MONOCLONAL ANTIBODIES SPECIFIC TO HUMAN

CAV-1 AND USES THEREOF

[0001] This application claims the benefit of United States Provisional Patent Application Nos. 61/618,305, filed March 30, 2012, which is incorporated herein by reference in its entirety.

[0002] The invention was made with government support under Grant No. ROl CA68814 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

1. Field of the Invention

[0003] The present invention relates generally to the field of molecular biology and oncology. More particularly, it concerns antibodies that specifically bind to human caveolin- 1 (Cav-1) and methods for using such antibodies.

2. Description of Related Art

[0004] Estimates were that there would be 341,290 newly diagnosed cancers of the genitourinary tract in men and 53,850 deaths caused by those cancers in 201 1; prostate cancer alone would account for 71% of the new cases of genitourinary cancer and 63% of cancer- related deaths in men (Siegel et ah, 201 1). PCa is androgen sensitive, and hormone therapy, mainly achieved by androgen deprivation, is one of the main treatment modalities used to manage advanced PCa. However, this therapy is only palliative and has numerous side effects (Loblaw et ah, 2007). Results of clinical studies have also indicated that docetaxel chemotherapy provides only modest survival benefits in castrate-resistant PCa (Petrylak et ah, 2004). When PCa progresses beyond the confines of the prostate gland and metastasizes to distant sites (predominantly bone marrow), the disease is very difficult to control. It is critically important to address this disease by development of sensitive diagnostics and effective therapies to control this devastating malignancy. SUMMARY OF THE INVENTION

[0005] Embodiments of the present invention address deficiencies in the art by providing agents for therapeutic targeting and detection of cancer cells, such an androgen insensitive prostate cancer cells. Specifically, aspects of the embodiments, provide novel Cav-1 -binding antibodies that are effective for detecting cancer cells, neutralizing Cav-1 activity and reducing cancer cell growth.

[0006] In a first embodiment, there is provided an isolated monoclonal antibody wherein the antibody specifically binds to the amino terminal portion a human Cav-1 polypeptide (SEQ ID NO: 1 ; NCBI accession no. NP_001744). For example, the antibody can bind to an epitope between amino acids 1-32, 19-32, 34-48 or 51-64 of the human Cav-1 polypeptide. Examples of such antibodies include, but are not limited to, antibodies that compete for binding of Cav-1 with a 1A3, 3E2 or 3H10 monoclonal antibody. In certain aspects, the antibody binds to an epitope between amino acids 34 and 48 of human Cav-1, such as an antibody that competes for binding Cav-1 with monoclonal antibody 3E2 or 3H10. [0007] In certain embodiments, therefore, provided a recombinant and/or isolated antibody, or antigen-binding portion thereof, that binds to a human Cav-1 polypeptide wherein the antibody comprises CDR sequences homologous or identical to a CDR from an antibody selected from the group consisting of 1A3, 3E2, 3H10 or 1E12. For example, the antibody can comprise (a) a first Vh CDR at least 80%, 85%, 90%, 95% or 99% identical to CDR1 of SEQ ID NO: 3, 7 or 11 ; (b) a second Vh CDR at least 80%, 85%, 90%, 95% or 99% identical to CDR2 of SEQ ID NO: 3, 7 or 1 1; (c) a third Vh CDR at least 80%, 85%, 90%, 95% or 99% identical to CDR3 of SEQ ID NO: 3, 7 or 1 1; (d) a first VI CDR at least 80%, 85%, 90%, 95% or 99% identical to CDR1 of SEQ ID NO: 5, 9 or 13; (e) a second VI CDR at least 80%, 85%, 90%, 95% or 99% identical to CDR2 of SEQ ID NO: 5, 9 or 13; and (f) a third VI CDR at least 80%, 85%, 90%, 95% or 99% identical to CDR3 of SEQ ID NO: 5, 9 or 13. In further aspects, the antibody comprises a first, second or third Vh CDR identical to CDR1, CDR2 or CDR3 of SEQ ID NO: 3, 7 or 1 1 or a sequence differing from the first, second or third Vh CDR of SEQ ID NO: 3, 7 or 1 1 by one, two or three amino acids (preferably differing from the first, second or third Vh CDR by not more than one amino acid). In yet further aspects, the antibody comprises a first, second or third VI CDR identical to CDR1, CDR2, or CDR3 of SEQ ID NO: 5, 9 or 13 or a sequence differing from the first, second or third VI CDR of SEQ ID NO: 5, 9 or 13 by one, two or three amino acids (preferably differing from the first, second or third VI CDR by not more than one amino acid). In some aspects an antibody or fragment thereof of the embodiments comprises a constant or J-region sequences that are substantially non-murine.

[0008] In certain aspects, a polypeptide or antibody of the embodiments comprises all or part of an amino acid sequence corresponding to the MAb 1 A3 variable heavy chain amino acid sequence of FIG. 6 (SEQ ID NO: 11). CDRs are indicated in solid underline. From amino to carboxy terminus the CDRs are CDRl, CDR2, and CDR3. In certain aspects, an antibody of the embodiments comprises a first, second and third Vh CDR at least 80%, 85%, 90% or 95% identical to CDRl, CDR2 and CDR3 of SEQ ID NO: 1 1. In certain aspects a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable heavy chain of MAb 1A3.

[0009] In a further aspect, a polynucleotide comprises all or part of a nucleic acid sequence corresponding to the MAb 1A3 variable heavy chain nucleotide sequence (SEQ ID NO: 10).

[0010] In certain aspects, a polypeptide or antibody of the embodiments comprises all or part of an amino acid sequence corresponding to the MAb 1A3 variable light chain amino acid sequence of FIG. 7 (SEQ ID NO: 13). CDRs are indicated in solid underline. From amino to carboxy terminus the CDRs are CDRl, CDR2, and CDR3. In certain aspects, an antibody of the embodiments comprises a first, second and third VI CDR at least 80%, 85%, 90% or 95% identical to CDRl, CDR2 and CDR3 of SEQ ID NO: 13. In still further aspects, a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable light chain of MAb 1A3.

[0011] In a further aspect, a polynucleotide comprises all or part of a nucleic acid sequence corresponding to the MAb 1A3 variable light chain nucleotide sequence (SEQ ID NO: 12).

[0012] In certain aspects, a polypeptide or antibody of the embodiments comprises all or part of an amino acid sequence corresponding to the MAb 3E2 or 3H10 variable heavy chain amino acid sequence FIG. 2 (SEQ ID NO: 3). CDRs are indicated in solid underline. From amino to carboxy terminus the CDRs are CDRl, CDR2, and CDR3. In certain aspects, an antibody of the embodiments comprises a first, second and third Vh CDR at least 80%, 85%, 90% or 95% identical to CDRl, CDR2 and CDR3 of SEQ ID NO: 3. In certain aspects a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable heavy chain of MAb 3E2 or 3H10. [0013] In a further aspect, a polynucleotide comprises all or part of a nucleic acid sequence corresponding to the MAb 3E2 or 3H10 variable heavy chain nucleotide sequence (SEQ ID NO: 2).

