MONOCLONAL ANTIBODY-BASED TARGETING OF FOLATE RECEPTORS

Cross-Reference to Related Application(s)

This application claims the benefit of U.S. Provisional Application Serial No. 61/047,636, filed on April 24, 2008, the disclosure of which is incorporated by reference herein.

Field of the Invention

The present invention relates generally to cancer therapy, and more particularly relates to cancers that over-express folate receptors.

Background of the Invention

Folate is an essential vitamin that participates in, for example, single carbon exchange reactions, DNA synthesis, de novo methionine synthesis and catabolism of homocysteine. Folate absorption by cells occurs via two pathways; namely, a transmembrane anion carrier (reduced folate carrier (RFC)) for reduced folates, and a pathway mediated by a folate receptor (FR). Functional defects in these proteins or any substance that prevents folate binding to these proteins could result in an intracellular folate deficiency.

FR is a cell surface glycoprotein that exists as three isoforms: alpha, beta and gamma. FRalpha and FRbeta are attached to the cell membrane via a glycosylphosphatidylinositol (GPI) adduct, whereas FRgamma is secreted due to a lack of the GPI moiety. FRalpha is found in KB cells (a human nasopharyngeal epidermal carcinoma cell line known for its elevated expression of FRalpha), placenta, choroid plexus and CaCo-2 cells. FRbeta is found in placenta, and hematopoietic stem and progenitor cells and FRgamma is primarily released from malignant hematopoietic cells.

Various tumors have been shown to over-express FRalpha (for example, ovary, kidney, lung, breast, brain, endometrium, and myeloid cells of hematopoietic origin) which probably represents a high folate requirement by these cells. Interference of folate uptake by these FRalpha over-expressing cells would be expected to reduce their proliferative activity and eventually cause their death. This over-expression of FR has been exploited for cancer therapy by conjugating various agents to folate for subsequent uptake by cancer cells. These conjugates have included imaging agents, radiopharmaceuticals, toxic peptides and proteins, haptens, anti-sense oligonucleotides, gene therapy vectors, chemotherapeutic agents, viruses and drugs encased in liposomes.

Summary of the Invention

Illustrative techniques of the present invention are directed to a beneficial cancer therapy involving monoclonal antibody-based targeting of folate receptors. Principles of embodiments of the invention, for example, have identified that monoclonal antibodies made against FRalpha prevent the binding and subsequent uptake of folate causing an intracellular folate deficiency in tumor cells that are over-expressing FRalpha. In addition, aspects of the invention have identified that monoclonal antibodies made against FRbeta can be used to prevent the proliferation of various hematological cancers, such as, for example, chronic myelogenous leukemia (CML). In accordance with one embodiment of the present invention, a method for identifying one or more cells that over-express one or more folate receptors includes the steps of: administering a therapeutically effective amount of one or more antibodies to one or more folate receptors; and identifying one or more cells that over-express one or more folate receptors by identifying increased binding of the one or more antibodies with one or more folate receptors.

In accordance with another embodiment of the present invention, a method for targeting one or more pharmaceutical agents to one or more cells that over-express one or more folate receptors includes the step of administering a therapeutically effective amount of one or more antibodies is administered to one or more folate receptors. The one or more antibodies are conjugated to carry one or more pharmaceutical agents targeted for one or more cells that over-express the one or more folate receptors.

In accordance with a third embodiment of the present invention, an exemplary method for blocking the uptake of folate into one or more cells that over-express one or more folate receptors includes the step of administering a therapeutically effective amount of one or more antibodies to one or more folate receptors, such that the therapeutically effective amount of one or more antibodies binds to the folate receptors and in doing so blocks the uptake of folate into one or more cells that over-express one or more folate receptors.

In accordance with a fourth embodiment of the invention, a pharmaceutical composition is provided which includes an antibody specifically adapted to bind to one or more folate receptors.

In accordance with a fifth embodiment of the present invention, a method of producing an antibody that specifically binds to one or more folate receptors includes the steps of isolating one or more cells secreting an antibody that specifically binds to one or more folate receptors and cloning the one or more cells secreting the antibody.

These and other objects, features and advantages of the present invention will become apparent from the following detailed description of illustrative embodiments thereof, which is to be read in connection with the accompanying drawings.

