WO2012032043A1

WO2012032043A1 - 212 pb imaging

Info

Publication number: WO2012032043A1
Application number: PCT/EP2011/065388
Authority: WO
Inventors: Michael Azure; Gilbert Andreoletti; Patrick Maquaire; Julien Torgue; Patrick Bourdet
Original assignee: Areva Med LLC
Current assignee: Orano Med LLC
Priority date: 2010-09-07
Filing date: 2011-09-06
Publication date: 2012-03-15
Anticipated expiration: 2013-03-07

Abstract

A radiopharmaceutical composition that comprises a radioactive isotope of lead (²¹²Pb) combination with a pharmaceutical or an antibody or antibody fragment and a chelating agent. These compositions are especially useful in the imaging and diagnosis of tumors and tumor metastases.

Description

Pb IMAGING

BACKGROUND OF THE INVENTION

Radioimmunotherapy in certain instances consists of irradiating cancerous cells present in an organism by injecting a monoclonal antibody conjugated to a radioisotope emitting alpha or beta particles which selectively binds to an antigen present predominantly on cancerous cells. Radioimmunotherapy is intended for the treatment of disseminated cancers, such as metastatic cancer, and for the treatment of micrometastases.

Certain descendants of 228 Th, in particular 212 Bi, which is an alpha-emitter, and 212 Pb, which is a beta-emitter and the radioactive parent of ²¹²Bi, can be used in alpha- radioimmunotherapy especially for treating cancers as set forth in WO2008113792, and other pending patents. However, the preparation of radiopharmaceutical products which comprise these descendants involves firstly being able to provide, and therefore to produce on an industrial scale,

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high-purity radioisotopes such as Ra, the parent of Pb.

Effective detection and diagnosis of certain disorders such as cancer have long been an object of intense research. The development of targeted pharmaceuticals and monoclonal antibodies has significantly improved the ability to target and deliver diagnostic agents to specific target cells, tissues or organs. Of particular interest in the diagnosis of many forms of carcinoma is the use of antibodies, both polyclonal and monoclonal, to specifically bind to tissues or molecules. The use of such antibodies continues to be an important tool in cancer detection through imaging. To be useful for such imaging purposes, pharmaceuticals and antibodies must first be labeled with an appropriate radionuclide. The present invention is based upon the finding that the highly purified agent ²¹²Pb allows imaging of tumors and pathogens in infected individuals.

SUMMARY OF THE INVENTION The invention relates to a pharmaceutical and/or diagnostic composition comprising of an

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amount of Pb effective for imaging. In some embodiments the Pb linked to a targeting molecule that specifically binds to a malignant cancer cell, cell surface marker or pathogen. In some embodiments, the targeting molecule is a polypeptide, including an antibody. The antibody may immunospecifically bind to one or more antigens selected from the group consisting of: HER2, HER1, CD45, CD25, CD20, CD22, CD19, CD33, VEGF, MET, CEA, BRCA1, BRCA2 and IGFR. In some embodiments of the pharmaceutical and/or diagnostic composition, the targeting molecule is a liposome. As used herein, effective amount is an amount effective to image a malignant cancer cell or pathogen. In some embodiments, the metastatic cancer cell is a micrometastatic cancer cell. The metastatic cancer cells may or may not be present in a solid tumor. In some embodiments, the pathogen is a virus or bacterium. In some embodiments, the pharmaceutical and/or diagnostic composition is substantially

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free of Ra. Substantially free may be defined such that the amount of Ra present in the composition is less than 0.5% of the total ²¹²Pb. Substantially free may be further defined such

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that 0.5% represents less than one Ra molecule per 200 molecules of Pb. In some embodiments, substantially free may be defined such that the amount of ²²⁴Ra present in the

212 224 composition is less than 0.25% of the total Pb where 0.25% represents less than one Ra molecule per 400 molecules of ²¹²Pb.

In some embodiments, the polypeptide present in the pharmaceutical and/or diagnostic composition is conjugated to said ²¹²Pb via a chelator. Such chelators include, but are not limited to, TCMC and DOTA. The invention also encompasses a method for imaging a malignant cancer cell or pathogen in a mammal, comprising administering any of the above pharmaceutical and/or diagnostic compositions in an amount sufficient to resolve gamma irradiation emitted by the ²¹²Pb and/or its decay products. In some embodiments, the malignant cancer cell is metastatic and includes, but is not limited to, a malignant cancer cell which is present in micrometastases. In some embodiments, the malignant cancer overexpresses a cell surface marker selected from the group consisting of HER2, HERl, CD45, CD25, CD20, CD22, CD 19, CD33, VEGF, MET, CEA, BRCA1, BRCA2 and IGFR whereas the pathogen may be a virus or bacterium. In some embodiments, the mammal is a human. The decay product referenced above may be selected from the group consisting of ²¹²Bi and ²⁰⁸T1. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: (a) NHP phantom organs; (b) assembled phantom placed on SPECT camera. The top 30 mL vial represents the heart, the center 250 mL saline bag the liver, and the two lower 15 mL vials the kidneys. Medium energy collimators are mounted on SPECT camera for all imaging. Figure 2: (a) NHP phantom images containing 2.8 MBq (75 μ^) in the heart, 3.2 MBq

(87 μθί) in the liver, 0.7 MBq (20 μθ) in the left kidney, and 0.8 MBq (23 μθ) in right kidney; (b) Discernable organs indicate the ²¹²Pb 238.6 keV can be isolated with a ± 20% window. Figure 3 : Planar gamma camera images of the phantom (peritoneal) with threshold adjustment together with a photograph of the phantom during imaging at 0 hour. The collimator is visible below. Small sources are located above the arrowheads to check orientation.