[0014] In certain aspects, a polypeptide or antibody of the embodiments comprises all or part of an amino acid sequence corresponding to the MAb 3E2 or 3H10 variable light chain amino acid sequence of FIG. 3 (SEQ ID NO: 5). CDRs are indicated in solid underline. From amino to carboxy terminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects, an antibody of the embodiments comprises a first, second and third VI CDR at least 80%, 85%, 90% or 95% identical to CDR1, CDR2 and CDR3 of SEQ ID NO: 5. In still further aspects, a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable light chain of MAb 3E2 or 3H10.

[0015] In a further aspect, a polynucleotide comprises all or part of a nucleic acid sequence corresponding to the MAb 3E2 or 3H10 variable light chain nucleotide sequence (SEQ ID NO: 4). [0016] In certain aspects, a polypeptide or antibody of the embodiments comprises all or part of an amino acid sequence corresponding to the MAb 1E12 variable heavy chain amino acid sequence of FIG. 4 (SEQ ID NO: 7). CDRs are indicated in solid underline. From amino to carboxy terminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects, an antibody of the embodiments comprises a first, second and third Vh CDR at least 80%, 85%, 90% or 95% identical to CDR1, CDR2 and CDR3 of SEQ ID NO: 7. In certain aspects a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable heavy chain of MAb 1E12.

[0017] In a further aspect, a polynucleotide comprises all or part of a nucleic acid sequence corresponding to the MAb IE 12 variable heavy chain nucleotide sequence (SEQ ID NO: 6). [0018] In certain aspects, a polypeptide or antibody of the embodiments comprises all or part of an amino acid sequence corresponding to the MAb 1E12 variable light chain amino acid sequence of FIG. 5 (SEQ ID NO: 9). CDRs are indicated in solid underline. From amino to carboxy terminus the CDRs are CDR1, CDR2, and CDR3. In certain aspects, an antibody of the embodiments comprises a first, second and third VI CDR at least 80%, 85%, 90% or 95% identical to CDR1, CDR2 and CDR3 of SEQ ID NO: 9. In still further aspects, a polypeptide can comprise 1, 2, and/or 3 CDRs from the variable light chain of MAb 1E12. [0019] In a further aspect, a polynucleotide comprises all or part of a nucleic acid sequence corresponding to the MAb 1E12 variable light chain nucleotide sequence (SEQ ID NO: 8).

[0020] In some aspects, an antibody of the embodiments is an IgG, IgM, IgA, IgE or an antigen binding fragment thereof. For example, the antibody can be a Fab', a F(ab')2, a F(ab')3, a monovalent scFv, a bivalent scFV, or a single domain antibody. In certain aspects, an antibody of the embodiments is recombinant. In still further aspects, the antibody is a human, humanized antibody or de-immunized antibody.

[0021] Thus, embodiments are directed to monoclonal antibody polypeptides, polypeptides having one or more segments thereof, and polynucleotides encoding the same. In certain aspects, a polypeptide can comprise all or part of the heavy chain variable region and/or the light chain variable region of Cav-1 specific antibody, such as the 1A3, 3E2, 3H10 or 1E12 monoclonal antibodies. In a further aspect, a polypeptide can comprise an amino acid sequence that corresponds to a first, second, and/or third complementary determining regions (CDRs) from the light variable chain and/or heavy variable chain of a Cav-1 specific antibody, such as the 1A3, 3E2, 3H10 or 1E12 monoclonal antibodies. For example, an antibody can comprises (a) a heavy chain comprising Vh CDR sequences from a 1A3, 3E2, 3H10 or 1E12 monoclonal antibody, and a human hinge, CHI, CH2, and CH3 regions from an IgGl, IgG2, IgG3 or IgG4 subtype; and (b) a light chain comprising said VI CDR sequences from a 1A3, 3E2, 3H10 or 1E12 monoclonal antibody, and either a human kappa CL or human lambda CL.

[0022] In still further aspects an antibody of the embodiments is conjugated to a second moiety. For example, the second moiety can be an imaging agent, a chemotherapeutic agent, a toxin or a radionuclide. In certain aspects, the conjugate can be a covalent conjugate, an affinity conjugate (e.g., biotin-avidin conjugation) or a chelation.

[0023] In additional embodiments, pharmaceutical compositions are provided comprising one or more antibodies or antibody fragments that are discussed herein in a pharmaceutically acceptable carrier. Such a composition may or may not contain additional active ingredients, such as a chemotherapeutic agent. [0024] In still a further embodiment there is provided a host cell comprising one or more polynucleotide molecule(s) encoding an antibody of the embodiments. Thus, in some aspects, a method of producing an antibody of the embodiments is provided comprising (a) expressing one or more polynucleotide molecule(s) encoding a VI and Vh chain of an antibody of the embodiments in a cell; and (b) purifying the antibody from the cell. Host cells for use according to the embodiments include, but are not limited to, mammalian cells, yeast cells, bacterial cells, ciliate cells or insect cells.

[0025] In yet a further embodiment there is provided a method for treating a subject having a cancer comprising administering and effective amount of an antibody of the embodiments to the subject. In some aspects, the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer. In certain aspects, the cancer is defined as an androgen insensitive cancer or is resistant to one or more chemotherapeutic agents.

[0026] An antibody or pharmaceutical composition of the embodiments may be administered locally or systemically. For example, administration can be intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneous ly, or locally.

[0027] In yet a further aspects, an antibody or pharmaceutical composition of the embodiments is administered in conjunction with at least a second anticancer therapy. For example, the second anticancer therapy can be a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy or cytokine therapy.

[0028] In still yet a further embodiment, there is provided a method for detecting a cancer in a subject comprising testing (e.g., testing in vitro) for the presence of elevated Cav- 1 relative to a control in a sample from the subject, wherein the testing comprises contacting the sample with an antibody of the embodiments. For example, the testing can be by performing an ELISA assay.

[0029] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0030] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0031] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0032] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0034] FIG. 1: Domain map of selected Cav-1 mAbs. Cav-1 mAbs are shown in relationship to their epitope domains on the Cav-1 polypeptide. [0035] FIG. 2: 3E2 and 3H10 antibody VH sequences. The polynucleotide (SEQ ID

NO: 2) and encoded polypeptide (SEQ ID NO: 3) sequence for the 3E2 and 3H10 antibody VH chain is shown. The signal peptide is indicated by a dashed underline. CDR1, CDR2 and CDR3 sequences are indicated by the solid underline.

[0036] FIG. 3: 3E2 and 3H10 antibody VL sequences. The polynucleotide (SEQ ID NO: 4) and encoded polypeptide (SEQ ID NO: 5) sequence for the 3E2 and 3H10 antibody VL chain is shown. The signal peptide is indicated by a dashed underline. CDR1, CDR2 and CDR3 sequences are indicated by the solid underline.

[0037] FIG. 4: 1E12 antibody VH sequence. The polynucleotide (SEQ ID NO: 6) and encoded polypeptide (SEQ ID NO: 7) sequence for the 1E12 antibody VH chain is shown. The signal peptide is indicated by a dashed underline. CDR1, CDR2 and CDR3 sequences are indicated by the solid underline.

