4239-111519-02 RADIOLABELED GLYPICAN-3 (GPC3)-SPECIFIC SINGLE DOMAIN ANTIBODY AND USE THEREOF AS AN IMAGING AGENT FOR DETECTION OF GPC3-POSITIVE TUMORS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63/570,344, filed March 27, 2024, which is herein incorporated by reference in its entirety. FIELD This disclosure concerns imaging agents that include a monoclonal antibody specific for tumor antigen glypican-3 (GPC3), which antibody is site-specifically bound to a chelator and the radionuclide 18F. Use of the imaging agent to detect GPC3-positive cancers, such as by positron emission tomography (PET) imaging, is also described. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT This invention was made with government support under project number Z01011800 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION OF ELECTRONIC SEQUENCE LISTING The electronic sequence listing, submitted herewith as an XML file named 4239-111519- 02.xml (13,461 bytes), created on March 21, 2025, is herein incorporated by reference in its entirety. BACKGROUND Glypican-3 (GPC3) is a heparan sulfate proteoglycan expressed by several different types of tumors, including hepatocellular carcinoma (HCC). Primary liver cancer, of which 80% are HCC, is the third leading cause of cancer-related deaths worldwide and has an 18% 5-year survival rate. In view of this, GPC3 is an attractive therapeutic target, as well as a tumor-selective marker for radiopharmaceutical imaging (Fayn et al., J Nucl Med 64(7):1017-1023, 2023). Positron emission tomography (PET) is a non-invasive imaging technique that has become one of the most widely used methods in diagnostic medicine and drug development, with high sensitivity (fmoles), high resolution (4-10 mm) and tissue accumulation that can be quantitated. The valuable in vivo functional information about biological processes in living subjects provided by PET imaging also provides a unique translational medical advantage in that the same tool can be used both preclinically and clinically. PET relies on the design and synthesis of molecules labeled with a positron-emitting radioisotopes, such as 18F, 64Cu, 11C, 150, 13N, 66Ga, 68Ga, 76Br, 89Zr, 94mTc, 86Y or 124I. In vivo, these radiotracers emit positrons from the nucleus of the isotope with different energies depending on the
4239-111519-02 isotope used. The energy of the ejected positron controls the average distance that it travels before it collides with an electron resulting in the emission of two gamma rays in opposite directions. The gamma rays produced by this positron annihilation event are detected by the PET imaging scanner to produce planar and tomographic images that reveal distribution of the radiotracer as a function of time. Accordingly, isotopes that are pure positron emitters with low ejection energy isotopes are typically used for PET imaging to minimize the distance traveled by the positron before annihilation and dosimetry problems caused by other emissions such as gamma rays, alpha particles, or beta particles. In addition, the half-life of the isotope used in PET imaging must be long enough to allow synthesis and analysis of the radiotracer molecule, injection into the patient, in vivo localization, clearance from non-target tissues and the production of a clear image.18F is commonly used as a PET emitting isotope because of its low positron emission energy, lack of side emissions and suitable half- life. SUMMARY Glypican-3 (GPC3) is a validated histochemical marker for hepatocellular carcinoma (HCC) and other types of cancer. The present disclosure describes a human single-domain monoclonal antibody (“ssHN3”) that specifically binds GPC3 with nanomolar affinity. The GPC3-specific single-domain antibody was functionalized with a chelating group and radiolabeled with a positron- emitting radionuclide. The radiolabeled GPC3-specific single-domain antibody exhibited high GPC3- positive tumor binding, but low level binding to most other organs. The tumor to liver ratio was exceptionally