Figure 4: Planar gamma camera images of dose residual syringe with threshold adjustment and regions of interest.

Figure 5 : Planar gamma camera images of NHP-1 with threshold adjustment.

Figure 6: (a) Planar Images Collected on μ8ΡΕΟΤ/ΟΤ of three separate mice post- intraperitoneal injection of ²¹²Pb-TCMC-Trastuzumab at time points of to, h, and t_8h; (b) μ8ΡΕΟΤ/ΟΤ images of a single mouse collected at post-intraperitoneal injection of ²¹²Pb-TCMC- Trastuzumab at time points of to, Uh, and t_8h.

DETAILED DESCRIPTION

Radiation is an effective cancer treatment, but it is difficult to direct and can be devastating to non-targeted parts of the body. Indeed, treatments are often limited by radiation's non-selectivity, resulting in toxicity on the normal tissues. On the other hand, agents which target malignant cancer cells selectively target diseased cells, limiting harmful exposure to nearby healthy cells. The ability of these agents to exploit antigenic differences between normal and malignant tissues and to exact a variety of anti-tumor responses offers significant advantages over conventional forms of imaging and therapy.

Radioimmunotherapy and radioimaging, which incorporate conjugating a radioisotope to an agent which selectively targets a malignant cancer cell, can solve both problems. These agents, such as monoclonal antibodies, are highly specific and can be used as vehicles to deliver substances to specific target sites. Thus, the conjugation of these agents to a radioisotope is an effective way of targeting specific cells, thus reducing the side effects of radiotherapy.

The present invention is a radiopharmaceutical composition that comprises a radioactive isotope of lead (²¹²Pb), in combination with a pharmaceutical or an antibody or antibody fragment, and a bifunctional chelating agent. The present invention includes the use of any monoclonal antibody which exhibits cell-binding or antigen-binding capability.

The invention described herein relates to compositions that comprise a targeting agent, which specifically binds to a cell surface marker on a malignant cancer cell, wherein said agent is

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conjugated to a highly purified Pb radioisotope. The use of highly purified Pb allows for more selective imaging of malignant cancer cells because such purified radioisotopes do not contain ²²⁴Ra that can interfere with resolution of gamma rays emitted from the radioisotope. The compositions of the invention can be used for imaging a tumor in a mammal, including a human, but can also be used for imaging a tumor in non-humans, including but not limited to, mice, rats, sheep, cattle, horses, monkeys, dogs and cats.

Conjugated antibodies

²¹²Pb has very favorable nuclear and chemical properties, and can be conjugated via a metal chelator to monoclonal antibodies. The resulting conjugate, administered through intravenous or intraperitoneal injection of the ²¹²Pb radioisotope antibody conjugate, has been used in vivo to image and assess the biodistribution of the antibody.

The development and production of monoclonal antibodies is well known and has been extensively discussed. The present invention includes the use of any monoclonal antibody which exhibits cell binding or antigen binding capability. The selection and production of suitable monoclonal antibodies is within the skill of the art.

The antibody or antibody fragment used in this invention may be any polyclonal or monoclonal antibody or antibody fragment which forms an immunochemical reaction with an antigen. An "antigen" as used herein includes any substance or molecule which induces the formation of an antibody (i.e. , that can trigger an immune response), and can be a virus, a bacterium, a fungus, a parasite, tissues or cells not naturally a member of a host's family of tissues or cells, or even a portion or product of any of these organisms. "Antigenic" or "immunogenic" are used to describe the capacity of a given substance to stimulate the production of antibodies.

The term "antibody" as used herein includes the soluble substance or molecule secreted or produced by an animal in response to an antigen, and which has the particular property of combining specifically with the antigen which induced its formation. Antibodies, also known as immunoglobulins, are classified into five distinct classes: IgG, IgA, IgM, IgD, and IgE. The basic IgG immunoglobulin structure consists of two identical light polypeptide chains and two identical heavy polypeptide chains (linked together by disulfide bonds). When IgG is treated with the proteolytic enzyme papain, an antigen binding fragment (or Fab) can be isolated. When IgG is treated with pepsin (another proteolytic enzyme), a larger fragment is produced, F(ab')₂. This fragment can be split in half by reduction to Fab'. The Fab' fragment is slightly larger than the Fab and contains one or more free sulfhydryls from the hinge region (which are not found in the smaller Fab fragment). It is well known in the art to treat antibody molecules with pepsin in order to produce antibody fragments.

