WO2012145125A1 - Targeted radionuclides - Google Patents

Abstract

The disclosure provides radiolabeled compounds suitable for in vivo imaging or as cytotoxic agents. These radiolabeled compounds can be used to image or treat cancer.

Description

Targeted Radionuclides

RELATED APPLICATIONS

This application claims the benefit of United States provisional application number 61/467,562, filed March 25, 2011. The disclosure of the foregoing application is hereby incorporated by reference in its entirety.

BACKGROUND

Radionuclides have multiple applications in medical science. For example, nuclear imaging technology allows in vivo, real time imaging of patient tissues. However, effective imaging requires that the radionuclides preferentially label certain tissues but not others.

Radionuclides may also be used therapeutically. This use is also based on the preferential targeting of the radionuclides to certain cells and tissues but not others. For example, high energy radionuclides may be targeted to tissue where the high energy has a cell damaging or cytotoxic effect on the targeted cells.

SUMMARY OF THE INVENTION

The present disclosure provides methods and compositions that may be used for in vivo imaging or radio-therapy. The present disclosure provides a targeting mechanism to help direct a radiolabel to particular cells and tissues using an internalizing moiety.

Specifically, the present disclosure provides various antibodies and antibody fragments labeled with a radionuclide and suitable for in vivo imaging or radio-therapy. In the context of these conjugates, the radionuclide is preferentially targeted to cells and tissue that express ENT2. In the context of, for example, rodent and human tissues, the conjugates are targeted to cancer cells, skeletal muscle, cardiac muscle, kidney and the diaphragm. Thus, by selecting the appropriate radionuclide, labeled reagents suitable for in vivo imaging of these tissues are readily made. Alternatively, by selecting the appropriate, high energy

radionuclide, labeled radio-therapeutic agents suitable as cell damaging or cytotoxic agents are readily made. Radio-therapeutic agents are also referred to herein as cell damaging or cytotoxic agent.

In one aspect, the disclosure provides an in vivo imaging reagent. The in vivo imaging reagent comprises an internalizing moiety for targeting purposes (e.g., polypeptides comprising: monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antibody that binds the same epitope as 3E10 and/or transits cell membranes via ENT2, or an antibody that has substantially the same cell penetrating activity as 3E10 and/or transits cell membranes via ENT2, or an antigen binding fragment of any of the foregoing); and a radionuclide suitable for in vivo imaging, such as gamma or positron emitting radionuclide or a radionuclide that decays by electron transfer.

In certain embodiments, the internalizing moiety, such as the 3E10 antibody, antigen binding fragment thereof, or variant thereof, comprises a heavy chain and a light chain, wherein the heavy chain variable domain comprises an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, or is a humanized variant of any of the foregoing. In certain embodiments, the 3E10 antibody, antigen binding fragment thereof, or variant thereof comprises a heavy chain and a light chain, wherein the light chain variable domain comprises an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is a humanized variant of any of the foregoing. In certain embodiments, the 3E10 antibody, antigen binding fragment thereof, or variant thereof comprises a heavy chain variable domain comprising an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, or is a humanized variant; and a light chain variable domain comprising an amino acid sequence at least 90%>, 92%, 93%>, 94%>, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is a humanized variant of any of the foregoing. In certain embodiments, the antibody fragment further includes a linker, such as a linker interconnecting a heavy chain variable domain (VH) and a light chain variable domain (VL).

In certain embodiments, the internalizing moiety, such as the 3E10 antibody, antigen binding fragment thereof, or variant thereof, comprises the six CDRs of 3E10 described herein. For example, the six CDRs of 3E10 provided in the context of a heavy chain variable domain and a light chain variable domain containing framework regions interspersed between the CDRs. In certain embodiments, the antibody or antigen binding fragment comprises:

VH CDR1 having the amino acid sequence NYGMH (SEQ ID NO: 4)

VH CDR2 having the amino acid sequence YISSGSSTIYYADTVKG (SEQ ID NO:5)

VH CDR3 having the amino acid sequence RGLLLDY (SEQ ID NO: 6)

VL CDR1 having the amino acid sequence RASKSVSTSSYSYMH (SEQ ID NO: 7)

VL CDR2 having the amino acid sequence YASYLES (SEQ ID NO: 8) VL CDR3 having the amino acid sequence QHSREFPWT (SEQ ID NO: 9).

As noted above, such CDRs are provided in the context of an antibody or antigen binding fragment, such as with interspersed framework regions. Any of these specific examples of antibodies and antigen binding fragments may be used in any of the methods described herein, such as diagnostic or therapeutic methods. Moreover, any of these specific examples of antibodies and antigen binding fragments may be conjugated or otherwise complexed with any of the radionuclides described herein.

In certain embodiments of any of the foregoing, the internalizing moiety comprises an antibody or antibody fragment. In certain embodiments, the antibody or antigen binding fragment is chimeric, humanized, or fully human.

In certain embodiments, the in vivo imaging reagent is labeled with a radionuclide detectable by positron emitting tomography (PET) or single photon emission computed tomography (SPECT). Thus, the radiolabeled compound can be imaged (the images collected and displayed) using an available PET or SPECT scanner, or other similar technology, and the accompanying computer hardware and software. In certain

embodiments, the in vivo imaging reagent is labeled with a radionuclide selected from the group consisting of carbon-11, nitrogen-13, oxygen-15, fluorine-18, gallium-67, gallium-68, krypton-81m, rubidium-82, technetium-99m, indium-11, iodine-123, iodine-124, iodine-125, iodine-131, xenon-133, thallium-201, zirconium-89, and copper-64. In other embodiments, the radionuclide is selected from technetium-99m, iodine- 123 , iodine- 124, iodine- 125.

In a related aspect, the disclosure contemplates that antibody or antibody fragment may be labeled with a radionuclide using any available method and chemistry. Association or conjugation of the radionuclide may be directly or via a coupling agent or linker. The disclosure contemplates methods of radiolabeling internalizing moiety (e.g., polypeptides comprising: monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10 and/or transits cell membranes via ENT2, or an antibody that binds the same epitope as 3E10 and/or transits cell membranes via ENT2, or an antibody that has substantially the same cell penetrating activity as 3E10 and/or transits cell membranes via ENT2, or an antigen binding fragment of any of the foregoing). The resulting radiolabeled compound is preferentially targeted to particular cells and tissues, such as skeletal muscle, cardiac muscle, kidney, neurons, Leydig cells and cancerous cells, upon administration. Further exemplary antibodies and antigen binding fragments are described above (e.g., antibodies and antigen binding fragments comprising SEQ ID NO: 1 and/or SEQ ID NO: 2; antibodies and antigen binding fragments comprising the six CDRs of 3E10 set forth in SEQ ID NOs 4-9; etc.).

In a further related aspect, the disclosure contemplates that the in vivo imaging reagent may be formulated in a physiologically or pharmaceutically acceptable carrier suitable for in vivo administration. In certain embodiments, such compositions are suitable for oral or intravenous administration. In certain embodiments, such compositions are suitable for subcutaneously administration

In another aspect, the disclosure provides a radio-therapeutic agent. The radio- therapeutic agent comprises an internalizing moiety for targeting purposes (e.g., polypeptides comprising: monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10 and/or transits cell membranes via ENT2, or an antibody that binds the same epitope as 3E10 and/or transits cell membranes via ENT2, or an antibody that has substantially the same cell penetrating activity as 3E10 and/or transits cell membranes via ENT2, or an antigen binding fragment of any of the foregoing); and a radionuclide that is high energy and/or a short range. Further exemplary antibodies and antigen binding fragments are described above (e.g., antibodies and antigen binding fragments comprising SEQ ID NO: 1 and/or SEQ ID NO: 2; antibodies and antigen binding fragments comprising the six CDRs of 3E10 set forth in SEQ ID NOs 4-9; etc.). Any of the antibodies and antigen binding fragments described above may be used in any of the methods disclosed herein.

In certain embodiments, the internalizing moiety comprises an antibody or antibody fragment. In certain embodiments, the antibody or antigen binding fragment is chimeric, humanized, or fully human.

In certain embodiments, the radio-therapeutic comprises a radionuclide that is damaging or otherwise cytotoxic to cells and the internalizing moiety targets the radio- therapeutic preferentially to cancerous cell. In certain embodiments, the radionuclide is an alpha or beta emitting radionuclide. In certain embodiments, radionuclide is selected from the group consisting of iodine-131, yttrium-90, lutetium-177, copper-67, astatine -211, bismuth-212, bismuth-213, actinium-225. In certain embodiments, the radionuclide is yttrium-90. These radio-therapeutics are used, for example, to target the damaging radionuclide to cancer tissue to preferentially damage or kill cancer cells.

In a related aspect, the disclosure contemplates that, in making a radio-therapeutic, antibody or antibody fragment may be labeled with a radionuclide using any available method and chemistry. Association or conjugation of the radionuclide may be directly or via a coupling agent or linker. The disclosure contemplates methods of radiolabeling

internalizing moiety (e.g., polypeptides comprising: monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antibody that binds the same epitope as 3E10, or an antibody that has substantially the same cell penetrating activity as 3E10, or an antigen binding fragment of any of the foregoing). Further exemplary antibodies and antigen binding fragments are described above (e.g., antibodies and antigen binding fragments comprising SEQ ID NO: 1 and/or SEQ ID NO: 2; antibodies and antigen binding fragments comprising the six CDRs of 3E10 set forth in SEQ ID NOs 4-9; etc.). The resulting radiolabeled compound is preferentially targeted to particular cells and tissues, such as cancerous cells, upon administration.