Brief Description of the Drawings

The following drawings are presented by way of example only and without limitation, wherein no limitations with respect to the specific embodiments shown and described herein are intended or should be inferred, and wherein:

FIG. 1 is a diagram illustrating an exemplary determination of the presence of blocking autoantibodies to folate receptors (FRs) in serum, according to an embodiment of the present invention;

FIG. 2 is a diagram illustrating an exemplary determination of the presence of autoantibodies to FRs in serum, according to an embodiment of the present invention;

FIG. 3 is a flow diagram illustrating exemplary techniques for identifying one or more cells that over-express one or more folate receptors, according to an embodiment of the present invention;

FIG. 4 is a flow diagram illustrating exemplary techniques for targeting one or more pharmaceutical agents to one or more cells that over-express one or more folate receptors, according to an embodiment of the present invention; FIG. 5A is a flow diagram illustrating exemplary techniques for blocking uptake of folate into one or more cells that over-express one or more folate receptors, according to an embodiment of the present invention; and

FIG. 5B is a diagram illustrating an exemplary prevention of folate uptake into KB cells by blocking autoantibodies to the folate receptor, according to an embodiment of the present invention.

Detailed Description of the Invention

Principles of the present invention will be described herein in the context of illustrative methodologies for monoclonal antibody-based targeting of the folate receptor in cancer therapy. However, it is to be understood that the teachings of the invention presented herein are more generally applicable to methodologies for diagnosing and/or treating one or more diseases in connection with folate regulation in a patient by administering a therapeutically effective amount of one or more antibodies directed to bind with one or more folate receptors in the patient. A "therapeutically effective amount" of a given compound in

a treatment methodology, as the term is used herein, is intended to refer broadly to an amount of compound sufficient to produce a measurable binding of antibody with one or more folate receptors in the patient. The term "patient" as used herein is intended to refer broadly to mammalian subjects, preferably humans, receiving medical attention (for example, diagnosis, monitoring, etc.), care or treatment.

Antibodies to the folate receptors (for example, FRalpha and/or FRbeta) can bind to these receptors expressed on cells. Because these receptors are over-expressed in certain types of cancers and other diseases, these antibodies can be used to identify these tumors, to target drugs to these tumors, and to block uptake of folate into these cells, without which the cell cannot survive.

One or more embodiments of the invention include using monoclonal antibodies that are made against FRalpha that prevent the binding and subsequent uptake of folate and cause an intracellular folate deficiency in tumor cells that are over-expressing FRalpha. Antibodies to human FRalpha, as described herein, are shown to block the binding and uptake of folate by KB cells in vitro. Additionally, one or more embodiments of the present invention include using monoclonal antibodies made against FRbeta to prevent the proliferation of various hematological cancers such as, for example, chronic myelogenous leukemia (CML).

As described herein, blocking (that is, antibodies that prevent the binding and cellular uptake of folate by the folate receptors) and binding autoantibodies (AuAbs) in women with neural tube defect pregnancy and in cerebral folate deficiency have been identified. Antibody producing cells from the blood of these patients can be isolated and immortalized using established techniques. Cells secreting monoclonal antibodies to the folate receptors can be identified and specific clones can be isolated. Additionally, such human antibodies can be used in the identification and treatment of certain cancers in patients (for example, humans). Monoclonal antibodies with similar properties can also be produced using conventional hybridoma technology in the mouse and then partially modified to render these suitable for use in humans, or also may be produced as human antibody in a transgenic mouse.

As noted above, existing approaches utilizing folate conjugates result in the conjugate entering cells that express either FRalpha or FRbeta isoforms. The use of monoclonal antibodies to FRalpha for cancer therapy, in contrast, leaves FRbeta expressing cells unaffected. One of the concerns of using a chemotherapeutic agent is the relative toxicity of the agent to neoplastic cells in relation to the toxic effect on bone marrow cells. The bone marrow contains both hematopoietic stem and progenitor cells that are undergoing rapid cell

division, and therefore likely have a high requirement for folate. These cells can be distinguished by a CD34 marker present on their surface.