An antibody is further defined to include a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of five of the basic heterotetramer units along with an additional polypeptide called J chain, and therefore contains ten antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages that comprise two to five of the basic four-chain units along with J chain). The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains, and the methods of the current invention include the use of antibodies with either a kappa or lambda L chain. Depending on the amino acid sequence of the constant domain of their heavy chains (C_H), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. The gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in C_H sequence and function, e.g. , humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The methods of the present invention include the use of antibodies, including monoclonal antibodies, from any of the above classes and/or subclasses. As used herein, the term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The variable domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the variable regions consist of relatively invariant stretches called framework regions (FR) of about fifteen to thirty amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each about nine to twelve amino acids long. The variable domains of native heavy and light chains each comprise four framework regions, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held in close proximity by the framework region and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Public Health Service, National Institutes of Health). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" which contributes to the specificity of the antibody. An antibody also refers to antibodies or fragments thereof that specifically bind to an antigen of interest. Preferably, antibodies or fragments that immunospecifically bind to a selected antigen do not non-specifically cross-react with other antigens (e.g. , binding cannot be competed away with a different protein, e.g., BSA in an appropriate immunoassay). Antibodies or fragments that immunospecifically bind to an antigen can be identified, for example, by immunoassays or other techniques known to those of skill in the art. Antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, diabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv) (including bi- specific scFvs), single chain antibodies, Fab' fragments, F(ab')₂ fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a malignant cancer cell marker (e.g., one or more complementarity determining regions (CDRs) of an antibody).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies present in the population are identical, except for possible naturally occurring mutations that may be present in minor amounts, and includes antibody fragments as defined herein. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a specified determinant on the antigen. The term "monoclonal antibody" also encompasses bispecific monoclonal antibodies (BsMAb, BsAb) which are composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al. (1975) Nature 256, 495, or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see U.S. Patent 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-628, and Marks et al. (1991) J. Mol. Biol. 222, pp. 581-597, for example.

As used herein, an "intact" antibody is one which comprises an antigen-binding site as well as a C_L and at least heavy chain constant domains, C_m and C_m and C_H3. The constant domains may be native sequence constant domains (e.g. , human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding CDR or variable region of the intact antibody. Examples of antibody fragments include Fab, Fv, Fab' and F(ab')₂ fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870 and Zapata et al. (1995) Protein Eng. 8, pp. 1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V_H), and the first constant domain of one heavy chain (C_HI). Each Fab fragment is monovalent with respect to antigen binding, i.e. , it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')₂ fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the C_M domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab') ₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which is also the part recognized by Fc receptors (FcR) found on certain types of cells.

As used herein, "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chains) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that comprises only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although at a lower affinity than the entire binding site. As used herein, "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the V_H and V_L antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding (see

Rosenburg et al. (1994) The Pharmacology of Monoclonal Antibodies, Springer- Verlag, pp. 269- 315).

As used herein, the term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5 to about 10 residues) between the V_H and V_L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the V_H and V_L domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, WO 93/11161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90, pp. 6444-6448. An "isolated antibody" is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous components. In preferred embodiments, the antibody will be purified to greater than 95% by weight of antibody, and most preferably more than 99% by weight. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

The conjugated antibodies used in the methods of the invention include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent 4,816,567 and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, pp. 6851-6855). Chimeric antibodies of interest herein include, but are not limited to "humanized" antibodies comprising variable domain antigen-binding sequences derived from a non-human mammal (e.g. , murine) and human constant region sequences.

Antibodies of the invention may also comprise a fully human antibody sequence. In another embodiment, antibodies of the invention may be a fully human antibody. The invention comprises antibodies, or fragments thereof, which immunospecifically bind to a selected antigen, wherein said antibody is conjugated to a radioisotope via a chelator linkage. Examples include a bifunctional chelant, which is a molecule that has, in addition to chelating functionality, the ability to be conjugated (linked) to a biotargeting molecule (e.g. monoclonal antibody). Examples include, but are not limited to, mono- or dicyclic anhydrides of DTPA (diethylenetriamine pentaacetic acid), TCMC (1, 4,7, 10-tetrakis(carbamoylmethyl)- 1,4,7, 10- tetraazacyclododecane), MeO-DOTA (a-(5-isothiocyanato-2-methoxyphenyl)-l,4,7, 10- tetraazacyclododecane-l,4,7, 10-tetraacetic acid which is a bifunctional chelant) and DOTA ( 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid), CYEDTA (trans- 1 ,2- diaminocyclohexane-N,N,N',N'-tetraacetic acid) and derivatives thereof. TCMC provides metal complexes with high stability, thereby reducing the incidence of background during imaging procedures or damage to non-targeted tissues in radioimmunotherapy.