[0038] FIG. 5: 1E12 antibody VL sequence. The polynucleotide (SEQ ID NO: 8) and encoded polypeptide (SEQ ID NO: 9) sequence for the 1E12 antibody VL chain is shown. The signal peptide is indicated by a dashed underline. CDR1, CDR2 and CDR3 sequences are indicated by the solid underline.

[0039] FIG. 6: 1A3 antibody VH sequence. The polynucleotide (SEQ ID NO: 10) and encoded polypeptide (SEQ ID NO: 1 1) sequence for the 1A3 antibody VH chain is shown. The signal peptide is indicated by a dashed underline. CDR1, CDR2 and CDR3 sequences are indicated by the solid underline.

[0040] FIG. 7: 1A3 antibody VL sequence. The polynucleotide (SEQ ID NO: 12) and encoded polypeptide (SEQ ID NO: 13) sequence for the 1A3 antibody VL chain is shown. The signal peptide is indicated by a dashed underline. CDR1, CDR2 and CDR3 sequences are indicated by the solid underline. [0041] FIG. 8: Cav-1 monoclonal antibody (mAb), 3E2, blocks the uptake of secreted Cav-1 in conditioned medium (CM) in LNCaP cells, suppressing the Wnt- -catenin signaling pathway.

[0042] FIG. 9A-B: Antitumor activity of Cav-1 mAb in DU145 xenografts. FIG. 9A, Cav-1 mAb 1E12 significantly reduced tumor volume in nude mice bearing DU145 tumor xenografts, relative to that with IgG control (P = 0.012). FIG. 9B, Cav-1 mAb 3H10, 3E2 and IE 12 significantly reduced the tumor weight in nude mice bearing DU145 tumor xenografts, relative to that in IgG control (P = 0.036, 0.036 and 0.016, respectively). All Cav-1 mAbs were administered intraperitoneally at 10 μg/q.o.d.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0043] Recently, targeted cancer therapies have been developed for treating various malignancies. By virtue of their specific targeting these agents can be both more effective and have fewer side effects than conventional chemotherapy. However, such targeted therapies are niot yet available for a wide range of malignanacies. One potential target for such therapies is Cav-1, a protein that is upregulated in a variety of malignancies. Notably, Cav-1 plays a critical role in androgen insensitive prostate cancer. In this, case the cancer cells produce a biologically active soluble form of the protein (sCav-1) that contributes to oncogenic progression. However, reagents for targeting Cav-1, and in particular sCav-1, have not been previously available. [0044] Embodiments of the invention address these limitations by providing reagents for specific targeting of Cav- 1. In particular, antibodies are provided that specifically bind to the amino terminus of Cav-1. Importantly these antibodies are able to neutralize the effects of sCav-1 in tissue culture (FIG. 8). Even more importantly, administration of the antibodies to mice bearing tumor explants results in significantly reduced tumor growth in the animals (FIG. 9A-B). Accordingly, these antibodies, and humanized versions thereof, can be employed for the treatment of Cav- 1 positive cancer, and particularly cancers that produce sCav-1 such as androgen insensitive prostate cancers. Likewise, antibodies of the embodiments can be used for detecting and charactering cancers in patient by detecting Cav- 1 in patient samples.

I. ANTIBODIES

[0045] In certain embodiments, one or more antibodies may be obtained or produced which has a specificity for human Cav-1 protein. These antibodies may be used in various diagnostic or therapeutic applications described herein. Among other things, the present embodiments provide compositions and methods for detecting and treating a cancer associated with elevated expression and/or secretion of Cav-1 (such as soluble Cav-1 fragments). It will be appreciated that any type of cancer may be associated with such elevated expression and/or thus, be targeted or detected by an anti-Cav-1 antibody of the embodiments. For example, in some embodiments, a cancer associated with elevated Cav-1 expression and/or secretion of soluble Cav-1 is a melanoma or a prostate cancer, such as an androgen-insensitive prostate cancer.

[0046] As used herein, the term "antibody" is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide, or fragments thereof. Suitable antibodies include, but are not limited to, human antibodies, primatized antibodies, de-immunized antibodies, chimeric antibodies, bi-specific antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular ImmunoPharmaceuticals ("SMIPs™ ), single chain antibodies, cameloid antibodies, antibody-like molecules (e.g., anticalins), and antibody fragments. As used herein, the term "antibodies" also includes intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies (e.g. bi-specific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. In some aspects, the antibody can be a VHH (i.e., an antigen-specific VHH) antibody that comprises only a heavy chain. For example, such antibody molecules can be derived from a llama or other camelid antibody (e.g., a camelid IgG2 or IgG3, or a CDR-displaying frame from such camelid Ig) or from a shark antibody. Antibody polypeptides for use herein may be of any type (e.g., IgG, IgM, IgA, IgD and IgE). Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.

[0047] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, Fc and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.

[0048] "Mini-antibodies" or "minibodies" are also contemplated for use with the present invention. Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region (Pack et ah, 1992). The oligomerization domain comprises self-associating a-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds. The oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein. Generally, minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992); Cumber ef a/. (1992). [0049] In some cases antibody-like molecules are protein scaffolds that can be used to display antibody CDR domains. The origin of such protein scaffolds can be, but is not limited to, the structures selected among: fibronectin (see, e.g., U.S. Patent Publn. No. 20090253899, incorporated herein by reference) and preferentially fibronectin type III domain 10, protein Z arising from domain B of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the "ankyrin repeat" (Kohl et ah, 2003), the "armadillo repeat", the "leucine-rich repeat" and the "tetratricopeptide repeat". The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Additional antibody-like molecules, such as anti-calins are described in detail in US Patent Publication Nos. 20100285564, 20060058510, 20060088908, 20050106660, PCT Publication No. WO2006/056464 and (Skerra, 2001), incorporated herein by reference.

[0050] Antibody-like binding peptidomimetics are also contemplated in the present invention. Liu et ah (2003) describe "antibody like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods. Likewise, in some aspects, antibodylike molecules are cyclic or bicyclic peptides. For example, methods for isolating antigen- binding bicyclic peptides (e.g., by phage display) and for using the such peptides are provided in U.S. Patent Publn. No. 20100317547, incorporated herein by reference. [0051] Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production. Embodiments of the invention provide monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred. [0052] "Humanized" antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. As used herein, the term "humanized" immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. Methods for humanizing antibodies such as those provided here are well known in the art, see, e.g., Harvey et ah, 2004, incorporated herein by reference.

A. Methods for Generating Antibodies [0053] Methods for generating and characterizing antibodies (e.g., polyclonal and/or monoclonal antibodies) are known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

[0054] Briefly, a polyclonal antibody is prepared by immunizing an animal with a Cav-1 polypeptide a portion thereof in accordance with the present embodiment and collecting antisera from that immunized animal. For example, a portion of the human Cav-1 amino terminus can be used as the immunogen can be used as an immunogen.

[0055] A wide range of animal species can be used for the production of antisera. Typically the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice of animal may be decided upon the ease of manipulation, costs or the desired amount of sera, as would be known to one of skill in the art. It will be appreciated that antibodies of the embodiments can also be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with the transgenic production in mammals, antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750, 172, and 5,741,957.