CD34+ cells specifically express the FRbeta and FRgamma isoforms and not the FRalpha isoform, and therefore would not be expected to be affected by a chemotherapeutic agent that is specifically delivered to cells via FRalpha. In addition, because cells of most normal tissue have a relatively low expression of FR, they would be expected to be relatively unaffected by the delivery of drugs and/or pharmaceutical agents via the folate receptor for the duration of the treatment. Therefore, monoclonal antibodies to FRalpha offer specific targeting of tumors that over-express FRalpha without adversely affecting bone marrow stem cells or normal tissue. Also, the use of monoclonal antibodies to FRbeta for cancer therapy (for example, CML) would leave FRalpha expressing cells unaffected.

As noted above, the observation of folate blocking autoantibodies in mothers with neural tube defect pregnancy and in children with cerebral folate deficiency resulted in the use of monoclonal antibodies with similar properties to block folate uptake into cells. As described herein, one or more embodiments of the present invention can be used to deplete intracellular folate in cancers over-expressing the folate receptor and that are dependent on this pathway for folate uptake. Because folate is essential for DNA synthesis and cell replication, blocking folate uptake in cells would lead to intracellular folate deficiency and ultimately cell death. FIG. 1 is a diagram illustrating an exemplary determination of the presence of blocking autoantibodies to folate receptors (FRs) in serum, according to an embodiment of the present invention. By way of illustration only, image 102 illustrates test serum, containing autoantibodies to FR, that has been acidified, charcoal treated (to remove endogenous folic acid (FA)), and neutralized, which is incubated with apo FR covalently bound to a well of a 96-well ELISA plate. The term "apo," as used herein, is preferably defined as the receptor protein without the ligand bound to it. The ligand in this case specifically refers to folic acid or its derivatives.

With continued reference to FIG. 1, image 104 illustrates control serum, lacking autoantibodies to FR, that has been acidified, charcoal treated (to remove endogenous FA), and neutralized, which is incubated with apo FR covalently bound to a well of a 96-well ELISA plate. Image 106 illustrates the apo FR coated wells incubated with folic acid coupled to X, where X can be an enzyme, a radioactive label, a fluorescent marker or a biotin. Image 108 illustrates the apo FR coated wells incubated with folic acid coupled to X, where X can be an enzyme, a radioactive label, a fluorescent marker or a biotin. In both image 106 and

image 108, FRs whose FA binding sites are not blocked by the autoantibody will bind to the folic acid coupled to X.

Image 110 illustrates test serum that has been washed to remove unbound folic acid coupled to X, where again X can be an enzyme, a radioactive label, a fluorescent marker or a biotin. Also, image 112 illustrates control serum that has been washed to remove unbound folic acid coupled to X.

Bound folic acid coupled to X can be detected, for example, by counting radioactivity, or by incubating with an avidin-enzyme complex followed by an appropriate substrate resulting in a color reaction, or by direct color substrate reaction, or by detecting immunofluorescence. The reduction in bound folic acid coupled to X obtained when compared to the control incubation conducted in the absence of serum containing autoantibodies to FRs (i.e., with serum lacking autoantibodies to FRs) identifies the presence of and provides a measure of the titer of the blocking autoantibody to the FRs.

FIG. 2 is a diagram illustrating a determination of the presence of autoantibodies to FRs in serum, according to an embodiment of the present invention. By way of illustration only, image 202 illustrates test serum, containing autoantibodies to FR, that has been acidified, charcoal treated (to remove endogenous folic acid (FA)), and neutralized, which is incubated in a well that was previously coated with apo FR. Image 204 illustrates control serum, lacking autoantibodies to FR, that has been acidified, charcoal treated (to remove endogenous FA), and neutralized incubated in a well that was previously coated with apo FR.

Image 206 illustrates the human antibodies present in the test serum bound to the apo

FR in the well following a wash to remove any unbound human antibody. Image 208 illustrates the lack of any human antibody binding with the control serum incubated with the apo FR in the well after a wash to remove any unbound human antibody. Image 210 illustrates binding of anti-human antibody conjugated with an enzyme, a radioactive label, a fluorescent marker or a biotin to the human antibody previously bound to the apo FR. Also, image 212 illustrates no binding of the anti-human antibody conjugated with an enzyme, a radioactive label, a fluorescent marker or a biotin in the well exposed to the control serum that lacked human antibodies to apo FR. Bound anti-human antibody conjugated with an enzyme, a radioactive label, a fluorescent marker or a biotin can be detected by incubating with an avidin-enzyme complex followed by an appropriate substrate resulting in a color reaction, or by direct color substrate reaction, or by detecting immunofluorescence. The increase in bound anti-human antibody conjugated with an enzyme, a radioactive label, a fluorescent marker or a biotin obtained

when compared to the control incubation conducted in the presence of serum lacking autoantibodies to FRs identifies the presence of and provides a measure the titer of the blocking autoantibody to the FRs.