Generally, the chelate conjugated to the monoclonal antibody is a derivative of the chelate bonded to an organic functional group which serves to link the chelate to the monoclonal antibody. Reacting the chelate derivative and the monoclonal antibody produces the chelate conjugated monoclonal antibodies which can be reacted with ²¹²Pb radioisotope to produce the ²¹²Pb-antibody conjugates. These conjugates retain sufficient activity and selectivity of the antibody or antibody fragment.

In one embodiment of the invention, the conjugated antibody binds to an epitope on the cytoplasmic domain of a protein specific to cancer cells (i.e. , a cancer cell marker). The term

"malignant cancer cell antigen" is defined herein as an antigen that is present in higher quantities on a tumor cell or in body fluids than unrelated tumor cells, normal cells, or in normal body fluid. The antigen presence may be tested by any number of assays known to those skilled in the art and include without limitation negative and/or positive selection with antibodies, such as an ELISA assay, a Radioimmunoassay, or by Western Blot.

In another embodiment, the conjugated antibody includes, but is not limited to, an antibody which binds to an epitope on the cytoplasmic domain of a tissue antigen selected from the group consisting of HER2 (Human Epidermal Growth Factor Receptor-2), HERl (Human Epidermal Growth Factor Receptor- 1 also known as Epidermal Growth Factor Receptor), CD45, CD25, CD20, CD22, CD 19, CD33, VEGF (Vascular Endothelial Growth Factor), MET (MNNG (N-Methyl-N'-nitro-N-nitroso-guanidine) HOS Tranforming gene also known as Hepatocyte Growth Factor Receptor), CEA (Carcinoembryonic antigen), BRCA1, BRCA2 and IGFR-1 (Insulin-like Growth Factor Receptor- 1). An example of an antibody is the Trastuzumab monoclonal antibody which binds to HER2 as described in U.S. Patent 6, 165,464 which is herein incorporated by reference in its entirety. Although tumor imaging and diagnosis is one application of the conjugated antibodies of the invention, conjugated antibodies to a number of other antigens also is encompassed in the invention. For example, antibodies to pathogens such as viruses can be conjugated to ²¹²Pb radioisotope to image and detect viral infections. In another example, antibodies to bacterial antigens can be conjugated to ²¹²Pb radioisotope to image and detect bacterial infections.

In some embodiments, the pathogens which can be detected include a virus (or an antigen present on the cell surface of infected cells) or bacteria. Exemplary pathogenic antigens can be found in Ansari (2010) Nucl. Acids Res. 38, D847-853. Exemplary viruses which can be detected according to the methods of the invention include human immunodeficiency virus (HIV), influenza virus, vaccinia virus, human papilloma virus, hepatitis virus (A, B or C) and human parvovirus. Exemplary bacteria that can be detected according to the methods of the invention include Mycobacterium, Plasmodium, Anaplasma, Brucella, Candida, Helicobacter,

Streptococcus, Staphylococcus, Haemophilus, Vibrio, Clostridium, Yersinia, Coccidioides, Neisseria, Borrelia, Chlamydia, Shigella, Bacillus, Legionella, Listeria, Pseudomonas, Boophilus, Treponema, Klebsiella, Salmonella and Echinococcus.

Free Radioisotope

The invention encompasses a formulation of highly purified ²¹²Pb for targeting a tumor site. Due to the absence of ²²⁴Ra in the highly purified radioisotope formulation, direct injection into a tumor site will allow for resolution of the tumor. Such formulations may include other agents which complex with ²¹²Pb to facilitate delivery to the tumor site. Such agents include, but are not limited to, liposomes or colloids. In some embodiments, free radioisotope can be administered directly to the tumor site for imaging purposes.

A unique property of liposomes is their natural ability to target tumors due to the enhanced permeability and retention effect in endothelial cells lining the blood vessels of tumors. Liposomes of certain sizes, typically less than 400 nm, can rapidly enter tumor sites from the blood, but are kept in the bloodstream by the endothelial wall in healthy tissue vasculature. Thus, encapsulation of highly purified ²¹²Pb in liposomes allows for delivery of the radioisotope directly to a tumor site, which can then be used for imaging and/or treating the tumor.

In certain embodiments, the ²¹²Pb radioisotope is suspended in solution phase to form a colloid. For example, in particular embodiments, a highly purified ²¹²Pb radioisotope is suspended in a continuous phase to form a colloid. In other particular embodiments, highly purified ²¹²Pb radioisotope is synthesized in a colloid and need not be subsequently suspended to form a colloid. In some embodiments, the colloid is a sulfur colloid as described in Rotmensch (1989)

Gynecologic Oncology 32, pp. 236-239.

Diagnostic methods

The present invention includes an in vivo diagnostic procedure that includes introducing the ²¹²Pb radioisotope conjugated antibody of the invention into the body of a mammal, and allowing sufficient time for the conjugated antibody to localize before imaging. The present invention also includes in vitro analytical procedures employing the conjugated antibody of the invention.