[0056] As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include any acceptable immunostimulatory compounds, such as cytokines, chemokines, cofactors, toxins, plasmodia, synthetic compositions or vectors encoding such adjuvants.

[0057] Adjuvants that may be used in accordance with the present embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also contemplated. MHC antigens may even be used. Exemplary, adjuvants may include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and/or aluminum hydroxide adjuvant.

[0058] In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/ Mead, NJ), cytokines such as γ-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

[0059] The amount of immunogen composition used in the production of antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen including but not limited to subcutaneous, intramuscular, intradermal, intraepidermal, intravenous and intraperitoneal. The production of antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.

[0060] A second, booster dose (e.g., provided in an injection), may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.

[0061] For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography, among others.

[0062] MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4, 196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide, peptide or domain, be it a wild-type or mutant composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.

[0063] The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. In some embodiments, Rodents such as mice and rats are used in generating monoclonal antibodies. In some embodiments, rabbit, sheep or frog cells are used in generating monoclonal antibodies. The use of rats is well known and may provide certain advantages (Goding, 1986, pp. 60 61). Mice (e.g., BALB/c mice)are routinely used and generally give a high percentage of stable fusions.

[0064] The animals are injected with antigen, generally as described above. The antigen may be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster administrations with the same antigen or DNA encoding the antigen may occur at approximately two- week intervals. [0065] Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Generally, spleen cells are a rich source of antibody-producing cells that are in the dividing plasmablast stage. Typically, peripheral blood cells may be readily obtained, as peripheral blood is easily accessible.

[0066] In some embodiments, a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 10 ⁷ to 2 x 10 ⁸ lymphocytes. [0067] The antibody producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma producing fusion procedures preferably are non antibody producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). [0068] Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65 66, 1986; Campbell, pp. 75 83, 1984). cites). For example, where the immunized animal is a mouse, one may use P3 X63/Ag8, X63 Ag8.653, NSl/l .Ag 4 1, Sp210 Agl4, FO, NSO/U, MPC 1 1, MPC11 X45 GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3 Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICR LON HMy2 and UC729 6 are all useful in connection with human cell fusions. See Yoo et al. (2002), for a discussion of myeloma expression systems.

[0069] One murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS- 1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8 azaguanine resistant mouse murine myeloma SP2/0 non producer cell line.

[0070] Methods for generating hybrids of antibody producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977). The use of electrically induced fusion methods is also appropriate (Goding pp. 71 74, 1986).

[0071] Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 ^"6 to 1 x 10 ^"8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. [0072] The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

[0073] This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

[0074] The selected hybridomas would then be serially diluted and cloned into individual antibody producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. First, a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. Second, the individual cell lines could be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.

[0075] Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase and DHFR gene expression systems are common approaches for enhancing expression under certain conditions. High expressing cell clones can be identified using conventional techniques, such as limited dilution cloning and Microdrop technology. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.

[0076] MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer. [0077] It is also contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. In one embodiment, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells. The advantages of this approach over conventional hybridoma techniques are that approximately 10 ⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies. Target- binding single domain antibodies can also be isolated by use of display libraries, see for example, U.S. Patent Appln. No. 20110183863, incorporated herein by reference. Ribosome expression libraries for isolation of target-bind Ig coding sequences are also described in U.S. Patent Appln. No. 20040161748; 20070299246 and 20080293591, each incorporated herein by reference.

[0078] Another embodiment of the invention for producing antibodies according to the present invention is found in U.S. Patent No. 6,091,001, which describes methods to produce a cell expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre-mediated site-specific recombination is disclosed. The method involves first transfecting an antibody-producing cell with a homology-targeting vector comprising a lox site and a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin loci of the genomic sequence which is to be converted to a modified region, so the first lox site is inserted into the genomic sequence via site-specific homologous recombination. Then the cell is transfected with a lox-targeting vector comprising a second lox site suitable for Cre-mediated recombination with the integrated lox site and a modifying sequence to convert the region of the immunoglobulin loci to the modified region. This conversion is performed by interacting the lox sites with Cre in vivo, so that the modifying sequence inserts into the genomic sequence via Cre-mediated site-specific recombination of the lox sites. [0079] Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.

B Antibody Conjugates

[0080] The present invention provides antibodies against Cav-1 proteins, polypeptides and peptides that are linked to at least one agent to form an antibody conjugate or payload. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins (e.g., TNF alpha or gelanin), anti-tumor agents, therapeutic enzymes, radio-labeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin. For example, an antibody can be conjugated to a maytansinoid (e.g., maytansinol or the DM1 maytansinoid, see, U.S. Pat. Nos. 5,208,020; 6,333,410; and 7,276,497), auriculin, calicheamicin, duocarmicin or tubulysin.

[0081] Any antibody of sufficient selectivity, specificity or affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art. Sites for binding to biological active molecules in the antibody molecule, in addition to the canonical antigen binding sites, include sites that reside in the variable domain that can bind pathogens, B-cell superantigens, the T cell co-receptor CD4 and the HIV-1 envelope (Sasso et al, 1989; Shorki et al, 1991; Silvermann et al, 1995; Cleary et al, 1994; Lenert et al, 1990; Berberian et al, 1993; Kreier et al, 1991). In addition, the variable domain is involved in antibody self- binding (Kang et al, 1988), and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler ei a/., 1989). [0082] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. "Detectable labels" are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti cellular agent, and may be termed "immunotoxins".

[0083] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and/or those for use in vivo diagnostic protocols, generally known as "antibody directed imaging".

[0084] Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patent Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference). The imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging.

[0085] In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).

[0086] In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might use astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt, copper67, 152Eu, gallium67, 3hydrogen, iodine 123, iodine 125, iodinel31, indiuml l l, 59iron, 32phosphorus, radium223, rheniuml 86, rheniuml 88, 75selenium, 35sulphur, technicium99m, thorium227 and/or yttrium90. 1251 is often used in certain embodiments, and technicium99m and/or indiuml l l are also often used due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present invention may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCI ₂ , a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA). [0087] Among the fluorescent labels contemplated for use as conjugates include

Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red, among others.

[0088] Antibody conjugates contemplated in the present invention include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149 and 4,366,241 ; each incorporated herein by reference. [0089] Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.

[0090] Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et ah, 1985). The 2- and 8- azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et ah, 1989; King et ah, 1989; and Dholakia et ah, 1989) and may be used as antibody binding agents.

[0091] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6 a-diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3 -(4-hydroxyphenyl)propionate.

[0092] In some embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5, 196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al, 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation. [0093] In some embodiments of the invention, anti-Cav-1 antibodies are linked to semiconductor nanocrystals such as those described in U.S. Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of which are incorporated herein in their entireties); as well as PCT Publication No. 99/26299 (published May 27, 1999). In particular, exemplary materials for use as semiconductor nanocrystals in the biological and chemical assays of the present invention include, but are not limited to those described above, including group II- VI, III-V and group IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, A1S, A1P, AlSb, PbS, PbSe, Ge and Si and ternary and quaternary mixtures thereof. Methods for linking semiconductor nanocrystals to antibodies are described in U.S. Patent Nos. 6,630,307 and 6,274,323.