high, an important feature for enabling visualization of liver tumors, such as by positron-emission tomography (PET). The radiolabeled GPC3-specific single-domain antibody disclosed herein can be used, for example, in point-of-care diagnostic applications and as an imaging agent for the detection of GPC3-positive tumors. Provided herein is a composition that includes a single-domain monoclonal antibody that specifically binds GPC3; a chelator site-specifically conjugated to the C-terminus of the single- domain monoclonal antibody; and a radiolabel that includes Al[18F]F in complex with the chelator. In some aspects, the monoclonal antibody includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of antibody ssHN3 (set forth herein as SEQ ID NO: 1). In some examples, the chelator includes restrained complexing agent (RESCA), 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), diethylenetriamine pentaacetate (DTPA), or mercaptoacetyltriglycine (MAG3). In specific non-limiting examples, the composition includes the single-domain monoclonal antibody conjugated to LPETGGGK((±)-Al[18F]F-RESCA). Also provided herein are kits that include a single-domain monoclonal antibody that specifically binds GPC3, wherein the single-domain monoclonal antibody includes a sortase
4239-111519-02 recognition sequence at the C-terminus; and a chelator conjugated to a glycine-containing amino acid sequence. In some aspects, the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of single-domain antibody ssHN3 (set forth as SEQ ID NO: 1). In some aspects, the sortase recognition sequence includes LPXTG (SEQ ID NO: 11) and the glycine-containing amino acid sequence includes GGGK (SEQ ID NO: 13). Further provided herein are kits that include a single-domain monoclonal antibody that specifically binds GPC3; and a chelator conjugated to the C-terminus of the single-domain monoclonal antibody via a peptide sequence that includes or consists of LPETGGGK (SEQ ID NO: 10). In some aspects, the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of single-domain antibody ssHN3 (set forth as SEQ ID NO: 1). In some aspects of the kits disclosed herein, the chelator includes RESCA and/or the sortase enzyme is sortase A. In other aspects of the kits, the chelator is DOTA, NOTA, DTPA, or MAG3. In some examples, the kits further include Al[18F]F. Also provided are methods of detecting a GPC3-positive tumor in a subject. In some aspects, the method includes administering to the subject a diagnostically effective amount of a composition disclosed herein, and performing positron emission tomography (PET) imaging on the subject. Further provided are methods of producing a monoclonal antibody that is site-specifically modified with a radiolabel at the C-terminus of the antibody. In some aspects, the method includes (a) providing a chelator comprising restrained complexing agent (RESCA) conjugated to GGGK (SEQ ID NO: 13) (GGGK-RESCA); (b) providing a single-domain monoclonal antibody conjugated at the C-terminus to a sortase recognition sequence including or consisting of the amino acid sequence LPXTG (SEQ ID NO: 11) (monoclonal antibody-LPXTG); (c) contacting the products of (a) and (b) with a sortase enzyme to generate the product monoclonal antibody-LPXTGGGK- RESCA; and (d) labeling the product of (c) with Al[18F]F to produce monoclonal antibody- LPXTGGGK-Al[18F]F-RESCA. In other aspects, the method includes (a) providing a chelator comprising restrained complexing agent (RESCA) conjugated to GGGK (SEQ ID NO: 13) (GGGK- RESCA); (b) labeling GGGK-RESCA with Al[18F]F to produce GGGK-Al[18F]F-RESCA; (c) providing a single-domain monoclonal antibody conjugated at the C-terminus to a sortase recognition sequence including or consisting of the amino acid sequence LPXTG (SEQ ID NO: 11) (monoclonal antibody-LPXTG); and (d) contacting the products of (b) and (c) with a sortase enzyme to generate the product monoclonal antibody-LPXTGGGK-Al[18F]F-RESCA. The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.