The invention encompasses use of any of the radioisotope compositions described herein for imaging a distribution of an antigen associated with a tumor, tissue or pathogen. In some embodiments of the invention, antibodies, or fragments thereof, can be used as diagnostic or detectable agents. In a certain embodiment, the antibody, or fragments thereof,

immunospecifically bind to a tissue antigen selected from HER2, HER1, CD45, CD25, CD20, CD22, CD 19, CD33, VEGF, MET, CEA, BRCA1, BRCA2 or IGFR-1, wherein said antibody is conjugated to a radioisotope via a chelator linkage. In some embodiments, the presence of binding to a tissue antigen indicates the presence of a malignant cancer cell. Antibodies of the invention can be useful for monitoring or prognosing the development or progression of a cancer as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Additionally, such antibodies can be useful for monitoring or prognosing the development or progression of cancerous conditions. Types of malignant cancer cells which can be detected using the methods of the invention include any malignant cancer cell which expresses one or more of the

aforementioned malignant cancer cell antigens.

Types of malignant cancer cells which can be detected using the compositions and methods of the invention include, but are not limited to malignant cancer cells which express of or more of the following antigens: breast (HER2, VEGF & BRCA), ovarian (HER2), stomach

(HER2), endometrial (HER2), lung (HER1), pancreatic (HER1), B and T cell lymphoma (CD45), T cell lymphoma (CD25), B cell lymphoma (CD20), B cell leukemia (CD22), B cell lymphoma and leukemia (CD19), acute myeloid leukemia (CD33), chronic myelogenous leukemia (CD33), pancreatic (MET), colorectal (CEA) and lung (IGFR-1). In another embodiment, following initial administration of the conjugated antibody of the invention, the cancer cells can be imaged, and the relative amount of malignant cancer cells can be determined by any available means. The invention includes diagnostic methods to detect cancer and/or assess the effect of therapeutic agents on cancer cells in an organ or body area of a mammal, including a human. Subsequent to administration of the therapeutic agent, an additional amount of detectable monoclonal antibody can be administered to determine the relative amount of cancer cells remaining following treatment. Comparison of the before- and after-treatment images can be used as a means to assess the efficacy of the treatment wherein a decrease in the number of cancer cells imaged following treatment is indicative of an efficacious treatment regimen.

In another embodiment, following initial administration of the conjugated antibody of the invention, pathogens such as viruses or bacteria can be imaged in an infected subject, and the relative amount of pathogens present can be determined by any available means. The invention includes diagnostic methods to detect pathogenic infection and/or assess the effect of therapeutic agents on the infection in an organ or body area of a mammal, including a human. Subsequent to administration of the therapeutic agent, an additional amount of detectable monoclonal antibody can be administered to determine the relative amount of pathogen remaining following treatment. Comparison of the before- and after-treatment images can be used as a means to assess the efficacy of the treatment wherein a decrease in the number of pathogens imaged following treatment is indicative of an efficacious treatment regimen.

In some embodiments, the pathogens that can be detected include a virus or bacteria. Exemplary pathogenic antigens can be found in Ansari (2010) Nucl. Acids Res. 38, D847-853. Exemplary viruses that can be detected according to the methods of the invention include human immunodeficiency virus (HIV), influenza virus, vaccinia virus, human papilloma virus, hepatitis virus (A, B or C) and human parvovirus. Exemplary bacteria that can be detected according to the methods of the invention include Mycobacterium, Plasmodium, Anaplasma, Brucella, Candida, Helicobacter, Streptococcus, Staphylococcus, Haemophilus, Vibrio, Clostridium, Yersinia, Coccidioides, Neisseria, Borrelia, Chlamydia, Shigella, Bacillus, Legionella, Listeria,

Pseudomonas, Boophilus, Treponema, Klebsiella, Salmonella and Echinococcus.

As used herein, the term "detectable amount" refers to the amount of labeled conjugated antibody that binds to a malignant cancer cell antigen when administered to a patient that is sufficient to enable detection of binding of the labeled monoclonal antibody to one or more malignant cancer cells in a tumor. As used herein, the term "imaging effective amount" refers to the amount of the labeled antibody administered to a patient that is sufficient to enable imaging of binding of the antibody to one or more malignant cancer cells in a tumor. In a specific embodiment, the term "imaging effective amount" refers to the amount of labeled antibody administered to a patent that is sufficient to enable imaging of micrometastases, including those of metastatic cancer. The methods of the invention comprise conjugated antibodies which, in conjunction with non-invasive imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), are used to identify and quantify abnormal cells in vivo including malignant cells in tumors. The term "/^'« vivo imaging" refers to any method that permits the detection of ²¹²Pb as described above. In some embodiments, detection of the decay products

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of Pb is also possible Bi and Tl using the methods described herein. For imaging, the radiation emitted from the tumor or area being examined is measured and expressed either as total binding, or as a ratio in which total binding in one tissue is normalized to (i.e. , divided by) the total binding in another tissue or the entire body of the same subject during the same in vivo imaging procedure. As used herein, the terms "subject" or "patient" refers to a mammal, including a human.