II. CAV-1 DIAGNOSTICS

[0094] Certain embodiments of the invention concern detecting, either in vivo or in a sample, Cav-1 expression or activation. For example, in some embodiments, Cav-1 expression can be detected by detecting protein in or on the surface of cells. In some embodiments, sCav-1 is detected, such as a serum sample from a subject.

[0095] In some embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying and/or otherwise generally detecting biological components such as Cav-1 protein components. Cav-1 antibodies prepared in accordance with the present invention may be employed to detect Cav-1 and/or sCav-1. Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev, 1999; Gulbis and Galand, 1993; De Jager et al, 1993; and Nakamura et al, 1987, each incorporated herein by reference.

[0096] In general, the immunobinding methods include obtaining a sample suspected of containing an Cav-1 protein, polypeptide and/or peptide, and contacting the sample with a first anti-Cav- 1 antibody in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes. Immunobinding methods can include methods for detecting and quantifying the amount of a Cav-1 or activated sCav-1 in a sample. Here, one would obtain a sample suspected of containing Cav-1 and contact the sample with an antibody and then detect and quantify the amount of immune complexes formed under the specific conditions.

[0097] In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing a cell expressing Cav-1, such as a skin or prostate tissue section or specimen, a homogenized tissue extract, or any biological fluid. Hyperproliferative diseases that may be suspected of expressing Cav-1 or sCav-1, for example can be detected as detailed herein.

[0098] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any Cav-1 protein antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0099] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. U.S. Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art. [00100] In some embodiments, Cav-1 antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. In some embodiments, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In some embodiments, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[00101] Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[00102] One method of immunodetection uses two different antibodies. A first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin. In that method the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible. [00103] Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.

[00104] In the clinical diagnosis and/or monitoring of patients with various forms of hyperproliferative disease, such as cancer, for example, leukemia, the detection of Cav-1, and/or an alteration in the expression or amount of soluble Cav-1, in comparison to the levels in a corresponding biological sample from a normal subject is indicative of a patient with hyperproliferative disease, such as cancer. However, as is known to those of skill in the art, such a clinical diagnosis would not necessarily be made on the basis of this method in isolation. Those of skill in the art are very familiar with differentiating between significant differences in types and/or amounts of biomarkers, which represent a positive identification, and/or low level and/or background changes of biomarkers. Indeed, background expression levels are often used to form a "cut-off above which increased detection will be scored as significant and/or positive.

A. ELISAs

[00105] As detailed above, immunoassays, in their most simple and/or direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and/or western blotting, dot blotting, FACS analyses, and/or the like may also be used. [00106] In some embodiments, the anti-Cav-1 antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing a Cav-1 protein antigen, such as a clinical sample, is added to the wells. After binding and/or washing to remove non-specifically bound immune complexes, the bound Cav-1 protein antigen may be detected. Detection is generally achieved by the addition of another anti- Cav- 1 antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second anti-Cav-1 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[00107] In some embodiments, the samples suspected of containing the Cav-1 protein antigen are immobilized onto the well surface and/or then contacted with the anti- Cav-1 antibodies of the invention. After binding and/or washing to remove non-specifically bound immune complexes, the bound anti-Cav-1 antibodies are detected. Where the initial anti-Cav- 1 antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-Cav-1 antibody, with the second antibody being linked to a detectable label.

[00108] In some embodiments, the Cav-1 proteins, polypeptides and/or peptides are immobilized. In certain embodiments, ELISA involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against Cav- 1 protein are added to the wells, allowed to bind, and/or detected by means of their label. The amount of Cav-1 protein antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against Cav-1 before and/or during incubation with coated wells. The presence of wild type and/or mutant Cav-1 protein in the sample acts to reduce the amount of antibody against wild type or mutant protein available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against Cav-1 protein in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.

[00109] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

[00110] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[00111] In some embodiments, a secondary or tertiary detection means is used rather than a direct procedure. In some such embodiments, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[00112] "Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

[00113] The "suitable" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25 °C to 27 °C, or may be overnight at about 4 °C or so. [00114] Following incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[00115] To provide a detecting means, the second or third antibody may have an associated label to allow detection. In some embodiments, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase- conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween). After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'- azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H202, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

B. Immunohistochemistry

[00116] Antibodies of the present invention may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et al, 1990; Allred et al, 1990). [00117] Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen

"pulverized" tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in 70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.

[00118] Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections.

C. Immunodetection Kits

[00119] In some embodiments, the present invention concerns immunodetection kits for use with the immunodetection methods described above. As Cav-1 antibodies are generally used to detect Cav-1 proteins, polypeptides and/or peptides, the antibodies will preferably be included in the kit. However, kits including both such components may be provided. Immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to a Cav-1 protein, polypeptide and/or peptide, and/or optionally, an immunodetection reagent and/or further optionally, a Cav-1 protein. [00120] In some embodiments, monoclonal antibodies will be used. In certain embodiments, the first antibody that binds to the Cav-1 protein may be pre-bound to a solid support, such as a column matrix and/or well of a microtitre plate.

[00121] Immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with and/or linked to the given antibody. Detectable labels that are associated with and/or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[00122] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and/or all such labels may be employed in connection with the present invention.

[00123] Kits in accordance with the present invention may further comprise a suitably aliquoted composition of the Cav-1 protein, whether labeled and/or unlabeled, as may be used to prepare a standard curve for a detection assay. Provided kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, and/or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media and/or in lyophilized form. [00124] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the antibody may be placed, and/or preferably, suitably aliquoted. Where Cav-1 protein and/or a second and/or third binding ligand and/or additional component is provided, the kit will also generally contain a second, third and/or other additional container into which this ligand and/or component may be placed. The kits of the present invention will also typically include a means for containing the antibody, antigen, and/or any other reagent containers in close confinement for commercial sale. Such containers may include injection and/or blow-molded plastic containers into which the desired vials are retained.

III. THERAPEUTIC METHODS

[00125] In order to increase the effectiveness of Cav-1 -targeted therapies of the embodiments, it may be desirable to combine these compositions with other agents effective in the treatment of the disease of interest. For example, in certain aspects an Cav-1 -targeted therapy is administered in conjunction with a chemotherapeutic agent

[00126] Treatment with an Cav- 1 -targeted therapy may precede or follow a chemotherapy treatment (or other anti-cancer agent treatment) by intervals ranging from minutes to weeks. In embodiments where the Cav-1 -targeted therapy and second anti-cancer treatment are applied separately to a cell or patient, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the Cav- 1 -targeted therapy would still be able to exert an advantageously combined effect on the cell with the second agent. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respective administrations.