4239-111519-02 BRIEF DESCRIPTION OF THE DRAWINGS FIG.1: Schematic showing the synthesis and structure of ssHN3-Al[18F]F-RESCA. FIG.2: A representative HPLC profile of ssHN3-Al[18F]F-RESCA. Dashed line, ultraviolet (UV) detector; solid line, radio detector. FIGS.3A-3E: Al18F-HN3 immunoPET agent specifically binds to GPC3+ tumors in heterotopic and orthotopic models of liver cancer. (FIG.3A) PET/CT images of mice bearing GPC3+ and (FIG.3B) GPC3- subcutaneous tumors (circled). (FIG.3C) Biodistribution shows GPC3 specificity with statistically significantly higher uptake in GPC3+ (HepG2) tumors compared to GPC3- tumors. (FIG.3D) PET/CT images of Al18F-HN3 in animals bearing orthotopic HepG2 tumors showing high uptake in tumors (T), as well as signal in kidney and bladder (clearance organs). (FIG. 3E) Biodistribution studies show persistence of tumor signal and excellent tumor:liver ratio. FIG.4: Imaging of GPC3-positive tumors following administration of ssHN3-Al[18F]F- RESCA. Nu/nu athymic mice were implanted with HepG2 orthotopic tumors, injected with 50 µCi of ssHN3-Al[18F]F-RESCA (~15 µg) and imaged 1, 2, and 3 hours post injection. Shown is a representative image at 1 hour post injection. FIG.5: Terminal biodistribution of ssHN3-Al[18F]F-RESCA. Nu/nu athymic mice were implanted with HepG2 orthotopic tumors, injected with ~15 µg of ssHN3-Al[18F]F-RESCA and imaged 1, 2, and 3 hours post injection. Shown is terminal biodistribution after the 3-hour imaging time point. FIGS.6A-6C: Al18F-HN3 immunoPET can assess treatment response and identify residual disease in orthotopic models of liver cancer. Mice bearing orthotopic HepG2 liver cancer tumors were imaged before and after (FIG.6A) total or (FIG.6B) partial thermal ablation of tumors. Results clearly show complete responses in total ablation and residual disease in the partial ablation. (FIG. 6C) Immunohistochemical staining shows responses in tumors collected following ablations. FIGS.7A-7D: Al18F-HN3 demonstrates binding corresponding to GPC3 expression. (FIG. 7A) PET/CT of agent shows uptake in xenograft tumors of liver cancer cell lines corresponding to expression of GPC3 where HepG2 >Hep3B > Huh7. (FIG.7B) Biodistribution and (FIGS.7C-7D) immunohistochemical staining confirms in vivo imaging results. SEQUENCES The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing: SEQ ID NO: 1 is the amino acid sequence of single-domain antibody ssHN3. SEQ ID NOs: 2-4 are the CDR1, CDR2 and CDR3 sequences of ssHN3 according to Kabat.
4239-111519-02 SEQ ID NOs: 5-7 are the CDR1, CDR2 and CDR3 sequences of ssHN3 according to IMGT. SEQ ID NO: 8 is an exemplary nucleic acid sequence encoding single-domain antibody ssHN3. SEQ ID NO: 9 is a peptide linker containing a sortase A recognition sequence motif. SEQ ID NO: 10 is a peptide linker containing an exemplary sortase A recognition sequence. SEQ ID NO: 11 is the amino acid sequence of a sortase A recognition sequence motif. SEQ ID NO: 12 is the amino acid sequence of an exemplary sortase A recognition sequence. SEQ ID NO: 13 is a glycine-containing amino acid sequence (GGGK). DETAILED DESCRIPTION I. Introduction There are currently no existing imaging agents for detecting glypican-3 (GPC3)-positive tumors. ssHN3-Al[18F]F-RESCA is a human single-domain antibody-based positron emission tomography (PET) imaging agent specific for GPC3, a tumor antigen expressed by the majority of hepatocellular carcinoma (HCC) tumors and other types of cancer. The present disclosure describes the development of an improved method to label ssHN3 using fluorine-18 (18F), a positron-emitting isotope used clinically that allows for same-day imaging. Testing of ssHN3-Al[18F]F-RESCA demonstrated a tumor to liver ratio of about 9:1 in subcutaneous and 17:1 in orthotopic xenograft models of liver cancer. These exceptionally high ratios allow for excellent resolution and the ability to locate GPC3-positive lesions in the liver. Applications of ssHN3-Al[18F]F-RESCA include diagnosis, treatment selection, treatment response assessment, and surveillance in patients with HCC and other GPC3-positive malignancies. II. Abbreviations DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DTPA diethylenetriamine pentaacetate GPC3 glypican-3 HCC hepatocellular carcinoma HPLC high-performance liquid chromatography PET positron emission tomography MAG3 mercaptoacetyltriglycine MS mass spectrometry NMR nuclear magnetic resonance NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid RESCA restrained complexing agent RFA radiofrequency ablation SE-HPLC size-exclusion high-performance liquid chromatography
4239-111519-02 III. Summary of Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided: Administration: To provide or give a subject an agent, such as a monoclonal antibody or composition disclosed herein, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen (such as GPC3). Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians. Antibody variable regions contain "framework" regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those
4239-111519-02 described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn/aligncdr/abrsa.php). A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains. A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies. A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species. A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Cervical intraepithelial neoplasia: Abnormal cells found on the surface of the cervix. Cervical intraepithelial neoplasia, also known as cervical dysplasia, is usually caused by infection with certain types of human papillomavirus (HPV).