In the present invention, conjugated antibodies are useful for in vivo detection and imaging of tumors. These compounds are to be used in conjunction with non-invasive imaging techniques such as positron emission tomography (PET), and single-photon emission computed tomography (SPECT). In accordance with this invention, the conjugated antibody may be labeled (complexed) with ²¹²Pb using techniques described below or known to those skilled in the art. In some embodiments, the inventor encompasses in vivo imaging simultaneous to or followed by treatment with the same conjugated antibody. Specific embodiments include in vivo imaging and treatment with a ²¹²Pb-TCMC-Trastuzumab. Generally, the dosage of the isotopically-labeled monoclonal antibody will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, concomitant therapies and other variables, to be adjusted by the skilled artisan. Dosage can vary from 7.4 MBq/m² (0.2 mCi/m²) to 370 MBq/m² (10.0 mCi/m²) of body surface. Administration to the patient may be local or systemic and accomplished intravenous, intra-arterial, intra-thecal (via the spinal fluid), intra-cranial or the like. Administration may also be intra-dermal or intra-cavitary, depending upon the body site under examination.

After a sufficient time has elapsed for the labeled monoclonal antibody to bind with the abnormal cells, for example thirty minutes to twenty-four hours, the area of the subject under investigation is examined by routine imaging techniques such as SPECT, planar scintillation imaging, PET, and emerging imaging techniques. The exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan. For breast tumor imaging, preferably, the amount (total or specific binding) of the bound isotopically-labeled monoclonal antibody is measured and compared (as a ratio) with the amount of isotopically-labeled monoclonal antibody bound to the tumor following chemotherapeutic treatment.

In another embodiment, the conjugated antibodies of the invention can be used for diagnosis and prognosis by using tissues and fluids distal to the primary tumor site (as well as methods using tissues and fluids of the primary tumor and/or tissue and fluids surrounding the tumor). Antibodies of the invention can be used to assay malignant cancer cell antigen levels in a biological sample using classical immunohistological methods as known to those of skill in the art (e.g. , see Jalkanen et al. (1985) J. Cell. Biol. 101, pp. 976-985; and Jalkanen et al. (1987) J. Cell. Biol. 105, pp. 3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).

In still further embodiments, the present invention provides diagnostic kits, including both immunodetection and imaging kits, for use with the immunodetection and imaging methods described above.

Method of Making the Antibody-Radioisotope Conjugates

The modification of antibodies for the addition of a chelator, in one embodiment, may be accomplished by formation of a covalent linkage with an amino acid residue of the protein and a functional group of the bifunctional chelator which is capable of binding proteins. The chelator could also be attached to the antibody via a linker molecule (such as biotin for avidin conjugates molecules or a hapten for bispecific mAbs). Suitable chelators include, but are not limited to, TCMC and DOTA.

Any suitable process that results in the formation of the conjugates of this invention is within the scope of this invention, but incorporation of radionucleotide will exceed 90% given the high purity of the starting radioisotope (e.g. , ²¹²Pb) material. However, one important aspect for an efficient reaction is the antibody to conjugate ratio. Once the conjugate and the antibody are purified and ready for conjugating both the antibody and the conjugate are added to a reaction mixture. The conjugate to antibody ratio should be approximately 7: 1, 10: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, 150: 1, 200: 1, 250: 1, respectively. The pH ofthe reaction mixture should be about 10 to about 5, preferably about 9 to about 8. More preferably the pH should be about 8.5. The conjugation reaction should be carried out at a temperature range from about 20°C to about 37°C. Up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chelates could be bound to the antibody following the described protocol. In one embodiment, the chelator to antibody ratio is about 5 : 1 or greater. In another embodiment, the chelator to antibody ratio is about 7: 1, or greater, with little loss, if any, of immunoreactivity.

Binding of a therapeutic radioactive isotope to the bifunctional chelator (complexing) may be accomplished either with the bifunctional chelator that is not complexed to an antibody or, alternatively, a composition consisting of a chelator conjugated to an antibody. The complexing reaction should be accomplished at a pH of about 7 to about 4, preferably about 6 to about 5. More preferably the pH should be about 5.5. The complexing reaction may be accomplished in an acetate buffer (e.g., ammonium acetate, sodium acetate, potassium acetate). Preferably the molar concentration should be about 100 mM. The complexing reaction should be carried out at a temperature range from about 20°C to about 37°C. The percent of radionucleotide complexed with chelator should be between about 70% and about 100%, preferably, above 90% and even above 95%.

Pharmaceutical Compositions

The conjugated antibodies of the invention may be administered in vivo in any pharmaceutically acceptable carrier. A physiologic normal saline solution can be used, and may optionally include an appropriate amount of carrier protein, such as human serum albumin (for antibody stabilization).

The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g. , impure or non-sterile compositions) and pharmaceutical compositions (i.e. , compositions that are suitable for administration to a human) that can be used in the preparation of unit dosage forms. Such compositions include an amount of agent disclosed herein or a combination of those agents effective for imaging and a

pharmaceutically acceptable carrier. Preferably, compositions of the invention include an effective amount of one or more antibodies of the invention and a pharmaceutically acceptable carrier.