[00127] Various combinations may be employed, where the Cav-1 -targeted therapy is "A" and the secondary agent, such as a chemotherapy, is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

[00128] Administration of the Cav-1 -targeted therapy of the present invention to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described hyperproliferative cell therapy. [00129] In some embodiments an Cav-1 -targeted therapy can be administered in conjunction with a range of other anti-cancer agents. An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with a Cav-1 -targeting agent and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the a Cav- 1 -targeting agent and the other includes the second agent(s). [00130] A wide variety of chemotherapeutic agents may be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, many chemotherapy agents are DNA damaging agents as outlined above. An agent may, for example, be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas. [00131] Examples of chemotherapeutic agents include without limitation alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[00132] Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines such as gene therapy vaccines and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[00133] In some embodiments, a DNA damaging agent for use according to the embodiments can be a radiation therapy. For example, a radition therapy can be administered by exposing a patient (or a part of the patient) to γ-rays, X-rays, and/or by the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. [00134] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[00135] In some embodiments, an Cav- 1 -targeted therapy can be administered in conjunction with an immunotherapy. Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[00136] In some embodiments, an immunotherapy can comprise administering one or more tumor antigens to a patient (e.g., a cancer vaccine) or exposing a patient's immune cells to such a cancer antigen (e.g., an autologous cellular immunotherapy). Antigens that can be used in such therapeutic approaches include, but are not limited to, prostatic acid phosphotase (PAP), MUC1, HER2/neu, 5T4 and whole killed cancer cells.

[00137] Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with a Cav- 1 -targeted therapy of the present invention. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pl55.

[00138] In some embodiments, an Cav- 1 -targeted therapy can be administered in conjunction with a gene therapy (e.g., a therapeutic polynucleotide composition). Viral vectors for the expression of a gene product are well known in the art, and include such eukaryotic expression systems as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia viruses, and papiloma viruses, including SV40. Alternatively, the administration of expression constructs can be accomplished with lipid based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these method are well known in the art (see, e.g. Sambrook et ah, 1989; Ausubel et ah, 1998; Ausubel, 1996).

[00139] Delivery of a vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on target tissues. [00140] In some embodiments, an Cav-1 -targeted therapy can be administered in conjunction with a surgical therapy (e.g., before after or during surgical resection of a tumor). Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.

[00141] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[00142] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well.

[00143] In some embodiments, Cav-1 -targeted therapies are administered in conjunction with an anti-inflammatory agent. An anti-inflammatory agent is defined herein to refer to an agent that is known or suspected to be of benefit in the treatment or prevention of inflammation in a subject. Corticosteroids are a major class of anti-inflammatory agent. The corticosteroids may be short, medium, or long acting, and may be delivered in a variety of methods. A non-limiting list of corticosteroids contemplated in the present invention include the oral corticosteroids such as: cortisone, hydrocortisone, prednisone, and dexamethasone.

[00144] Another major class of anti-inflammatory agents are non-steroidal antiinflammatory agents. Non-steroidal anti-inflammatory agents include a class of drugs used in the treatment of inflammation and pain. The exact mode of action of this class of drugs is unknown. Examples of members of this class of agents include, but are not limited to, ibuprofen, ketoprofen, flurbiprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, flufenamic acid, diflunisal, oxaprozin, rofecoxib, and celecoxib. One of ordinary skill in the art would be familiar with these agents. Included in this category are salicylates and derivates of salicylates, such as acetyl salicylic acid, sodium salicylate, choline salicylate, choline magnesium salicylate and diflunisal. [00145] Other anti-inflammatory agents include anti-rheumatic agents, such as gold salts (e.g., gold sodium thiomalate, aurothioglucose, and auranofin), anti-rheumatic agents (e.g., chloroquine, hydroxychloroquine, and penicillamine), antihistamines (e.g., diphenhydramine, chlorpheniramine, clemastine, hydroxyzine, and triprolidine), and immunosuppressive agents (e.g., methotrexate, mechlorethamine, cyclophosphamide, chlorambucil, cyclosporine, and azathioprine). Other immunosuppressive agent contemplated by the present invention is tacrolimus and everolimus. Tacrolimus suppresses interleukin-2 production associated with T-cell activation, inhibits differentiation and proliferation of cytotoxic T cells. Today, it is recognized worldwide as the cornerstone of immunosuppressant therapy. One of ordinary skill in the art would be familiar with these agents, and other members of this class of agents, as well as the mechanism of actions of these agents and indications for use of these agents.

[00146] It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adehesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, ΜΓΡ- lbeta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti- hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adehesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy. [00147] Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

IV. ADMINISTRATION

[00148] A Cav-1 -targeted therapy of the embodiments can be administered in a number of ways depending upon whether local or systemic treatment is desired, and upon the area to be treated. In some embodiments, an Cav-1 -targeted therapy is administered in a formulation comprising a DNA damaging agent as detailed above.

[00149] Administration may be topical (including opthalmic, vaginal, rectal, intranasal, transdermal), oral, nasal, bronchial or parenteral, for example, by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. In the case a patient having a tumor a composition can be administered proximal to the site of a tumor, such as by an intratumoral injection. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulations. [00150] In some embodiments Cav- 1 -targeting agents may be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. For example, cationic lipids may be included in the formulation to facilitate uptake of the agents.

[00151] The actual dosage amount of a composition of the present invention administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions (e.g., use of DNA damaging agents), idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[00152] An effective amount of the therapeutic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired. In some embodiments, dosages can be determined by measuring for example changes in Cav-1 expression in a subject. For example, an effective amount of a Cav- 1 -targeting agent can be an amount that reduces sCav-1 in the blood or in a tissue sample by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more.

[00153] Precise amounts of the therapeutic composition may also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus attaining a particular serum insulin or glucose concentration) and the potency, stability and toxicity of the particular therapeutic substance. [00154] In some embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active agent, such as an Cav-1 -targeting agent and/or a DNA damaging agent. In some embodiments, the an active agent may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein of an active agent. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

V. EXAMPLES

[00155] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 - Production of murine anti-Cav-1 monoclonal antibodies

[00156] Murine anti-Cav-1 monoclonal antibodies were produced by immunization of Cav-1 knock-out mice with various portions of human Cav-1 polypeptide ( CBI accession no. NP_001744; SEQ ID NO: 1). Briefly:

[00157] For the 1A3 antibodies - a peptide having the sequence (REQGNIYKPNNKAMC (SEQ ID NO: 19)) was conjugated with KLH as immunogen (the c-terminal cys residue was used for conjugation) and BALB/c mice were immunized. All mice showed satisfactory immune response and the optimal mice were used for cell fusion and hybridoma production. Total 6 hybridoma cell lines are produced, 5 mice ascites production was completed for 1 selected cell line and the produced monoclonal antibodies in the ascites were purified by Protein G affinity column. Antibodies were found to be murine IgGl.

[00158] For the 1E12 antibodies - Peptide (LV RDPKHLNDDVVC (SEQ ID NO:20)) was conjugated with KLH as immunogen (the c-terminal cys residue was used for conjugation) and BALB/c mice were immunized. All mice showed satisfactory immune response and the optimal mice were used for cell fusion and hybridoma production. Total 4 hybridoma cell lines are produced, 5 mice ascites production is done for 1 selected cell line and the produced monoclonal antibodies in the ascites were purified by Protein G affinity column. Antibodies were found to be murine IgGl.