In a further related aspect, the disclosure contemplates that the radio-therapeutic may be formulated in a physiologically or pharmaceutically acceptable carrier suitable for in vivo administration. In certain embodiments, such compositions are suitable for oral or intravenous administration. In other embodiments, such compositions are suitable for local administration directly to the site of a tumor. In certain embodiments, such compositions are suitable for subcutaneous administration.

In another aspect, the disclosure provides an in vivo imaging method. The method comprises administering to a subject, such as a human or non-human subject, an effective amount of an in vivo imaging reagent, as described herein. The effective amount is the amount sufficient to label the desired cells and tissues so that the labeled structures are detectable over the period of time of the analysis. The method further comprises collecting one or more images of the subject and displaying the one or more images of the subject. This collecting and displaying is done by a commercially available scanner and the accompanying computer hardware and software. For example PET and SPECT scanners may be used. Moreover, to further improve the usefulness of the images generated, CT, X-ray or MRI may be simultaneously or consecutively used to provide additional information, such as depiction of structural features of the subject. For example, dual PET/CT scanners can be used to collect the relevant data, and display images that overlay the data obtained from the two modalities.

Any of the radionuclides suitable for in vivo imaging and the corresponding radiolabeled compounds can be used in these methods.

In certain embodiments, the subject is a human. In certain embodiments, the subject is a patient having or suspected of having cancer, and the method is use to help diagnose the presence and location of the cancer. In certain embodiments, the in vivo imaging method is used to follow a patient's progression over time (e.g., over the course of treatment). In certain embodiments, the patient has or is suspected of having muscle cancer (e.g. rhabdomyosarcoma), testicular cancer (e.g. Leydig cell cancer), colon cancer, breast cancer, or ovarian cancer. In certain embodiments, the patient has or is suspected of having leukemia. In certain embodiments, the subject is healthy and the method is used to image normal tissue. In other embodiments, the method is used to observe potential irregularities in skeletal muscle, kidney, heart muscle, diaphragm, the testicles, the spinal cord or other nervous system tissue.

In certain embodiments, the in vivo imaging agent is administered orally or intravenously.

The images may be taken over a period of time, including multiple images over a period of time. In certain embodiments, the collecting and the displaying of the images are done using a PET scanner.

In another aspect, the disclosure provides a method for damaging cells, such as cancer cells, in a patient in need thereof. The method comprises administering to the patient an effective amount of any of the radio-therapeutic described herein. The radio-therapeutic is labeled with a high energy emitting radionuclide which is targeted to the cancer cells to damage the cancer cells.

In certain embodiments, the radio-therapeutic agent is administered orally or intravenously. In other embodiments, the agent is administered locally to the tumor.

The disclosure contemplates all combinations of any of the foregoing aspects and embodiments, as well as combinations with any of the embodiments set forth in the detailed description and examples.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure provides reagents and methods useful for in vivo imaging or for inducing cell damage. The disclosure is based on making certain radiolabeled compounds. The radiolabeled compounds comprise a suitable radionuclide (suitable for either in vivo imaging or for inducing cell damage) associated with certain antibodies or antibody fragments that serve to preferentially target the radionuclide to particular tissues.

Specifically, the antibodies or antibody fragments mediate cell targeting and internalization via ENT2 (equilibrative nucleoside transporter 2). It will appreciated that preferential targeting does not mean that the antibodies or antibody fragments, such as 3E10 and antigen binding fragments thereof, exclusively target a particular tissue or set of tissues. What is meant is that targeting and uptake is not ubiquitous, and that cell penetration is preferential for cells and tissues that express ENT2. In the present case, 3E10-related antibodies and antibody fragments preferentially transit ENT2 expressing cells and tissues, including skeletal muscle, cardiac muscle, kidney, diaphragm, cells of the testicle, and cancer cells. The ability to non-ubiquitously target radionuclides makes this approach particularly suitable for diagnostic, research and certain therapeutic approaches.

In addition to uptake by ENT2 expressing cells, 3E10 and 3E10-related antibody and antibody fragments are taken-up by the liver, despite the fact that the liver does not highly express ENT2. This is not surprising and is commonly observed following administration of biologies because of the high level of blood flow through the liver and the role of the liver in metabolism. However, the level of liver uptake seen following administration of conjugates of the disclosure is lower than that observed following administration of other biologies, including those conjugated to other targeting moieties (e.g., TAT, which does not selectively target certain cell types). Thus, the use of an internalizing moiety that targets cells non- ubiquitously and with a level of selectivity based on ENT2 expression results in a significant decrease in non-specific liver uptake relative to that observed for other biologies.

I. Internalizing Moieties

As used herein, the term "internalizing moiety" refers to a moiety capable of interacting with a target tissue or a cell type to effect delivery of the attached molecule into the cell (i.e., penetrate desired cell; transport across a cellular membrane; deliver across cellular membranes to, at least, the cytoplasm). In certain embodiments, this disclosure relates to an internalizing moiety which selectively, although not necessarily exclusively, targets and penetrates muscle cells (skeletal and cardiac), neurons, Leydig cells, cancer cells, and kidney cells. The internalizing moiety preferentially targets a particular cell or tissue type. In certain embodiments, suitable internalizing moieties include, for example, antibodies, monoclonal antibodies, or derivatives or analogs thereof. In certain embodiments, the internalizing moiety mediates transit across cellular membranes via an ENT2 transporter.

As used herein, the internalizing moiety is associated (conjugated, linked or otherwise coupled) with a radionuclide for use in vivo as an imaging reagent or a therapeutic agent. The associated moiety is referred to as a radiolabeled internalizing moiety or a radiolabeled compound. In certain embodiments, the radiolabeled internalizing moiety is a radiolabeled antibody or antibody fragment, such as 3E10 or an antigen binding fragment thereof, that transits cellular membranes via ENT2.

(a) Antibodies

In certain aspects, an internalizing moiety may comprise an antibody, including a monoclonal antibody, a polyclonal antibody, and a humanized antibody. Without being bound by theory, such antibody mediates delivery into a target tissue (e.g., muscle, cancer cells, etc.). In the context of the present disclosure, the ability to deliver radionuclides non- ubiquitously makes this approach and the compounds of the disclosure suitable for diagnostics, research, and certain therapeutic applications. By way of example, in the diagnostic context, if a detectable agent or other form of imaging agent is ubiquitously taken up by cells, it may be difficult to effectively use that agent. Similarly, in certain therapeutic contexts, it may be desirable to administer compounds systemically, and yet achieve a level of selectivity regarding which cells and tissues the compound actually acts upon. The present disclosure provides compositions and methods that address, for example, these needs.

In some embodiments, internalizing moieties may comprise antibody fragments, derivatives or analogs thereof, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, human antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent internalizing moieties including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((scFv)₂ fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments; receptor molecules which naturally interact with a desired target molecule. In certain embodiments, the antibodies or variants thereof, may be modified to make them less immunogenic when administered to a subject. For example, if the subject is human, the antibody may be humanized. In some

embodiments, the internalizing moiety is any peptide or antibody-like protein having the complementarity determining regions (CDRs) of the 3E10 antibody sequence, or of an antibody that binds the same epitope as 3E10. Also, transgenic mice, or other mammals, may be used to express humanized or human antibodies. Such humanization may be partial or complete. In certain embodiments, the internalizing moiety comprises the monoclonal antibody 3E10 or an antigen binding fragment thereof. For example, the antibody or antigen binding fragment thereof may be monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antigen binding fragment of 3E10 or said 3E10 variant. Additionally, the antibody or antigen binding fragment thereof may be an antibody that binds to the same epitope as 3E10, or an antibody that has substantially the same cell penetrating activity as 3E10, or an antigen binding fragment thereof. These are exemplary of agents that penetrate cells via ENT2. In certain embodiments, the antigen binding fragment is an Fv or scFv fragment thereof. Monoclonal antibody 3E10 can be produced by a hybridoma placed permanently on deposit with the American Type Culture Collection (ATCC) under ATCC accession number PTA-2439 and is disclosed in US Patent No. 7,189,396. Additionally or alternatively, the 3E10 antibody can be produced by expressing in a host cell nucleotide sequences encoding the heavy and light chains of this antibody. The term "3E10 antibody" or "monoclonal antibody 3E10" are used to refer to the antibody, regardless of the method used to produce the antibody. Similarly, when referring to variants or antigen-binding fragments of 3E10, such terms are used without reference to the manner in which the antibody was produced.

As used herein, 3E10Fv also refers to an antibody or antigen binding fragment thereof comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 2. Moreover, this antibody comprises the following CDRs, presented in the context of variable heavy and light chains comprising framework regions interspersed between the CDRs:

VH CDR1 having the amino acid sequence NYGMH (SEQ ID NO: 4);

VH CDR2 having the amino acid sequence YISSGSSTIYYADTVKG (SEQ ID

NO:5);

VH CDR3 having the amino acid sequence RGLLLDY (SEQ ID NO: 6);

VL CDR1 having the amino acid sequence RASKSVSTSSYSYMH (SEQ ID NO: 7);

VL CDR2 having the amino acid sequence YASYLES (SEQ ID NO: 8); and

VL CDR3 having the amino acid sequence QHSREFPWT (SEQ ID NO: 9).

