for discovery, development and validation.
HUE mice generated in accordance with Example 1 were injected with B16F10T1 cells to the flanks subcutaneously. The animals received 5 doses at 40 mg/kg of MORAb-004 or a control of PBS only consecutively starting on day 3 post tumor cell implantations. To assess the tumor vasculature upon treatment, X-ray micro-CT was used. Micro-CT provides an overall analysis of tumor vasculature in the entire tumor and thus may overcome some limitations inherent in some other approaches such as immunohistochemistry.
Seventeen days post implantation; the tumor-bearing animals received a 50 μl intraperitoneal injection of heparin 10 min before being euthanized. The thoracic cavity was opened, a small nick was made in the left atrium of the heart using a 20 G needle, and a polyethylene cannula (inner diameter, 0.58 mm; outer diameter, 0.96 mm) was passed through the left ventricle and manually secured in place. 15-20 ml of PBS was manually perfused at a rate of 6 ml min-1 to remove blood. MICROFIL (Carver, commercially available lead chromate latex), was prepared as recommended by the manufacturer. Mice were then manually perfused with 10 ml of MICROFIL at a rate of 2 ml min-1. The infused latex mixture was allowed to polymerize at room temperature for sixty minutes then the animals were refrigerated at 4-8 degrees Celsius overnight before dissection of tissues of interest. Dissected tumors were immersed in 10% neutral buffered formalin.
The tumors were then imaged with an X-ray micro-CT system performed by Numira Biosciences, and analyzed using image analysis software package (Altaview, Numira). The results can be seen in
This application claims the benefit of U.S. Application No. 61/387,898, filed Sep. 29, 2010, which application is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/53651 | 9/28/2011 | WO | 00 | 6/11/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61387898 | Sep 2010 | US |