[00159] For the 3E2 and 3H10 antibodies - a mixture of peptides derived from human Cav-1 were conjugated with immunogen and Cav-1 knockout mice were immunized. Resulting antibodies were specific for Cav-1 amino acids 34-48 (DELSEKQVYDAHTKE (SEQ ID O:21)).

[00160] Thus, of the hybridoma clones that were isolated, 3E2 and 3H10 were induced by peptide from amino acid 34 to 48 of human Cav-1, and 1E12 from amino acids 51 to 64. Clone 1A3 was induced by amino acids 19-32 of human Cav-1. Resulting monoclonal antibodies were further analyzed to determine their coding sequence and functional properties as detailed below.

Example 2 - Sequencing of monoclonal antibodies

[00161] 3E2.H5, 3E2.H6 and 3H10.6.3.4 cells were grown in RPMI-1640 media containing 10% fetal bovine serum (FBS; HyClone, Logan, UT), 1 mM sodium pyruvate (Mediatech, Herndon, VA), lx gentamicin (Sigma, St. Louis, MO) and lx penicillin- streptomycin mix (Mediatech) at 37°C in a 7.5% C0 ₂ incubator. 1A3F9 and 1E12C9 cells were grown in DME media containing 10% FBS and lx penicillin-streptomycin mix (Mediatech) at 37°C in a 7.5% CO ₂ incubator. Frozen cell lysates prepared by treating approximately 4 x 10 ⁶ of each of 3E2.1 and 3H10.6.3.4 hybridoma cells. Antibody isotyping

[00162] Isotype of mouse monoclonal antibody produced by the above mentioned hybridoma cells was determined by ELISA as follows. An ELISA plate was coated with 100 μΐ/well of one of the following five goat polyclonal antibodies (all from SouthemBiotech, Birmingham, AL), 1/2,000-diluted in PBS, at 4°C overnight.

1. Anti-mouse IgG, γ chain-specific

2. Anti-mouse IgG, γΐ chain-specific

3. Anti-mouse IgG, y2a chain-specific

4. Anti-mouse IgG, y2b chain-specific

5. Anti-mouse IgM, μ chain-specific

[00163] After washing wells with Wash Buffer (PBS containing 0.05% Tween 20), blocking for 30 min at room temperature with 300 μΐ/well of Block Buffer (PBS containing 2% Skim Milk and 0.05% Tween 20), and washing with Wash Buffer, 100 μΐ/well of culture supernatant of each hybridoma was applied in duplicate to the ELISA plate. After incubating the ELISA plate for 1 hr at room temperature and washing with Wash Buffer, bound antibodies were detected using 100 μΐ/well of one of the following two goat polyclonal antibodies (both from SouthemBiotech), 1/2,000-diluted in ELISA buffer (PBS containing 1% Skim Milk and 0.025% Tween 20) for 30 min at room temperature.

A. HRP-conjugated goat anti-mouse kappa chain polyclonal antibody

B. HRP-conjugated goat anti-mouse lambda chain polyclonal antibody

[00164] After washing with Wash Buffer, color development was performed by adding 100 μΐ/well of ABTS substrate (AMRESCO, Solon, OH) and stopped by adding 100 μΐ/well of 2% oxalic acid. Absorbance was read at 405 nm.

[00165] The result of isotyping shown below was consistent independent isotyping studies.

3E2.H6 IgG2a/kappa

3H10.6.3.4 IgG2a/kappa

1E12C9 IgG 1 /kappa 1A3F9 IgGl/kappa

Cloning and sequencing of mouse immunoglobulin variable region genes

[00166] Total RNA was extracted from approximately 5 x 10 ⁶ of each of 3E2.H5, 1E12C9 and 1A3F9 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the supplier's protocol. Similarly, total RNA was extracted from TRIzol-treated 3H10.6.3.4 and 3E2.1 cells. Oligo dT-primed cDNA for 5'-RACE was synthesized for each of 3E2.H5, 3E2.1, 3H10.6.3.4, 1E12C9 and 1A3F9 using the SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA) following the supplier's protocol. The variable region cDNAs for heavy and light chains were amplified by polymerase chain reaction (PCR) with Phusion DNA polymerase (New England Biolabs, Beverly, MA) using 3' primers that anneal specifically to the mouse heavy and light chain constant regions, and the 5'-RACE primer (Universal Primer A Mix or Nested Universal Primer A) provided in the SMARTer RACE cDNA Amplification Kit.

[00167] For PCR amplification of heavy chain variable region (VH), the 3' primers have the sequences shown below.

MCG1 : 5 '-GCCAGTGGATAGACAGATGG-3 ' (for γΐ chain; SEQ ID NO: 14) MCG2A: 5 '-GCCAGTGGATAGACCGATGG-3 ' (for y2a chain; SEQ ID NO: 15)

[00168] For PCR amplification of kappa light chain variable region (VL), the 3' primers have the sequences shown below.

MCK: 5'-GATGGATACAGTTGGTGCAGC-3' (SEQ ID NO: 16)

MCK2: 5 ' -AGATGTTAACTGCTCACTGG-3 ' (SEQ ID NO: 17)

MCK3: 5'-ACACGACTGAGGCACCTC-3 ' (SEQ ID NO: 18) [00169] The amplified VH and VL cDNAs were subcloned into the pCR4-Blunt vector (Invitrogen) for sequence determination. DNA sequencing was carried out at Tocore (Menlo Park, CA). Several heavy and light chain clones were sequenced and unique sequences homologous to typical mouse heavy and light chain variable regions were identified. The consensus cDNA sequence for each V gene was obtained with at least three independent clones. Sequences of VH and VL genes

[00170] Nucleotide sequences of the VH and VL regions of 3E2.H5, 3E2.1, 3H10.6.3.4, 1E12C9 and 1A3F9 hybridomas are shown alongside with deduced amino acid sequences in Figs. 1 through 6. In each figure, amino acid residues are shown in single letter code. The predicted signal peptide sequence is underlined with dotted line. CDR1, 2 and 3 sequences according to the definition of Kabat et al. (1991), incorporated herein by reference, are underlined with solid line. Table 1 is the chart for the assignment of figures to the VH and VL sequences. 3E2.H5, 3E2.1 and 3H10.6.3.4 share the same VH and VL nucleotide sequences.

[00171] Table 1. Assignment of figures to VH and VL sequences

Hybridoma VH VL

3E2.H5 FIG. 2 FIG. 3

3E2.1 FIG. 2 FIG. 3

3H10.6.3.4 FIG. 2 FIG. 3

1E12C9 FIG. 4 FIG. 5

1A3F9 FIG. 6 FIG. 7

Example 3 - Characterizing Cav-1 monoclonal antibodies [00172] Using Cav-1 -secreting RM-9 mouse PCa cells, it has been reported that systemic polyclonal Cav-1 antiserum can dramatically reduced the number of experimental metastases without any obvious signs of toxicity (Watanabe et al, 2009). Preliminary studies also showed that treating Cav-1 -negative LNCaP PCa cells with sCav-1 -containing conditioned medium (CM) in vitro leads to uptake of sCav-1 and induction of LRP6 phosphorylation and β-cat signaling (FIG. 8). Combined treatment with Cav-1 CM and a Cav-1 mAb (3E2) resulted in suppression of sCav-l-stimulated uptake, LRP6 phosphorylation, and β-cat signaling in vitro (see e.g., FIG. 8). Owing to constitutive expression of the Wnt ligand in these PCa cells, induction of LRP6 phosphorylation by Cav- 1-containing CM is marginal. However, treatment with Cav-1 mAb suppressed LRP6 phosphorylation and β-cat signaling in the presence of Cav-1 -containing CM in vitro. Suppression of sCav-1 uptake from the CM and reduction of P-Akt (T308) by Cav-1 mAb was also demonstrated.