The specific 3E10Fv construct used in the Examples is an scFv where the variable light and variable heavy chain domains are interconnected via a linker (see, SEQ ID NO: 3).

The internalizing moiety may also comprise variants of mAb 3E10, such as variants of 3E10 which retain the same cell penetration characteristics as mAb 3E10, as well as variants modified by mutation to improve the utility thereof (e.g., improved ability to target specific cell types, improved ability to penetrate the cell membrane, improved ability to localize to the cellular DNA, convenient site for conjugation, and the like). Such variants include variants wherein one or more conservative substitutions are introduced into the heavy chain, the light chain and/or the constant region(s) of the antibody. Such variants include humanized versions of 3E10 or a 3E10 variant. In some embodiments, the light chain or heavy chain may be modified at the N-terminus or C-terminus. Moreover, the antibody or antibody fragment may be modified to facilitate conjugation to a radionuclide. Similarly, the foregoing description of variants applies to antigen binding fragments. Any of these antibodies, variants, or fragments may be made recombinantly by expression of the nucleotide sequence(s) in a host cell.

Monoclonal antibody 3E10 has been shown to penetrate cells to deliver proteins and nucleic acids into the cytoplasmic or nuclear spaces of target tissues (Weisbart RH et al, J Autoimmun. 1998 Oct;l l(5):539-46; Weisbart RH, et al. Mol Immunol. 2003

Mar;39(13):783-9; Zack DJ et al, J Immunol. 1996 Sep l;157(5):2082-8.). Further, the VH and Vk sequences of 3E10 are highly homologous to human antibodies, with respective humanness z-scores of 0.943 and -0.880. Thus, Fv3E10 is expected to induce less of an anti- antibody response than many other approved humanized antibodies (Abhinandan KR et al., Mol. Biol. 2007 369, 852-862). A single chain Fv fragment of 3E10 possesses all the cell penetrating capabilities of the original monoclonal antibody, and proteins such as catalase, dystrophin, HSP70 and p53 retain their activity following conjugation to Fv3E10 (Hansen JE et al, Brain Res. 2006 May 9; 1088(1): 187-96; Weisbart RH et al, Cancer Lett. 2003 Jun 10;195(2):211-9; Weisbart RH et al, J Drug Target. 2005 Feb;13(2):81-7; Weisbart RH et al, J Immunol. 2000 Jun 1;164(11):6020-6; Hansen JE et al, J Biol Chem. 2007 Jul

20;282(29):20790-3). The 3E10 is built on the antibody scaffold present in all mammals; a mouse variable heavy chain and variable kappa light chain. 3E10 gains entry to cells via the ENT2 nucleotide transporter that is particularly enriched in skeletal muscle and cancer cells, and in vitro studies have shown that 3E10 is nontoxic. (Weisbart RH et al., Mol Immunol. 2003 Mar;39(13):783-9; Pennycooke M et al, Biochem Biophys Res Commun. 2001 Jan 26;280(3):951-9).

The internalizing moiety may also include mutants of mAb 3E10, such as variants of 3E10 which retain the same or substantially the same cell penetration characteristics as mAb 3E10, as well as variants modified by mutation to improve the utility thereof (e.g., improved ability to target specific cell types, improved ability to penetrate the cell membrane, improved ability to localize to the cellular DNA, improved binding affinity, and the like). Such mutants include variants wherein one or more conservative substitutions are introduced into the heavy chain, the light chain and/or the constant region(s) of the antibody. Numerous variants of mAb 3E10 have been characterized in, e.g., US Patent 7,189,396 and WO 2008/091911, the teachings of which are incorporated by reference herein in their entirety. In certain embodiments, the internalizing moiety comprises an antibody or antigen binding fragment having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 99%, or 100% identical to the amino acid sequence of 3E10, or at least 80%, 85%, 90%, 95%, 96%, 97%, 99%, or 100% identical to the amino acid sequence of a single chain Fv of 3E10 (for example, a single chain Fv comprising SEQ ID NOs: 1 and 2). In certain embodiments, the internalizing moiety comprises an antigen binding fragment, such as asingle chain Fv of 3E10, comprising a V_H domain comprising an amino acid sequence at least 90%>, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO : 1 , and comprising a V_L domain comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. The variant 3E10 or fragment thereof retains the function of an internalizing moiety. In certain embodiments, the internalizing moiety comprises at least 1 , 2, 3, 4, or 5 of the CDRs of 3E10. In certain embodiments, the internalizing moiety comprises all six CDRs of 3E10. For any of the foregoing, in certain embodiments, the internalizing moiety is an antibody that binds the same epitope as 3E10 and/or the

internalizing moiety competes with 3E10 for binding to antigen. Exemplary internalizing moieties target and transit cells via ENT2.

The present disclosure utilizes the cell penetrating ability of 3E10 or 3E10 fragments or variants to target radionuclides in vivo. In the imaging context, this provides improved methods for in vivo, nuclear imaging, and is particularly advantageous for imaging cancer cells and tissues, skeletal muscle, cardiac muscle, diaphragm and kidney. In the therapeutic context, coupling of 3E10 or 3E10 fragments or variants to a high energy radionuclide, such as a high energy alpha or beta emitter, provides a way to target cytotoxic radionuclides to cancer tissue. By targeting cancer tissue, such methods decrease the damage that occurs to healthy cells and tissue during cancer therapy.

As described further below, a recombinant 3E10 or fragment can be conjugated, linked or otherwise joined to a radionuclide. Methods of joining radionuclides to proteins are well known in the art and depend on the radionuclide, protein, and application.

Preparation of antibodies or fragments thereof (e.g., a single chain Fv fragment encoded by VH-linker-VL or VL-linker-Vn) is well known in the art. In particular, methods of recombinant production of mAb 3E10 antibody fragments have been described in WO 2008/091911. Further, methods of generating scFv fragments of antibodies are well known in the art. When recombinantly producing an antibody or antibody fragment, a linker may be used. For example, typical surface amino acids in flexible protein regions include Gly, Asn and Ser. One exemplary linker is provided in SEQ ID NO: 3. Permutations of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the criteria (e.g., flexible with minimal hydrophobic or charged character) for a linker sequence. Other near neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Moreover, linkers may also be used to provide a convenient site for labeling with a radionuclide.

The disclosure contemplates the use of one linker, multiple linkers or no linkers to, for example, interconnect the radionuclide to the internalizing moiety and to interconnect portions of the internalizing moiety (e.g., Vh and VI domains). The disclosure contemplates chimeric polypeptides that include 0 linkers, 1 linker, or 2 linkers. In certain embodiments, the chimeric polypeptides include more than 2 linkers.

Preparation of antibodies may be accomplished by any number of well-known methods for generating monoclonal antibodies. These methods typically include the step of immunization of animals, typically mice, with a desired immunogen (e.g., a desired target molecule or fragment thereof). Once the mice have been immunized, and preferably boosted one or more times with the desired immunogen(s), monoclonal antibody-producing hybridomas may be prepared and screened according to well known methods (see, for example, Kuby, Janis, Immunology, Third Edition, pp. 131-139, W.H. Freeman & Co.

(1997), for a general overview of monoclonal antibody production, that portion of which is incorporated herein by reference). Over the past several decades, antibody production has become extremely robust. In vitro methods that combine antibody recognition and phage display techniques allow one to amplify and select antibodies with very specific binding capabilities. See, for example, Holt, L. J. et al., "The Use of Recombinant Antibodies in Proteomics," Current Opinion in Biotechnology, 2000, 11 :445-449, incorporated herein by reference. These methods typically are much less cumbersome than preparation of hybridomas by traditional monoclonal antibody preparation methods. In one embodiment, phage display technology may be used to generate an internalizing moiety specific for a desired target molecule. An immune response to a selected immunogen is elicited in an animal (such as a mouse, rabbit, goat or other animal) and the response is boosted to expand the immunogen-specific B-cell population. Messenger RNA is isolated from those B-cells, or optionally a monoclonal or polyclonal hybridoma population. The mRNA is reverse- transcribed by known methods using either a poly-A primer or murine immuno globulin- specific primer(s), typically specific to sequences adjacent to the desired V_H and V_L chains, to yield cDNA. The desired VH and VL chains are amplified by polymerase chain reaction (PCR) typically using VH and VL specific primer sets, and are ligated together, separated by a linker. VH and VL specific primer sets are commercially available, for instance from

Stratagene, Inc. of La Jolla, California. Assembled V_H-linker-V_L product (encoding an scFv fragment) is selected for and amplified by PCR. Restriction sites are introduced into the ends of the VH-linker-VL product by PCR with primers including restriction sites and the scFv fragment is inserted into a suitable expression vector (typically a plasmid) for phage display. Other fragments, such as an Fab' fragment, may be cloned into phage display vectors for surface expression on phage particles. The phage may be any phage, such as lambda, but typically is a filamentous phage, such as fd and Ml 3, typically Ml 3.