FIG. 3 is a flow diagram illustrating techniques for identifying one or more cells that over-express one or more folate receptors, according to an embodiment of the present invention. Step 302 includes administering a therapeutically effective amount of one or more antibodies to one or more folate receptors. The quantity of antibody that may be administered varies widely and is preferably controlled as a function of, for example, an affinity of the antibody, location and/or type of cancer being targeted, blood supply to a tumor of the cancer being targeted, etc. Step 304 includes identifying one or more cells that over-express one or more folate receptors by identifying increased binding of the one or more antibodies with one or more folate receptors. This increase in binding of the one or more antibodies with the one or more folate receptors can be detected, either as part of step 304 or as a separate step, by use of an appropriately labeled secondary antibody. The cells that over-express folate receptors can include a tumor and/or one or more cancerous cells. The one or more cancerous cells can include, for example, chronic myelogenous leukemia (CML). Also, the folate receptors can include FRalpha, FRbeta and/or FRgamma.

FIG. 4 is a flow diagram illustrating techniques for targeting one or more pharmaceutical agents to one or more cells that over-express one or more folate receptors, according to an embodiment of the present invention. Step 402 includes administering a therapeutically effective amount of one or more antibodies to one or more folate receptors, wherein the one or more antibodies carry one or more pharmaceutical agents targeted for one or more cells that over-express one or more folate receptors. Additionally, the cells that over-express folate receptors can include a tumor and/or one or more cancerous cells. The one or more cancerous cells can include, for example, CML. Also, the folate receptors can include FRalpha, FRbeta and/or FRgamma.

FIG. 5A is a flow diagram illustrating techniques for blocking uptake of folate into one or more cells that over-express one or more folate receptors, according to an embodiment of the present invention. Step 502 includes administering a therapeutically effective amount of one or more antibodies to one or more folate receptors, wherein the therapeutically effective amount of the antibodies bind to the FR and in doing so blocks the uptake of folate into cells that over-express one or more folate receptors.

FIG. 5B is a diagram illustrating the prevention of folate uptake into KB cells by human blocking autoantibodies to the folate receptor, according to an embodiment of the present invention. In connection with FIG. 5B, KB cells were incubated overnight at 37 degrees Celsius ( ⁰ C) with the autoantibodies isolated from either a control subject (lacking autoantibodies) or an index subject (containing autoantibodies). The uptake of [ ³ H]folic acid by the KB cells was then determined. By way of illustration, FIG. 5B depicts line 504, which denotes control incubation lacking the isolated fraction of serum, line 506, which denotes an isolation fraction of serum from a control subject without autoantibodies, and line 508, which denotes an isolated fraction of serum from an index subject containing autoantibodies. Blocking uptake of folate into cells that over-express one or more folate receptors prevents proliferation of the cells that over-express the folate receptors.

As above, the cells that over-express folate receptors can include a tumor and/or one or more cancerous cells. The one or more cancerous cells can include, for example, a leukemia (for example chronic myelogenous leukemia (CML)), and tumors of a reproductive system that over-express a folate receptor. Also, the folate receptors can include FRalpha, FRbeta and/or FRgamma.

Additionally, as described herein, one or more embodiments of the invention can include a purified antibody that specifically binds to a folate receptor, a cell that expresses such an antibody, and/or a pharmaceutical composition including an antibody that specifically binds to a folate receptor. Further, the techniques described herein can include producing an antibody that specifically binds to a folate receptor by isolating cells secreting an antibody that specifically binds to folate receptors and cloning such cells, and/or using hybridoma technology in a mouse and partially modifying the antibody to render it suitable for use in a human. Although illustrative embodiments of the present invention have been described herein with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various other changes and modifications may be made therein by one skilled in the art without departing from the scope of the appended claims.