4239-111519-02 Chelator: A chemical compound that binds tightly to metal ions. In some aspects of the present disclosure, the chelator is restrained complexing agent (RESCA). In other aspects, the chelator is DOTA, NOTA, DTPA, or MAG3. Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. In one aspect, a chemotherapeutic agent is an agent of use in treating a GPC3-positive tumor. In one aspect, a chemotherapeutic agent is a radioactive compound. A skilled person can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.17 in Abeloff, Clinical Oncology 2nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). In one example, a chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., therapeutic monoclonal antibody), such as anti-GPC3, anti-PD1 or anti-PDL1 (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti-CTLA-4). Complementarity determining region (CDR): A region of hypervariable amino acid sequence that defines the binding affinity and specificity of an antibody. The light and heavy chains of a mammalian immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. A single-domain antibody contains three CDRs, referred to herein as CDR1, CDR2 and CDR3. Conjugate: In the context of the present disclosure, a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to another molecule, such as an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, toxin, therapeutic agent, detectable label, a radiolabel, protein, nucleic acid, lipid, nanoparticle, photon absorber, carbohydrate or recombinant virus. An antibody conjugate is often referred to as an “immunoconjugate.” When the conjugate includes an antibody linked to a drug (such as a cytotoxic agent), the conjugate is often referred to as an “antibody-drug conjugate” or “ADC.” Other antibody conjugates include, for example, multi-specific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs). In a specific example, a conjugate includes a GPC3 antibody (such as ssHN3), a chelating group (such as RESCA) and a positron- emitting radionuclide (such as 18F). Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody to GPC3. For example, a monoclonal antibody that specifically binds GPC3 can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and
4239-111519-02 specifically bind the GPC3 polypeptide. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody specifically binds GPC3. Non-conservative substitutions are those that reduce an activity or binding to GPC3. Conservative amino acid substitution tables providing functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Diagnostically effective amount: A quantity of a specific substance sufficient to enable diagnosis of a particular disease or disorder. For instance, this can be the amount of a radiolabeled antigen-specific antibody necessary to detect a tumor expressing the antigen, such as the dose of ssHN3-Al[18F]F-RESCA capable of detecting GPC3-positive tumors by PET imaging. In some aspects, the dose of ssHN3-Al[18F]F-RESCA is about 5 mCi to about 10 mCi, such as about 5, about 6, about 7, about 8, about 9 or about 10 mCi. Framework region: Amino acid sequences interposed between CDRs. Framework regions of an immunoglobulin molecule include variable light and variable heavy framework regions. Glypican-3 (GPC3): A member of the glypican family of heparan sulfate (HS) proteoglycans that are attached to the cell surface by a glycosylphosphatidylinositol anchor (Filmus and Selleck, J Clin Invest 108:497-501, 2001). The GPC3 gene codes for a core protein of approximately 70 kD, which can be cleaved by furin to produce an N-terminal 40 kD fragment and a C-terminal 30 kD fragment. Two HS chains are attached on the C-terminal portion of GPC3. GPC3 and other glypican family proteins play a role in cell division and cell growth regulation. GPC3 is highly expressed in HCC and some other human cancers including melanoma, squamous cell carcinomas of the lung, neuroendocrine prostate cancer, and clear cell carcinomas of the ovary (Ho and Kim, Eur J Cancer 47(3):333-338, 2011), but is not expressed in normal tissues. GPC3 is also known as SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1 and GTR2-2. There are four known isoforms of human GPC3 (isoforms 1-4). Nucleic acid and amino acid sequences of the four isoforms of GPC3 are known, including GenBank Accession numbers: NM_001164617 and NP_001158089 (isoform 1); NM_004484 and NP_004475 (isoform 2); NM_001164618 and NP_001158090 (isoform 3); and NM_001164619 and NP_001158091 (isoform 4).