In certain embodiments, an antibody or antibody fragment is made recombinantly in a host cell. In other words, once the sequence of the antibody or fragment is known (for example, using the methods described above), the antibody can be made recombinantly using standard techniques.

In certain embodiments, the internalizing moieties may be modified to make them more resistant to cleavage by proteases. For example, the stability of an internalizing moiety comprising a polypeptide may be increased by substituting one or more of the naturally occurring amino acids in the (L) configuration with D-amino acids. In various embodiments, at least 1%, 5%, 10%, 20%, 50%, 80%, 90% or 100% of the amino acid residues of internalizing moiety may be of the D configuration. The switch from L to D amino acids neutralizes the digestion capabilities of many of the ubiquitous peptidases found in the digestive tract. Alternatively, enhanced stability of an internalizing moiety comprising an peptide bond may be achieved by the introduction of modifications of the traditional peptide linkages. For example, the introduction of a cyclic ring within the polypeptide backbone may confer enhanced stability in order to circumvent the effect of many proteolytic enzymes known to digest polypeptides in the stomach or other digestive organs and in serum. In still other embodiments, enhanced stability of an internalizing moiety may be achieved by intercalating one or more dextrorotatory amino acids (such as, dextrorotatory phenylalanine or dextrorotatory tryptophan) between the amino acids of internalizing moiety. In exemplary embodiments, such modifications increase the protease resistance of an internalizing moiety without affecting the activity or specificity of the interaction with a desired target molecule.

II. Radionuclides

The present disclosure provides radiolabeled internalizing moieties, particularly radiolabeled antibodies, particularly radiolabeled 3E10-related antibodies and antibody fragments. As used herein, the terms "radionuclide" and "radioisotope" may be used interchangeably to refer to atoms with an unstable nucleus, which is a nucleus characterized by excess energy which is available to be imparted either to a newly-created radiation particle within the nucleus, or else to an atomic electron. The radionuclide, in this process, undergoes radioactive decay. Radionuclides may occur naturally, but can also be artificially produced.

Radionuclides vary based on their characteristics, which include half-life, energy emission characteristics, and type of decay. This allows one to select radionuclides that have the desired mixture of characteristics suitable for use diagnostically and/or therapeutically. For example, gamma emitters are generally used diagnostically and beta emitters are generally used therapeutically. However, some radionuclides are both gamma emitters and beta emitters, and thus, may be suitable for both uses by altering the amount of radioactivity used (the total and/or specific activity).

Exemplary radionuclides used to radiolabel include, but are not limited to, ²H, ³H, ⁿC,

^;C, ¹⁴C, ¹⁵N, ¹⁸0, ¹⁷0, ¹⁸F, ³⁶C1, ³²P, ³³P, ⁴³K, ⁴⁷Sc, ⁵²Fe, ⁵⁷Co, ⁶⁴Cu, ⁶⁷Ga, ⁶⁷Cu, ⁶⁸Ga, ⁷¹Ge, Br, ⁷⁶Br, ⁷⁷Br, ⁷⁷As, ⁷⁷Br, ⁸¹Rb, ^81mKr, ^87mSr, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ^99mTc, ¹⁰⁰Pd, ¹⁰¹Rh, ¹⁰³Pb,

211 212 212 213

At, Pb, Bi and Bi. These radionuclides, as well as their characteristics (e.g., half- life, emission, etc) are well known in the art, as are methods of making them and labeling proteins with them. Thus, one can select amongst available radionuclides to select the radionuclide with the appropriate combination of characteristics based on the particular application.

By way of example, when selecting a radionuclide for in vivo imaging, a gamma or positron emitting radionuclide or a radionuclide that decays by electron transfer may be preferred. Emissions can then be readily detected using, for example, positron emission tomography (PET) or single photon emission computed tomography (SPECT). Generally, it is desirable that the half-life of the radionuclide is long enough to be made and used in testing, but not so long that radioactivity lingers in the patient for a considerable period of time after the test has been performed. Moreover, the amount of radioactivity used to label can be modulated so that the minimum amount of total radiation is used to achieve the desired effect.

In certain embodiments, the radionuclide for in vivo imaging is selected from the group consisting of carbon-11, nitrogen-13, oxygen-15, fluorine-18, gallium-67, gallium-68, krypton-81m, rubidium-82, technetium-99m, indium-11, iodine-123, iodine-124, iodine-125, iodine-131, xenon-133, thallium-201 , zirconium-89, and copper-64. Note that iodine-131, for example, may also be useful therapeutically. However, by altering the dose and specific activity used, it can also be used diagnostically. In certain embodiments, the radionuclide for in vivo imaging is selected from technetium-99m, iodine-123, iodine-124, and iodine-125.

In certain embodiments, the radionuclide is selected to facilitate detection by PET scan or SPECT scan technologies.

Exemplary radionuclides that can be used to damage cells, such as cancer cells, are high energy emitters. For example, a high energy radionuclide is selected and targeted to cancer cells. The high energy radionuclide preferably acts over a short range so that the cytotoxic effects are localized to the targeted cells. In this way, radio-therapy is delivered in a more localized fashion to decrease damage to non-cancerous cells. In certain embodiments, the suitable radionuclide is an alpha or beta emitting radionuclide.

In certain embodiments, the radionuclide suitable for use as a radio-agent to damage cells is selected from the group consisting of iodine-131, yttrium-90, lutetium-177, copper- 67, astatine-211, bismuth-212, bismuth-213, and actinium-225. In certain embodiments, the radionuclide is yttrium-90.

As detailed herein, the present disclosure provides radiolabeled internalizing moieties. The internalizing moiety portion of the molecule serves to help target the radionuclide to particular tissues. This improves the use of the radionuclides in diagnostics and therapeutics. The disclosure contemplates that any such radionuclides, as well as other radionuclides suitable for diagnostic or therapeutic use may be complexed with an internalizing moiety to provide compositions of the disclosure. Moreover, such compositions can be used, for example, for research, diagnostic and therapeutic purposes. III. Radiolabeled Internalizing Moieties

The disclosure provides radiolabeled internalizing moieties. The disclosure contemplates that any of the internalizing moieties described in section I may be labeled with any of the radionuclides described in section II. The selection of the appropriate radionuclide is based on the desired application of the technology. Throughout the application, the terms "radiolabeled compound" or "radiolabeled agent" or "radiolabeled internalizing moiety" will be used to refer to radiolabeled compounds of the disclosure which are capable of selectively targeting radionuclides to particular tissues (e.g., skeletal muscle, cardiac muscle, diaphragm, kidney, testicles, nervous tissue and cancer cells). In certain embodiments, such a radiolabeled compound that selectively targets to particular tissues is a radiolabeled antibody or antibody fragment that transits cell membranes via ENT2. In certain embodiments, such a radiolabeled compound that selectively targets to particular tissues is a radiolabeled monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antibody that binds the same epitope as 3E10, or an antibody that has

substantially the same cell penetrating activity as 3E10, or an antigen binding fragment of any of the foregoing.

It should be noted that numerous methods for labeling proteins with radionuclides exist in the art and the particular method used depends on the protein, the radionuclide, and the application. In certain embodiments, the radionuclide may be incorporated into the protein during synthesis (either chemical synthesis or recombinant synthesis). In certain embodiments, the radionuclide may be appended via a reactive-thiol of a cysteine in the protein. In certain embodiments, an amino acid substitution may be introduced into the protein to provide a site for labeling. Numerous other chemistries exist by which proteins may be labeled with radionuclides, either directly or via linkers. One of skill can readily select amongst available chemistries and methodologies. It should be noted that, depending on the particular methodology used to label a protein, the protein may be labeled at one position or at multiple positions. The disclosure contemplates the making and use of radiolabeled compounds, including compounds that are labeled at a single position or at multiple positions. Moreover, in a sample containing numerous molecules of a particular protein, generally, only a subset of the molecules are appended with radionuclide. See Snook et al. (199) British Journal of Cancer 62 Supp X, 89-91 for a sample calculation. However, prior to use, unbound radiolabel is removed so that the radiolabel in a particular sample is bound to the protein of interest. Example 1 provides an example of this. In some embodiments, the polypeptide is isotopically labeled such that one or more atoms in the peptide is replaced by one or more atoms having specific atomic mass or mass numbers. Examples of isotopes that can be incorporated into proteins include isotopes of hydrogen, carbon, nitrogen, oxygen, and sulfur, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O.

Certain isotopically-labeled polypeptides, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated (i.e., ³H), and carbon-14 (i.e., ¹⁴C), isotopes are easily prepared and detected. Further, substitution with heavier isotopes such as deuterium (i.e., ²H), can afford certain advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances.

Isotopically labeled polypeptides can generally be prepared by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. Methods for doing so are well known in the art. Exemplary additional methods for labeling proteins with radionuclides are summarized below. However, numerous methods exist and are well know in the art.