In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water, saline, or buffers containing any combination of the following components: sugars, amino acids, detergents, antioxidants, metal chelators, alcohol and/or inorganic salts. The compositions of the invention can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

Various delivery systems are known and can be used to administer the agents of the invention useful for imaging malignant cancer cells, e.g. , encapsulation in liposomes, microparticles or microcapsules. Methods of administering agents of the invention include, but are not limited to, parenteral administration (e.g. , intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous). In a specific embodiment, agents of the invention are

administered intravenously. The agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In a specific embodiment, it may be desirable to administer the agents of the invention locally directly to the tumor site; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples describe embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES Example 1 : Production of Purified ²¹²Pb-TCMC-Trastuzumab

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Pb is produced and purified from Th which is obtained as set forth in

WO20081 13792. Purified ²¹²Pb is then used as follows to produce ²¹²Pb-TCMC-Trastuzumab. The glass vial containing the ²¹²Pb generator eluate is placed on a stir/heat plate set at 100°C under a 250 watt heating lamp shining directly on the borosilicate glass vial. Allow the generator eluate in the borosilicate glass to evaporate completely with gentle stirring. After eluate evaporation, the glass vial is placed in a lead pig and allowed to cool down for approximately one minute. Add 1.5 ml of 8N HN0₃ to the borosilicate glass vial containing the ²¹²Pb. Place the borosilicate glass vial on the stir plate under the 250 watt heating lamp, allowing the contents of the borosilicate glass vial to gently evaporate with stirring on the hot plate. This step is repeated two more times followed by cooling in a lead pig for approximately five minutes.

Add 300 μΐ of 0.1 N HN0₃ to the glass vial containing the ²¹²Pb, then vortex to dissolve

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the Pb. Add 50 μΐ of chelexed 220 mg/ml ascorbic acid solution to the Pb solution in the glass vial. Add 30 μΐ of chelexed 5M NH₄OAc solution to the glass vial, followed by vortexing for three seconds. Verify that the pH of the mixture is 5 to 5.5. If necessary, adjust the pH with 0.1 N HN0₃ or 5M NH₄OAc.

Add 1 mg of 5.25 mg/ml cGMP TCMC-Trastuzumab per 37 MBq (1 mCi) of ²¹²Pb to the glass vial. Vortex for three seconds and move the reaction mixture to the 37°C incubator for one hour. Stop the reaction by adding 4 μΐ of 0.1M EDTA to the reaction vial. Vortex for three seconds. Purify ²¹²Pb-TCMC-Trastuzumab using a PD-10 column with phosphate buffered saline. Add 6.8 μΐ. of 25% Human Serum Albumin (HSA) and 5.7 μΐ. of 0.1M EDTA.

QC samples are removed and tests conducted by ITLC, HPLC, HER2 binding and gel electrophoresis. The final solution is sterile filtered into a sterile vial. Samples are submitted for endotoxin and sterility testing. The radioactivity content of the purified drug is evaluated using an AREVA Canberra HpGe detector.

The TCMC-Trastuzumab starting material enables 90+% yields based on ²¹²Pb content (decay-corrected) using the radiosynthetic procedure outlined above. QC of the ²¹²Pb-TCMC- Trastuzumab can be achieved relatively quickly, and the material is stable over the time period required prior to injection.

Example 2: Imaging Experiments

Planar imaging feasibility studies were conducted with a gamma camera equipped with a medium-energy collimator (Picker Axis Variable Technology camera) set at 238 keV with a ± 20% window. Gamma camera imaging detected primarily the 238.6 keV gamma ray associated

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with ^zlzPb, and not the 727.3 keV gamma ray associated with decay of Bi. Planar gamma camera images were analyzed using ImageJ (version 1.43).

A first phantom was built by assembling a 30 mL vial as the heart containing 2.8 MBq (75 μ^) of ²¹²Pb, a 250 mL saline bag with 3.2 MBq (87 μθ) as the peritoneal cavity, two 15 mL vials with 0.7 and 0.8 MBq (20 and 23 μθ) for the kidneys (Figure 1) and imaged to confirm that the discernable phantom organs could be detected (Figure 2). Once confirmed, a new phantom study was conducted to match the size of the peritoneal phantom organ with that of the NHPs used in the study. The average weight of the peritoneal organs for the NHPs used in the studies was 385 grams. It was determined that a 1000 ml plastic i.v. saline bag could be shaped to a peritoneal phantom that would approximate the peritoneal space of the NHPs (Figure 3, bottom right). The phantom volume was 400 ml of saline to match the weight. It was reasoned that while the ²¹²Pb-TCMC-Trastuzumab was not uniformly distributed in the peritoneal organs, it was distributed throughout the cavity coating the organs. Therefore, for the purpose of planar imaging calibration studies, the peritoneal phantom with 400 ml of water with a dose of ²¹²Pb (212 μθ) mixed in and imaged in the same position for the NHPs.

Phantom images are presented in Figure 3 and NHP images in Figure 5. All images were collected with a gamma camera equipped with a parallel-hole, high-energy collimator (Picker Axis SPECT camera) until 500,000 counts were detected, and for that reason, they appear similar over the 48-hour experiment.