4239-111519-02 GPC3-positive cancer: A cancer that expresses or overexpresses GPC3. Examples of GPC3-positive cancers include, but are not limited to, HCC, melanoma, ovarian clear-cell carcinomas, yolk sac tumors (YST), neuroblastoma, hepatoblastoma, Wilms' tumors, squamous cell carcinoma of the lung, liposarcoma, cervical intraepithelial neoplasia, adenoma of the adrenal gland, schwannoma and embryonal tumors (Ho and Kim, Eur J Cancer 47(3):333-338, 2011; Baumhoer et al., Am J Clin Pathol 129(6):899-906, 2008; Saikali and Sinnett, Int J Cancer 89(5):418-422, 2000). Hepatoblastoma: A type of liver tumor that occurs in infants and children. Hepatocellular carcinoma (HCC): A primary malignancy of the liver typically occurring in patients with inflammatory livers resulting from viral hepatitis, liver toxins or hepatic cirrhosis (often caused by alcoholism). HCC is also called malignant hepatoma. Liposarcoma: A rare type of cancer that begins in fat cells. Liposarcoma, a type of soft tissue sarcoma, usually forms in the layer of fat just under the skin or in the deep soft tissues of the legs (especially in the thigh or back of the knee) or in the abdomen. It may also form in other parts of the body. Most liposarcomas are painless and tend to grow slowly, but some may grow quickly and spread to nearby tissue or to other parts of the body. Liposarcomas usually occur in adults, and are rare in children and adolescents. Melanoma: A form of cancer that originates in melanocytes (cells that make the pigment melanin). Melanocytes are found primarily in the skin, but are also present in the bowel and eye. Melanoma in the skin includes superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo maligna (melanoma). Any of the above types may produce melanin or can be amelanotic. Similarly, any subtype may show desmoplasia (dense fibrous reaction with neurotropism) which is a marker of aggressive behavior and a tendency to local recurrence. Other melanomas include clear cell sarcoma, mucosal melanoma and uveal melanoma. Neuroblastoma: A solid tumor arising from embryonic neural crest cells. Neuroblastoma commonly arises in and around the adrenal glands, but can occur anywhere that sympathetic neural tissue is found, such as in the abdomen, chest, neck or nerve tissue near the spine. Neuroblastoma typically occurs in children younger than 5 years of age. Neuroendocrine prostate cancer: An aggressive subtype of cancer arising from prostate that can arise via transformation of prostate adenocarcinoma. Ovarian cancer: Cancer that forms in tissues of the ovary (one of a pair of female reproductive glands in which the ova, or eggs, are formed). Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells). Ovarian clear cell carcinoma: A distinct histopathologic subtype of epithelial ovarian cancer with an incidence of less than 5% of all ovarian malignancies. When viewed under a microscope, the insides of the cells of this type of tumor appear clear.
4239-111519-02 Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Positron emission tomography (PET): An imaging technique that utilizes radiolabeled molecules to visualize and/or measure metabolic activity or the presence of tumors or other diseases. Measurement of the tissue concentration of a positron emitting radionuclide is based on coincidence detection of the two gamma photons arising from positron annihilation. When a positron is annihilated by an electron, two 511 keV gamma photons are simultaneously produced and travel in approximately opposite directions. Gamma photons produced by an annihilation event can be detected by a pair of oppositely disposed radiation detectors capable of producing a signal in response to the interaction of the gamma photons with a scintillation crystal. Radiolabel: A label (such as a label for proteins, e.g., antibodies) that includes a radionuclide, such as, e.g., 18F, 35S, 11C, 13N, 15O, 19F, 99mTc, 124/125/131I, 3H, 14C, 15N, 64Cu, 67Cu,90Y, 99mTc, 203Pb 212Pb, 223Ra, 225Ac, 227Th. In some aspects herein, the radiolabel comprises or is Al[18F]F. Sequence identity: The similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide or polynucleotide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are known. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math.2:482, 1981; Needleman & Wunsch, J. Mol. Biol.48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res.16:10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio.24:307-31, 1994. Altschul et al., J. Mol. Biol.215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