One can use cross-linking agents such as heterobifunctional cross-linkers, which can be used to link molecules in a stepwise manner. Heterobifunctional cross-linkers provide the ability to design more specific coupling methods, thereby reducing the occurrences of unwanted side reactions. A wide variety of heterobifunctional cross-linkers are known in the art, including succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1-carboxylate (SMCC), m- Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a- methyl-a-(2-pyridyldithio)-tolune (SMPT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl 6-[3-(2-pyridyldithio) propionate] hexanoate (LC-SPDP). Those cross- linking agents having N-hydroxysuccinimide moieties can be obtained as the N- hydroxysulfosuccinimide analogs, which generally have greater water solubility. In addition, those cross-linking agents having disulfide bridges within the linking chain can be

synthesized instead as the alkyl derivatives so as to reduce the amount of linker cleavage in vivo. In addition to the heterobifunctional cross-linkers, there exist a number of other cross- linking agents including homobifunctional and photoreactive cross-linkers. Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH) and dimethylpimelimidate-2 HC1 (DMP) are examples of useful homobifunctional cross-linking agents, and bis-[B-(4 - azidosalicylamido)ethyl] disulfide (BASED) and N-succinimidyl-6(4'-azido-2'- nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive cross-linkers. One useful class of heterobifunctional cross-linkers, included above, contain the primary amine reactive group, N-hydroxysuccinimide (NHS), or its water soluble analog N- hydroxysulfosuccinimide (sulfo-NHS). Another reactive group useful as part of a

heterobifunctional cross-linker is a thiol reactive group. For a review of protein coupling techniques, see Means et al. (1990) Bioconjugate Chemistry. 1 :2-12. Example 1 provides an exemplary method of radiolabeling a protein, in this case with an iodine label.

In certain embodiments, the radiolabeled internalizing moieties, such as 3E10 and variants and fragments thereof, are formulated with a physiologically or pharmaceutically acceptable carrier. One or more labeled antibodies or antibody fragments may be

administered alone or as a component of a formulation (composition). The labeled compounds may be formulated for administration in any convenient way for use in human or veterinary medicine, for example, to a human or non-human subject. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Formulations include, for example, those suitable for oral, nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. For example, the specific activity of the radiolabeled antibody or antibody fragment may be modulated based on the particular application. In certain embodiments, the formulation is suitable for intravenous or oral administration.

Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a subject labeled compound as an active ingredient. Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxy ethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more subject labeled compounds may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example,

carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol

monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and

emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

Pharmaceutical compositions suitable for parenteral administration may comprise one or more radiolabeled compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.

When making and formulating a radiolabeled compound, one must select the activity and specific activity appropriate for the particular radionuclide and application. In the imaging context, it is preferable that the activity and specific activity is sufficient to effectively image the subject over the desired period of time. However, it is preferable to limit unnecessary exposure to radioactivity (which is also influenced by the half-life).

In a therapeutic context where the goal is to damage or be cytotoxic to cells, such as cancer cells, the activity and specific activity must be sufficient to have that cytotoxic effect.

In either case, the dose of the radiolabeled compound, as well as the activity and specific activity of the radionuclide itself, is selected based on factors, such as, the particular application of the technology, the patient, the radionuclide, etc.

Methods of introduction can be enteral or parenteral, including but not limited to, intradermal, intramuscular, intraperitoneal, intramyocardial, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes. In certain embodiments, the radiolabeled antibodies or antibody fragments are administered orally or intravenously.

However, local delivery is also contemplated (e.g., direct delivery to a tumor, such as injection into a tumor).

IV. In vivo Imaging

In one aspect, the disclosure provides compositions and methods for in vivo imaging.

The disclosure provides radiolabeled internalizing moieties. By associating a radionuclide useful for in vivo imaging with an internalizing moiety that preferentially targets to skeletal muscle, cardiac muscle, neurons, Leydig cells, kidney, diaphragm, and cancer cells, the disclosure provides improved methods and compositions suitable for in vivo imaging of these cells and tissues.

In certain embodiments, the in vivo imaging method is used to image healthy tissue, particularly healthy skeletal muscle, cardiac muscle, and kidney. Imaging analysis may be of the whole body of the subject, or on particular regions of the body. In certain embodiments, the in vivo imaging method is used to image skeletal muscle, cardiac muscle, kidney or diaphragm to observe potential or suspected abnormality. Imaging analysis may be of the whole body of the subject, or on particular regions of the body. As detailed in the Examples, we have successfully used radiolabeled 3E10Fv to image mice. Because the radiolabeled compositions of the disclosure preferentially label certain cells and tissues (rather than ubiquitously labeling cells and tissues), these compositions are particularly suitable to use in the in vivo imaging context.

In certain embodiments, the in vivo imaging method is used to detect cancer cells in a patient who has or is suspected of having cancer. In addition to use as a diagnostic tool, the method may also be used after diagnosis to follow the progress of the patient during treatment. For example, image analysis is performed at specified intervals (e.g., 3 months, 6 months, 1 year, etc.) to evaluate responsiveness to treatment, regression, spread, etc.

As detailed herein, 3E10 and related fragments transit cells via ENT2. ENT2 is expressed preferentially in certain tissues. ENT2 is also expressed in cancer cells. In certain embodiments, the subject is suspected of having colon, lung, ovarian, or breast cancer.

Journal of Experimental Therapeutics and Oncology (2002) 2: 200-212. In certain embodiments, the subject is suspected of having leukemia. In this context, radiolabeled compositions of the disclosure are particularly suitable to diagnosing and monitoring cancer. The diagnostic methods may be used as part of initial detection. Additionally or

alternatively, the diagnostic methods may be used to follow patients over time, such as to monitor spread of the disease or responsive of the disease to treatment.

In certain embodiments, the internalizing moiety used for the radiolabeled compounds suitable for diagnostic use is an antibody or antigen binding fragment comprising a heavy chain and a light chain, wherein the heavy chain variable domain comprises an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, or is a humanized variant of any of the foregoing. In certain embodiments, the internalizing moiety used for the radiolabeled compounds suitable for diagnostic use is an antibody or antigen binding fragment comprising a heavy chain and a light chain, wherein the light chain variable domain comprises an amino acid sequence at least 90%, 92%, 93%>, 94%>, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is a humanized variant of any of the foregoing. In certain embodiments, the internalizing moiety used for the radiolabeled compounds suitable for diagnostic use is an antibody or antigen binding fragment comprising a heavy chain variable domain comprising an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 , or is a humanized variant thereof; and a light chain variable domain comprising an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is a humanized variant of any of the foregoing. In certain embodiments, the antibody fragment further includes a linker, such as a linker interconnecting a heavy chain variable domain (VH) and a light chain variable domain (VL). In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3 and the antigen binding fragment is an scFv.

In certain embodiments, the internalizing moiety used for the radiolabeled compounds suitable for diagnostic use is an antibody or antigen binding fragment comprising the six CDRs of 3E10Fv described (SEQ ID NOs 4-9). For example, the six CDRs of 3E10 provided in the context of a heavy chain variable domain and a light chain variable domain containing framework regions interspersed between the CDRs. In certain embodiments, the antibody or antigen binding fragment comprises:

VH CDR1 having the amino acid sequence NYGMH (SEQ ID NO: 4)

VH CDR2 having the amino acid sequence YISSGSSTIYYADTVKG (SEQ ID NO:5)

VH CDR3 having the amino acid sequence RGLLLDY (SEQ ID NO: 6)

VL CDR1 having the amino acid sequence RASKSVSTSSYSYMH (SEQ ID NO: 7) VL CDR2 having the amino acid sequence YASYLES (SEQ ID NO: 8)

VL CDR3 having the amino acid sequence QHSREFPWT (SEQ ID NO: 9).

As noted above, such CDRs are provided in the context of an antibody or antigen binding fragment, such as with interspersed framework regions. In certain embodiments, the antigen binding fragment is an scFv, and the VH and VL domains are interconnected via a linker.

Numerous imaging technologies are known in the art. In the imaging context, gamma or positron emitting radionuclides or radionuclides that decay by electron transfer are typically used. These are detected using scanning equipment readily available, such as PET scanners and SPECT scanners. The scanning systems include detectors for detecting the emitted radiation, as well as computer equipment to process and interpret the emitted radiation and display it to the user. These systems include software that helps cancel background and improve the overall clarity of the image.

In certain embodiments, the internalizing moiety is labeled with a radionuclide suitable for in vivo imaging and selected from the group consisting of carbon- 11, nitrogen- 13, oxygen-15, fluorine-18, gallium-67, gallium-68, krypton-81m, rubidium-82, technetium-99m, indium-11, iodine-123, iodine-124, iodine-125, iodine-131, xenon-133, thallium-201, zirconium-89, and copper-64. In other embodiments, the internalizing moiety is labeled with a radionuclide suitable for in vivo imaging and selected from technetium-99m, iodine-123, iodine-124, and iodine-125.

In certain embodiments, the radionuclide is selected to facilitate detection and analysis using positron emission tomography or single photon emission computed

tomography. These are exemplary of imaging systems readily available and used

diagnostically, and are thus exemplary of the systems that can be used to perform in vivo imaging according to the present disclosure. These exemplary techniques are described briefly below.

Positron emission tomography (PET) is a nuclear medicine imaging technique which produces an image or picture of functional processes in the body. The system detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide (tracer), which is introduced into the body. Images of tracer concentration within the body are then constructed by computer analysis. In modern scanners, three dimensional imaging is often accomplished with the aid of a CT (computed tomography) scan performed on the patient during the same session, in the same machine. This is often referred to as PET/CT scanning.

PET is both a medical and research tool. It is used heavily in clinical oncology. PET is also an important tool to map normal human function, such as heart function.