Gamma Camera Image Data of syringes. Representative data of the images obtained from nine separate experiments are presented in Figures 4. The same regions of interest used for the phantom analyses were applied to the images from the NHPs and clearly show that the ROIs were appropriate for the peritoneal regions for all nine animals. From the data, it is apparent that the change in detected radioactivity (IP and syringe ROIs) over time followed first-order kinetics (i.e. radioactive decay). In fact, using mean NHP IP ROI data for the 8, 24, and 48 hour time points, the half-life of ²¹²Pb was determined to be 10.53 hours, consistent with the accepted value of 10.64 hours.

Figure 6 shows (top) planar images of three C57BL/6 female mice imaged simultaneously in planar mode on the μSPECT/CT camera with medium energy collimators at to, h, and t_8h post IP injection of ²¹²Pb-TCMC-Trastuzumab; (bottom) single C57BL/6 female mouse imaged on the μSPECT/CT camera at to, h, and t_8h post IP injection of ²¹²Pb-TCMC-Trastuzumab.

Despite the plethora of ionizing radiation emanations from the ²¹²Pb and its daughters; a and β particles and their associated bremsstrahlung coupled with a number of various gamma and x rays reasonable planar and μSPECT images were able to be obtained. Through the use of medium energy collimators and a ± 20% window around the 238.6 keV ²¹²Pb gamma ray peak discernable phantom organs were observed. After correlation with the phantom imaging study, imaging of the NHPs was used to assess that the average radioactivity retained in the NHP's IP region was 95.54% (SD=4.96), using corrected ROI analyses of the 4, 8, 24, and 48 hour NHP imaging data.

Although the present invention has been described in detail, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.

Claims

1. A pharmaceutical composition comprising of an amount of ²¹²Pb effective for imaging.

2. The pharmaceutical composition of claim 1, wherein the ²¹²Pb is linked to a targeting molecule that specifically binds to a malignant cancer cell, cell surface marker or pathogen.

3. The pharmaceutical composition of claim 2, wherein said targeting molecule is a polypeptide.

4. The pharmaceutical composition of claim 3, wherein said polypeptide is an antibody.

5. The pharmaceutical composition of claim 4, wherein said antibody which

immunospecifically binds to an antigen selected from the group consisting of HER2, HER1, CD45 , CD25 , CD20, CD22, CD 19, CD33 , VEGF, MET, CEA, BRCA 1 , BRCA2 and IGFR.

6. The pharmaceutical composition of claim 1, wherein the composition is substantially free of ²²⁴Ra.

7. The pharmaceutical composition of claim 6, wherein amount of ²²⁴Ra present in the composition is less than 0.5% of the total ²¹²Pb.

8. The pharmaceutical composition of claim 6, wherein 0.5% represents less than one

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Ra molecule per 200 molecules of Pb.

9. The pharmaceutical composition of claim 6, wherein amount of ²²⁴Ra present in the composition is less than 0.25% of the total ²¹²Pb.

10. The pharmaceutical composition of claim 9, wherein 0.25% represents less than one ²²⁴Ra molecule per 400 molecules of ²¹²Pb.

11. The pharmaceutical composition of any of claims 3 to 10, wherein said polypeptide is conjugated to said ²¹²Pb via a chelator.

12. The pharmaceutical composition of claim 11, wherein the chelator is selected from the group consisting of TCMC and DOTA.

13. The pharmaceutical composition of claim 2 wherein said targeting molecule is a liposome.

14. The pharmaceutical composition of claim 2, wherein said effective amount is an amount effective to image a malignant cancer cell or pathogen.

15. The pharmaceutical composition of claim 14, wherein the malignant cancer cell is a metastatic cancer cell.

16. The pharmaceutical composition of claim 15, wherein the metatstatic cancer cell is a micrometastatic cancer cell.

17. The pharmaceutical composition of claim 14, wherein the malignant cancer cells are present in a solid tumor.

18. The pharmaceutical composition of claim 14, wherein the pathogen is a virus or bacterium.

19. A method for imaging a malignant cancer cell or pathogen in a mammal, comprising administering the pharmaceutical composition of any of claims 1 to 16 in an amount sufficient to resolve gamma irradiation emitted by the ²¹²Pb and/or its decay products.

20. The method of claim 19, wherein said malignant cancer cell is metastatic.

21. The method of claim 19, wherein said malignant cancer cell is present in micrometastases.

22. The method of claim 19, wherein said malignant cancer overexpresses a cell surface marker selected from the group consisting of HER2, HERl, CD45, CD25, CD20, CD22, CD 19, CD33, VEGF, MET, CEA, BRCA1, BRCA2 and IGFR.

23. The method of claim 19, wherein the pathogen is a virus or bacterium.

24. The method of claim 19, wherein the mammal is a human.

25. The method of claim 19, wherein the decay product is selected from the group consisting of ²¹²Bi and ²⁰⁸T1.