[00173] It was previously shown that Cav-1 overexpression maintains activated Akt in PCa cells through CSD binding site interactions with and inhibition of the serine/threonine phosphatases PP1 and PP2A (Li et ah, 2003). Further, it was demonstrated that uptake of rCav-1 by endothelial cells results in increased levels of P-Akt (T308) (Tahir et ah, 2008). Thus, neutralization of sCav-1 with Cav-1 mAb appears to suppress both the stimulatory effects of internalized Cav-1 on P-Akt (T308) and the stimulation of P-LRP6- FZD-Wnt- -cat signaling. [00174] Studies with newly produced Cav-1 mAbs show that systemic Cav-1 mAb treatment in male nude mice bearing subcutaneous DU145 PCa tumors which secret Cav-1 was able to significantly suppressed tumor growth (FIG. 9A; 1E12, P = 0.012) and tumor wet weight in vivo (FIG. 9B; 3H10, 3E2 and 1E12; P = 0.036, 0.036, and 0.016, respectively).

* * * [00175] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

U.S. Patent 3,817,837

U.S. Patent 3,850,752

U.S. Patent 3,939,350

U.S. Patent 3,996,345

U.S. Patent 4, 196,265

U.S. Patent 4,275, 149

U.S. Patent 4,277,437

U.S. Patent 4,366,241

U.S. Patent 4,472,509

U.S. Patent 4,472,509

U.S. Patent 4,938,948

U.S. Patent 4,938,948

U.S. Patent 5,021,236

U.S. Patent 5, 196,066

U.S. Patent 5,208,020

U.S. Patent 5,262,357

U.S. Patent 5,505,928

U.S. Patent 5,690,807

U.S. Patent 5,741,957

U.S. Patent 5,750, 172

U.S. Patent 5,756,687

U.S. Patent 5,827,690

U.S. Patent 5,990,479

U.S. Patent 6,048,616

U.S. Patent 6,091,001

U.S. Patent 6,274,323

U.S. Patent 6,333,410

U.S. Patent 6,630,307 U.S. Patent 7,276,497

U.S. Patent Appln. 20040161748

U.S. Patent Appln. 20050106660

U.S. Patent Appln. 20060058510

U.S. Patent Appln. 20060088908

U.S. Patent Appln. 20070299246

U.S. Patent Appln. 20080293591

U.S. Patent Appln. 20100285564

U.S. Patent Appln. 20100317547

U.S. Patent Appln. 201 10183863

U.S. Patent Appln. 20110183863

U.S. Patent Publn. 20090253899

Abbondanzo et al., Am. J. Pediatr. Hematol Oncol, 12(4), 480-489, 1990.

Allred et al, Pro Natl Acad. Sci. USA, 87:3220-3224, 1990.

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor

Press, Cold Spring Harbor, New York, 1988.

Atherton et al, Biol. Reprod. , 32(1): 155- 171, 1985.

Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y., 1996.

Ausubel et al , In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, New York, 1998

Berberian et al, Science, 261(5128): 1588-1591, 1993.

Brown et al. Immunol. Ser., 53 :69-82, 1990.

Campbell, In: Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology, Burden and Von Knippenberg (Eds.), Elseview, Amsterdam, 13 :71-74/75-83, 1984.

Cleary et al, J. Biol. Chem. , 269(29): 18747-9, 1994.

Cumber et al, J. Immunology., 149B: 120-126, 1992.

De Jager et al, Semin. Nucl Med., 23(2): 165-179, 1993.

Dholakia e a/., J Biol. Chem., 264(34):20638-20642, 1989.

Doolittle and Ben-Zeev, Methods Mol, Biol,, 109:215-237, 1999.

Eur. Patent Appln. 89303964.4

Eur. Patent No. 0 216 846 Eur. Patent No. 0 256 055

Eur. Patent No. 0 323 997

Gefter et al , Somatic Cell Genet, 3 :231-236, 1977.

Goding, In: Monoclonal Antibodies: Principles and Practice, 2d ed., Orlando, Fla., Academic Press, 60-61, 65-66, 71-74, 1986.

Gulbis and Galand, Hwm. Pathol. 24(12), 1271 -1285, 1993.

Kabat et al , In: Sequences of Proteins of Immunological Interest, 5 ^th Ed., Public Health Service, National Institute of Health, Bethesda, Md., 91-3242(I):647-669, 1991.

Kang e? al, Science, 240: 1034-1036, 1988.

Khatoon et al, Ann. Neurol, 26(2):210-215, 1989.

King et al, J. Biol Che . , 264( 17): 10210-10218, 1989.

Kohl et al, Proc. Natl. Acad. Set, USA, 100(4): 1700-1705, 2003.

Kohler and Milstein, Eur. J. Immunol, 6:511-519, 1976.

Kohler and Milstein, Nature, 256:495-497, 1975.

Kohler et al , Methods Enzymol, 178:3-35, 1989.

Kreier et al , In: Infection, Resistance and Immunity, Harper and Row, New York, 1991.

Lenert et al, Science, 248(4963): 1639-1643, 1990.

Li et al, Mol. Cell Biol, 23 :9389-9404, 2003

Liu et al Cell Mol. Biol, 49(2):209-216, 2003.

Loblaw et al, J. Clin. Oncol, 25: 1596-605, 2007.

Nakamura et al, In: Handbook of Experimental Immunology (4 ^th Ed.), Weir et al. (Eds.), 1 :27, Blackwell Scientific Publ, Oxford, 1987.

O'Shannessy et al, Anal Biochem, 163(l):204-209, 1987.

Owens and Haley, J. Biol. Chem., 259: 14843- 14848, 1987.

Pack et/., Biochem., 31 : 1579-1584, 1992.

PCT Publn. WO06/056464

PCT Publn. W099/26299

Petrylak et al, N. Engl J. Med. , 351 : 1513-1520, 2004.

Potter and Haley, Meth. Enzymol, 91 :613-633, 1983.

Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, N.Y., 1989.

Sasso et al, J. Immunol, 142:2778-2783, 1989.

Shorki ei a/., J. Immunol , 146:936-940, 1991.

Siegel et al, Ca Cancer J. Clin., 61(4):212-236, 2011. Silvermann et al, J. Clin. Invest., 96:417-426, 1995. Skerra, J. Biotechnol, 74(4):257-75, 2001.

Tahir et al, Cancer Res., 68:731-739, 2008.

Watanabe et al, Mol. Cancer Res., 7: 1446-1455, 2009. Yoo et al, J. Immunol Methods, 261(l-2): l-20, 2002.