PET is also used in pre-clinical studies using animals, where it allows repeated investigations into the same subjects. This is particularly valuable in cancer research, as it results in an increase in the statistical quality of the data and substantially reduces the numbers of animals required for a given study.

PET imaging is best performed using a dedicated PET scanner. However, it is possible to acquire PET images using a conventional dual-head gamma camera fitted with a coincidence detector. In either case, commercially available scanners are typically used in the context of systems that include the computer hardware and software necessary to interpret and display the data to the end user. Such systems include algorithms that decrease or eliminate background.

Generally, the radiolabeled compound is administered intravenously or swallowed. Over some period of time, the radiolabeled compound accumulates, in the case of the present disclosure, the radiolabeled compound preferentially accumulates in muscle, kidney, or cancer cells. The radionuclide emits energy which is detected by, for example, a gamma camera or a PET scanner. Once the radionuclide has been administered, the subject is positioned on a table in the scanner which collects data over some period of time. The period of time, as well as the frequency with which data is collected during the examination may vary depending on the particular application of the technology. Note that this technology may be used to perform whole body imaging, as well as to provide image analysis that focuses on a more particular portion of the body.

In some centers, nuclear medicine images may be superimposed with computed tomography (CT) or magnetic resonance imaging (MRI) to produce special views, a practice known as image fusion or co-registration. These views allow the information from two different studies to be correlated and interpreted on one image, leading to more precise information and accurate diagnoses. In addition, manufacturers are now making single photon emission computed tomography/computed tomography (SPECT/CT) and positron emission tomography/computed tomography (PET/CT) units that are able to perform both imaging studies at the same time.

CT imaging uses special x-ray equipment, and in some cases a contrast material, to produce multiple images or pictures of the inside of the body. These images can then be interpreted by a radiologist on a computer monitor as printed images. CT imaging provides excellent anatomic information.

In certain embodiments, the in vivo imaging methods are performed using instruments that are combined PET and CT scanners. Exemplary PET and PET/CT systems, including computer workstations are available, for example, from GE Healthcare and Siemens.

Single photon emission computed tomography (SPECT, or less commonly, SPET) is a nuclear medicine tomographic imaging technique using gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera. However, it is able to provide true 3D information. This information is typically presented as cross-sectional slices through the patient, but can be freely reformatted or manipulated as required. The basic technique requires injection of a gamma-emitting radionuclide into the bloodstream of the patient. In the same way that a plain X-ray is a 2-dimensional (2-D) view of a 3-dimensional structure, the image obtained by a gamma camera is a 2-D view of 3-D distribution of a radionuclide.

SPECT imaging is performed by using a gamma camera to acquire multiple 2-D images (also called projections), from multiple angles. A computer is then used to apply a tomographic reconstruction algorithm to the multiple projections, yielding a 3-D dataset. This dataset may then be manipulated to show thin slices along any chosen axis of the body, similar to those obtained from other tomographic techniques, such as MRI, CT, and PET. Exemplary SPECT systems, including computer workstations are available, for example, from GE Healthcare and Siemens.

SPECT is similar to PET in its use of radioactive tracer material and detection of gamma rays. In contrast with PET, however, the tracer used in SPECT emits gamma radiation that is measured directly, whereas PET tracer emits positrons which annihilate with electrons up to a few millimeters away, causing two gamma photons to be emitted in opposite directions. A PET scanner detects these emissions "coincident" in time, which provides more radiation event localization information and thus higher resolution images than SPECT (which has about 1 cm resolution). SPECT scans, however, are significantly less expensive than PET scans, in part because they are able to use longer-lived more easily-obtained radioisotopes than PET.

To acquire SPECT images, the gamma camera is rotated around the patient.

Projections are acquired at defined points during the rotation, typically every 3-6 degrees. In most cases, a full 360 degree rotation is used to obtain an optimal reconstruction. The time taken to obtain each projection is also variable, but 15-20 seconds is typical. This gives a total scan time of 15-20 minutes.

Multi-headed gamma cameras can provide accelerated acquisition. For example, a dual headed camera can be used with heads spaced 180 degrees apart, allowing 2 projections to be acquired simultaneously, with each head requiring 180 degrees of rotation. Triple-head cameras with 120 degree spacing are also used.

The foregoing are exemplary of the commercially available systems available in nuclear medicine to detect and display images. These are exemplary of the systems that can be used in diagnostic, prognostic and research methods of the present disclosure. V. Therapeutics

The disclosure also contemplates radiolabeled internalizing moieties suitable for use in inducing cell damage. In this aspect, an internalizing moiety, such as 3E 10 or a fragment thereof, is labeled with a high energy radionuclide. The radiolabeled compound is administered to a subject in need thereof. Given that 3E10 preferentially targets cancer cells, the subject in need thereof is a subject diagnosed with cancer. Following administration, such as oral or intravenous administration, the radiolabeled compound preferentially localizes to cancer cells to deliver cytotoxic emission to the cells. Suitable radionuclides are high energy but have a short range. In certain embodiments, the radiolabeled compounds are administered locally to the site of a tumor.

In certain embodiments of any of the therapeutic methods described herein, the internalizing moiety is monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antibody that binds the same epitope as 3E10, or an antibody that has substantially the same cell penetrating activity as 3E10, or an antigen binding fragment of any of the foregoing

The suitable radionuclide is preferably a high energy, short range radionuclide. In certain embodiments, the radionuclide is an alpha or beta emitter. In certain embodiments, the radionuclide additionally or alternatively is a gamma emitter.

In certain embodiments, the radionuclide is selected from the group consisting of iodine- 131, yttrium-90, lutetium- 177, copper-67, astatine-211 , bismuth-212, bismuth-213 , actinium-225. In certain embodiments, the radionuclide is yttrium-90.

As detailed herein, 3E10 and related fragments transit cells via ENT2. ENT2 is expressed preferentially in certain tissues. ENT2 is also expressed in cancer cells. In certain embodiments, the subject has colon, lung, ovarian, muscular, testicular or breast cancer, and the radio-agents provided herein are administered as part of a method of treatment. Journal of Experimental Therapeutics and Oncology (2002) 2: 200-212. In certain embodiments, the subject has leukemia, and the radio-agents provided herein are administered as part of a method of treatment.

The terms "treatment", "treating", and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. "Treating" a condition or disease refers to curing as well as ameliorating at least one symptom of the condition or disease. "Treatment" as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing symptoms of the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet begun experiencing symptoms; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms). For example, "treatment" of cancer encompasses decreasing tumor size, slowing progression, slowing metastases, decreasing pain, and the like. In the context of cancer therapy, it is understood that these and other therapeutic benefits are achieved by inflicting damage or cytotoxicity on cancer cells.

By the term "effective dose" is meant a dose that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

In certain embodiments, the internalizing moiety used for the radiolabeled compounds suitable for therapeutic use is an antibody or antigen binding fragment comprising a heavy chain and a light chain, wherein the heavy chain variable domain comprises an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, or is a humanized variant of any of the foregoing. In certain embodiments, the internalizing moiety used for the radiolabeled compounds suitable for therapeutic use is an antibody or antigen binding fragment comprising a heavy chain and a light chain, wherein the light chain variable domain comprises an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is a humanized variant of any of the foregoing. In certain embodiments, the internalizing moiety used for the radiolabeled compounds suitable for therapeutic use is an antibody or antigen binding fragment comprising a heavy chain variable domain comprising an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 , or is a humanized variant thereof; and a light chain variable domain comprising an amino acid sequence at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, or is a humanized variant of any of the foregoing. In certain embodiments, the antibody fragment further includes a linker, such as a linker interconnecting a heavy chain variable domain (VH) and a light chain variable domain (VL). In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3 and the antigen binding fragment is an scFv. In certain embodiments, the internalizing moiety used for the radiolabeled compounds suitable for therapeutic use is an antibody or antigen binding fragment comprising the six CDRs of 3E10Fv described (SEQ ID NOs 4-9). For example, the six CDRs of 3E10 provided in the context of a heavy chain variable domain and a light chain variable domain containing framework regions interspersed between the CDRs. In certain embodiments, the antibody or antigen binding fragment comprises:

VH CDR1 having the amino acid sequence NYGMH (SEQ ID NO: 4)

VH CDR2 having the amino acid sequence YISSGSSTIYYADTVKG (SEQ ID

NO:5)

VH CDR3 having the amino acid sequence RGLLLDY (SEQ ID NO: 6)

VL CDR1 having the amino acid sequence RASKSVSTSSYSYMH (SEQ ID NO: 7) VL CDR2 having the amino acid sequence YASYLES (SEQ ID NO: 8)

VL CDR3 having the amino acid sequence QHSREFPWT (SEQ ID NO: 9).

As noted above, such CDRs are provided in the context of an antibody or antigen binding fragment, such as with interspersed framework regions. In certain embodiments, the antigen binding fragment is an scFv, and the VH and VL domains are interconnected via a linker. The VH and VL domains may be in either orientation (e.g., VH-linker-VL or VL-linkerVH).

The radiolabeled compounds of the disclosure provide a mechanism for preferentially directing a cancer therapeutic to a subset of cells and tissues in a patient, such as a human or animal subject. In doing so, the risk of side effects associated with damaging healthy cells and tissues is diminished.

VI. Kits

In certain embodiments, the disclosure also provides a pharmaceutical package or kit comprising one or more containers filled with at least a radiolabeled compound of the disclosure. Exemplary containers include, but are not limited to, vials, bottles, pre-filled syringes, IV bags, blister packs (comprising one or more pills). Optionally associated with such container(s) is a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, or both.

In certain embodiments, the package is provided with shielding to limit exposure to radioactivity. In certain embodiments, the kit comprises a therapeutic agent and is packaged and labeled in a manner indicating its intended use in human or animal patients. In other embodiments, the kit comprises a diagnostic agent and is packaged and labeled in a manner indicating its intended use in diagnostics. In still another embodiment, the kit comprises agents intended for research purposes only, and the kit is packaged and labeled in a manner indicating that it may only be used for research purposes.

In certain embodiments, a kit comprises more than one of the radiolabeled compounds of the disclosure. When multiple compounds are provided in the same kit, the compounds may be provided in separate containers, each comprising a single such compound.

Alternatively, the compounds may be provided as a mixture in the same container.

EXEMPLIFICATION

The disclosure now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure. For example, the particular constructs and experimental design disclosed herein represent exemplary tools and methods for validating proper function. As such, it will be readily apparent that any of the disclosed specific constructs and experimental plan can be substituted within the scope of the present disclosure.

Example 1 : Radiolabeling of 3E10Fv

A 3E10 scFv fragment was produced recombinantly and is exemplary of the internalizing moieties that may be used. In this example, the fragment comprises the heavy and light chain sequences represented in SEQ ID NOs: 1 and 2 joined by a glycine-serine linker represented in SEQ ID NO: 3. Optionally, the internalizing moiety may have additional sequence, such as a tag to facilitate production and/or purification. The CDRs of the heavy and light chain variable domains are underlined and bolded below.

For this radiolabeling experiment, 3E10Fv, having a molecular weight of

approximately 25,000, was provided at a concentration of 1.4 mg/ml. A radioisotope of iodine was selected as the radionuclide. The resulting material was to be used for in vivo imaging, and thus a radioisotope suitable for in vivo labeling was selected. In this example, I- 124 was selected. However, other radioisotopes of iodine, such as 1-123 or 1-125 could similarly have been used. Moreover, other radioisotopes suitable for detection by, for example, PET or SPECT scanning may be used. 1-124 has a half-life of 4.2 days. 3 mCi of activity was ordered and shipped. Due to the half-life of 1-124, approximately 2 mCi in 50 microliters arrived for use.

The 3E10Fv protein was radioiodinated in vitro using commercially available Iodogen Tubes. Briefly, 100 micrograms of 3E10Fv in 71 microliters was aliquoted to an Iodogen Tube. The Iodogen Tube, such as is available from ThermoFisher, is pre-coated with an iodination reagent that activates the radioisotopes of iodine to facilitate the introduction of the radioisotope into proteins, typically via introduction into tyrosine residues.

In a separate tube, buffers and potassium iodide were combined. For these experiments, two labeling reactions were conducted. For reaction 1, 2 microliters of 0.2M HC1, 5 microliters of 0.5M phosphate buffer, and 20 microliters KI were combined. For reaction 2, 2 microliters of 0.2 M HC1, 5 microliters of 0.5M phosphate buffer, and 30 microliters KI were combined. For each of reactions 1 and 2, 20 microliters of the iodine radioisotope were added to the buffers, and then all of the reagents were combined with the 3E10Fv. This produced a total volume of 118 microliters for reaction 1 and 128 microliters for reaction 2. The reactions were incubated at room temperature for 15 minutes. The total activity in reaction 1 was 639 microCi and the total activity in reaction 2 was 612 microCi.

Labeling efficiency was calculated as 63.49% for reaction 1 and 84.8% for reaction 2. Unincorporated label was separated using a spin column, and the labeling efficiency was 99% following removal of unincorporated label. The specific activity of reaction 1 was calculated to be 4.06 microCi/ug and the specific activity of reaction 2 was calculated to be 5.19 microCi/ug. The two reactions were pooled, and the total activity following pooling was 731 microCi. Radiolabeled 3E10Fv was available for further use.

Example 2: Radiolabeled 3E10Fv Retains Cell Targeting Ability

As an initial control, we confirmed that radiolabeled 3E10Fv retains its cell penetrating ability. To do this, we evaluated binding of radiolabeled 3E10Fv to COS-7 cells in vitro. This experiment was performed two times and indicated 6.15% and 7.79%, respectively, of radiolabeled protein penetrated the COS-7 cells. This result is high relative to other internalizing technologies which typically yield less than 1% cell penetration.

Example 3 : Imaging and Biodistribution

Imaging analysis was performed using five mice (strain Nu/Nu). Radiolabeled antibody (250 microliters) was diluted in HSA (44 microliters) and saline (806 microliters) for a total volume of 1100 microliters. Approximately 200 microliters (approximately 133 microCi) was injected into each of five mice. Two of the mice were imaged for two hours each using PET followed by CT to obtain dynamic data over time. For two mice, 10 minute images were acquired at 2 hours post injection and for one mouse such images were acquired at 2 hours and 4 hours post injection.

PET imaging was conducted using both Siemens Focus 220 and Inveon microPET systems, with two hour or 10 minute acquisitions, filtered back projection image

reconstruction to 1.8 mm resolution and no attenuation correction. CT images were acquired using a Siemens microCAT II with 360 steps, 70 kVp, 500 ms exposure time and 2 mm aluminum filtration, and reconstructed using Feldkamp cone beam to a voxel size of 200 microns.

For PET data analysis, each mouse had a whole body (WB) region of interest (ROI) defined using the CT image to cover the entire animal. Applying this WB ROI to the PET data, we obtained the mean value and size of the animal and multiplying the two provided the total activity that was located in the body. This method explicitly excludes any activity remaining in the tail and only accounts for activity that was bio-available in the animals.

We observed the following from the PET/CT live-animal analysis:

• Significant uptake into muscle, including cardiac muscle, diaphragm, and skeletal muscle (2.46 ID/g average in skeletal muscle; 2.66 ID/g in heart; 2.98 in diaphragm)

• Significant motor neuron uptake (2.76 ID/g in sciatic nerve)

• No uptake into the brain - the radiolabeled antibody does not cross the blood-brain barrier in these studies

• Low liver uptake, in relation to that observed following administration of other

biologies

• Slow kidney clearance

• Radiolabeled 3E10Fv clears through the kidneys to the bladder with minimal liver uptake.

These results indicated that radiolabeled 3E10Fv is distributed in a manner consistent with expression of ENT2 in mice and is suitable for in vivo imaging by, for example, PET scan. In human tissue, high levels of ENT2 expression have previously been observed in skeletal muscle, cardiac muscle, and cancer cells. Published results indicate that ENT2 expression may be even more selective in humans. For example, expression in human skeletal muscle is approximately 20 fold higher than baseline (Biochemical and Biophysical Research Communications, 2001, 280: 951-959; Journal of Biological Chemistry, 1998, vol 273: 5288-5293).

Moreover, radiolabeled 3E10Fv compares very favorably in these experiments to other targeting molecules examined previously. We estimated that muscle uptake of radiolabeled 3E10Fv is 6-7 times better than TAT and 10-20x better than the general cell internalizing antibody, anti-PMSA (Cancer Biother Radiopharm, 2007, 22: 33-39;

Bioploymers (Pept Sci), 2007, 88: 98-107). As noted above, in addition to better targeting to muscle, we observed less uptake of 3E10Fv to liver that what has been documented in the literature for liver uptake of TAT.

We note that radiolabeling for in vivo imaging of humans can be easily modified. One of skill can select the appropriate dosage, specific activity, and total activity based on the imaging equipment being used.

Example 4: Diagnostic Imaging to Detect Cancer

Radiolabeled compounds, such as radiolabeled monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antibody that binds the same epitope as 3E10, or an antibody that has substantially the same cell penetrating activity as 3E10, or an antigen binding fragment of any of the foregoing are prepared for use as in vivo imaging reagents to detect cancer. The antibody or antibody fragment is radiolabeled with an appropriate radionuclide, such as a gamma or positron emitter or other emitter suitable for detection by PET scan or SPECT scan. The antibody or antibody fragment is radiolabeled with an appropriate activity and specific activity for diagnostic imaging purposes, based on the equipment used, the period of time over which images will be detected, and the like.

Radiolabeled compound is administered to a patient having or suspected of having cancer. Following administration, the patient is imaged using, for example a PET scanner. In certain embodiments, a combination PET/CT scanner is used. The detectors of the PET scanner collect the emitted radiation at specified intervals over some period of time.

Associated computer hardware and software are used to generate images, including images that overlay the PET and CT data, where applicable.

Given that ENT2 is highly expressed in cancer cells and tissues, cancer tissue, if present, will be heavily labeled. Additionally, given that cancer cells have a higher metabolic activity relative to healthy cells, cancer tissue is distinguishable from healthy tissue based on the rate at which the radiolabeled compound is metabolized by such tissue. Thus, by taking images over time, cancer tissue can be further distinguished from healthy tissue that may also take-up label. Moreover, even without differences in metabolic activity, cancerous tissue will have a different appearance and intensity relative to healthy tissue.

Radio-imaging may be used to diagnose the cancer, as well as to follow progression or improvement over time or in response to therapy.

Example 5 : Radiolabeled Compounds to Promote Cell Damage

Radiolabeled compounds, such as radiolabeled monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antibody that binds the same epitope as 3E10, or an antibody that has substantially the same cell penetrating activity as 3E10, or an antigen binding fragment of any of the foregoing are prepared for use as cytotoxic agents. Such agents are administered to a patient in need thereof to cause localized cell damage. This is particularly suitable in cancer patients.

The antibody or antibody fragment is radiolabeled with an appropriate radionuclide, such as a high energy emitter that cause localized cell damage. This radionuclide may be an alpha or beta emitter capable of having a cytotoxic effect over a small distance. The antibody or antibody fragment is radiolabeled with an appropriate activity and specific activity for cell damaging purposes, based on the radionuclide used, the particular cancer, the patient and the like.

Radiolabeled compound is administered to a patient in need thereof. Administration is oral, intravenous, or local administration directly to a tumor cite. Radiolabeled compound localizes preferentially to the cancer tissue so that the cell damaging effects of the

radionuclide are not ubiquitous across all patient tissues. Targeting of the cytotoxic radionuclide to the cancer tissue helps minimize side effects in comparison to administration of non-targeted cytotoxic agents (e.g., chemotherapeutics).

SEQUENCE LISTING

SEQ ID NO: 1 = 3E10 Variable Heavy Chain

EVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPE GLEWVAYISSGSS

TIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTT

LTVSS

SEQ ID NO: 2 = 3E10 Variable Light Chain

DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLI YASY LESGVPARFSGSGSGTDFHLNIHPVEEEDAATYYCOHSREFPWTFGGGTKLEL

SEQ ID NO: 3 = GS3 linker = (G₄S)₃

GGGGSGGGGSGGGGS

SEQ ID NO: 4 = variable heavy chain CDR1 of exemplary 3E10 molecule

NYGMH

SEQ ID NO: 5 = variable heavy chain CDR2 of exemplary 3E10 molecule

YISSGSSTIYYADTVKG

SEQ ID NO: 6 = variable heavy chain CDR3 of exemplary 3E10 molecule

RGLLLDY SEQ ID NO: 7 = variable light chain CDR1 of exemplary 3E10 molecule

RASKSVSTSSYSYMH

SEQ ID NO: 8 = variable light chain CDR2 of exemplary 3E10 molecule

YASYLES

SEQ ID NO: 9 = variable light chain CDR3 of exemplary 3E10 molecule

QHSREFPWT

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below.

The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

We Claim:

1. An in vivo imaging reagent, comprising

(a) monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antibody that binds the same epitope as 3E10 and transits cells via ENT2, or an antibody that has substantially the same cell penetrating activity as 3E10 and transits cells via ENT2, or an antigen binding fragment of any of the foregoing; and

(b) a radionuclide suitable for in vivo imaging selected from a gamma or positron emitting radionuclide or a radionuclide that decays by electron transfer.

2. The in vivo imaging reagent of claim 1, wherein (a) comprises an antibody or antigen binding fragment comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1, or a humanized antibody or antigen binding fragment thereof.

3. The in vivo imaging reagent of claim 1 or 2, wherein (a) comprises an antibody or antigen binding fragment comprising a heavy chain and a light chain, wherein the light chain comprises a variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, or a humanized antibody or antigen binding fragment thereof.

4. The in vivo imaging reagent of any of claims 1-3, wherein (a) comprises an antibody or antigen binding fragment comprising a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1, or a humanized variant thereof, and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, or a humanized antibody or antigen binding fragment thereof.

5. The in vivo imaging reagent of any of claims 1-4, wherein (a) comprises an antibody or antigen binding fragment comprising a heavy chain and a light chain, and wherein the heavy chain and light chain variable domains comprise:

VH CDRl having the amino acid sequence NYGMH (SEQ ID NO: 4)

VH CDR2 having the amino acid sequence YISSGSSTIYYADTVKG (SEQ ID

NO:5)

VH CDR3 having the amino acid sequence RGLLLDY (SEQ ID NO: 6)

VL CDRl having the amino acid sequence RASKSVSTSSYSYMH (SEQ ID NO: 7) VL CDR2 having the amino acid sequence YASYLES (SEQ ID NO: 8) VL CDR3 having the amino acid sequence QHSREFPWT (SEQ ID NO: 9).

6. The in vivo imaging reagent of any of claims 1-5, wherein the antibody or antigen binding fragment is chimeric, humanized, or fully human.

7. The in vivo imaging reagent of any of claims 1-6, wherein the reagent is detectable by PET or SPECT.

8. The in vivo imaging reagent of any of claims 1-7, wherein the radionuclide is selected from the group consisting of carbon-11, nitrogen-13, oxygen-15, fluorine-18, gallium-67, gallium-68, krypton-81m, rubidium-82, technetium-99m, indium-11, iodine-123, iodine-124, iodine-125, iodine-131, xenon-133, thallium-201, zirconium-89, and copper-64.

9. The in vivo imaging reagent of any of claims 1-7, wherein the radionuclide is selected from technetium-99m, iodine-123, iodine-124, and iodine-125.

10. A composition, comprising

the in vivo imaging reagent of any of claims 1-9 formulated in a physiologically acceptable carrier.

11. The composition of claim 10 suitable for oral or intravenous administration.

12. A radio-therapeutic agent, comprising

(a) monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antibody that binds the same epitope as 3E10 and transits cells via ENT2, or an antibody that has substantially the same cell penetrating activity as 3E10 and transits cells via ENT2, or an antigen binding fragment of any of the foregoing; and

(b) a high energy, short range radionuclide.

13. The radio-therapeutic agent of claim 12, wherein (a) comprises an antibody or antigen binding fragment comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1, or a humanized antibody or antigen binding fragment thereof.

14. The radio-therapeutic agent of claim 12 or 13, wherein (a) comprises an antibody or antigen binding fragment comprising a heavy chain and a light chain, wherein the light chain comprises a variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, or a humanized antibody or antigen binding fragment thereof.

15. The radio-therapeutic agent of any of claims 12-14, wherein (a) comprises an antibody or antigen binding fragment comprising a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1, and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2; or a humanized antibody or antigen binding fragment thereof.

16. The radio-therapeutic agent of any of claims 12-15, wherein (a) comprises an antibody or antigen binding fragment comprising a heavy chain and a light chain, and wherein the heavy chain and light chain variable domains comprise:

VH CDRl having the amino acid sequence NYGMH (SEQ ID NO: 4)

VH CDR2 having the amino acid sequence YISSGSSTIYYADTVKG (SEQ ID NO:5)

VH CDR3 having the amino acid sequence RGLLLDY (SEQ ID NO: 6)

VL CDRl having the amino acid sequence RASKSVSTSSYSYMH (SEQ ID NO: 7)

VL CDR2 having the amino acid sequence YASYLES (SEQ ID NO: 8)

VL CDR3 having the amino acid sequence QHSREFPWT (SEQ ID NO: 9).

17. The radio-therapeutic agent of any of claims 12-16, wherein the antibody or antigen binding fragment is chimeric, humanized, or fully human.

18. The radio-therapeutic agent of any of claims 12-17, wherein the radionuclide is an alpha or beta emitting radionuclide.

19. The radio-therapeutic agent of any of claims 12-18, wherein the radionuclide is selected from the group consisting of iodine-131, yttrium-90, lutetium-177, copper-67, astatine-211 , bismuth-212, bismuth-213, actinium-225.

20. The radio-therapeutic agent of any of claims 12-18, wherein the radionuclide is yttrium-90.

21. A composition, comprising

the radio-therapeutic agent of any of claims 12-20 formulated in a physiologically acceptable carrier.

22. The composition of claim 21 suitable for oral or intravenous administration.

23. A in vivo imaging method, comprising

administering to a subject the in vivo imaging reagent of any of claims 1-9;

collecting one or more images of the subject; and

displaying the one or more images of the subject.

24. A in vivo imaging method, comprising

administering to a subject the composition of claim 10 or 11;

collecting one or more images of the subject; and

displaying the one or more images of the subject.

25. The method of claim 23 or 24, wherein the subject is a human.

26. The method of any of claims 23-25, wherein the subject is a patient having or suspected of having cancer.

27. The method of any of claims 23-25, wherein the subject is a healthy.

28. The method of any of claims 23-27, wherein the method is used to image one or more of skeletal muscle, heart muscle, diaphragm, or kidney.

29. The method of any of claims 23-28, wherein the method comprises taking images over a period of time.

30. The method of any of claims 23-29, wherein the collecting and the displaying of the images are done using a PET scanner.

31. A method for damaging cancer cells in a patient in need thereof, comprising

administering to the patient the radio-therapeutic agent of any of claims 12-20, wherein the agent is targeted to the cancer cells to damage the cancer cells.

32. A method for damaging cancer cells in a patient in need thereof, comprising

administering to the patient the composition of claim 21 or 22, wherein the agent is targeted to the cancer cells to damage the cancer cells.

33. The method of claim 31 or 32, wherein the agent is administered intravenously.

34. The method of claims 31 or 32, wherein the agent is administered orally.