4239-111519-02 Variants of a polypeptide or nucleic acid sequence are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid or nucleotide sequence of interest. Sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids (or 30-60 nucleotides) and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. As used herein, reference to “at least 90% identity” (or similar language) refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence. Sortase: A type of enzyme found in certain types of bacteria, such as Staphylococcus species (e.g., S. aureus), that recognizes and cleaves short peptide sequences (recognition sequences) at the C- terminal end of a protein. Sortase A, first identified in S. aureus, binds the LPXTG (SEQ ID NO: 11) sortase recognition sequence and cleaves this motif between the threonine (T) and glycine (G) residues, forming a thioester-linked acyl enzyme intermediate with its active site cysteine thiol (Mazmanian et al., Science 285:760-763, 1999; Ton-That et al., Proc Natl Acad Sci USA 96:12424- 12429, 1999; Schneewind and Missiakas, Microbiol Spectr 7(1):10.1128/microbiolspec.PSIB-0004- 2018, 2019). The active-site cysteine of sortase A forms a bond with the carbonyl of the threonine residue of the target protein. This intermediate is resolved by nucleophilic attack by a glycine- containing peptide (e.g., GGGK, SEQ ID NO: 13) (Guimaraes et al., Nat Protoc 8(9):1787-1799, 2013). This mechanism can be harnessed to label biomolecules of interest, such as for labeling of monoclonal antibodies with a radiolabel. Squamous cell carcinoma: A type of cancer that originates in squamous cells, thin, flat cells that form the surface of the skin, eyes, various internal organs, and the lining of hollow organs and ducts of some glands. Squamous cell carcinoma is also referred to as epidermoid carcinoma. One type of squamous cell carcinoma is squamous cell carcinoma of the lung. Squamous cell carcinoma is the most common type of skin cancer. Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. Wilms tumor: A type of malignant tumor of the kidney, which can spread to other organs, including the lungs, liver or nearby lymph nodes. Wilms tumor typically occurs in children younger than five years old.
4239-111519-02 Yolk sac tumor: A rare type of cancer that begins in germ cells. Yolk sac tumors, also known as endodermal sinus tumors, occur most frequently in the ovary or testicle, but may also occur in other parts of the body, such as the chest, abdomen or brain. This type of tumor tends to growth quickly and spread to other parts of the body if not treated. Yolk sac tumors are the most common germ cell tumor in children. IV. Radiolabeled GPC3-Specific Single Domain Monoclonal Antibody GPC3 is a validated histochemical marker for hepatocellular carcinoma (HCC) and other types of cancer that express or overexpress GPC3. The present disclosure describes a human single- domain monoclonal antibody (ssHN3) that specifically binds GPC3 with nanomolar affinity. The GPC3-specific single-domain antibody was functionalized with a chelating group and radiolabeled with a positron-emitting radionuclide. The radiolabeled GPC3-specific single-domain antibody exhibited high GPC3-positive tumor binding, but low level binding to most other organs. The tumor to liver ratio was exceptionally high, an important feature for enabling visualization of liver tumors, such as by positron-emission tomography (PET). The radiolabeled GPC3-specific single-domain antibody disclosed herein can be used, for example, in point-of-care diagnostic applications and as an imaging agent for the detection of GPC3-positive tumors. The amino acid and nucleotide sequences of single-domain monoclonal antibody ssHN3 are provided below. The positions of the complementarity determining region 1 (CDR1), CDR2 and CDR3 residues, according to the Kabat and IMGT numbering schemes, are provided below in Table 1A and Table 1B, respectively. ssHN3 Protein Sequence (SEQ ID NO: 1) QVQLVQSGGGLVQPGGSLRLSCAASYFDFDSYEMSWVRQAPGKGLEWIGSIYHSGSTYYNP SLKSRVTISRDNSKNTLYLQMNTLRAEDTATYYCARVNMDRFDYWGQGTLVTVSSS Table 1A. Locations of the CDRs in the ssHN3 sequence according to Kabat CDR Residues of SEQ ID NO: 1 Sequence SEQ ID NO:
Table 1B. Locations of the CDRs in the ssHN3 sequence according to IMGT CDR Residues of SEQ ID NO: 1 Sequence SEQ ID NO:
4239-111519-02 ssHN3 DNA Sequence (SEQ ID NO: 8) CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACT CTCCTGTGCAGCCTCTTATTTCGATTTCGATTCTTATGAAATGAGCTGGGTCCGCCAGGCT CCAGGGAAGGGCCTAGAGTGGATTGGGAGTATCTATCATAGTGGGAGCACCTACTACAA CCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCT GCAAATGAACACCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAGAGTAAATA TGGACCGATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAAGT Provided herein are compositions that include a single-domain monoclonal antibody that specifically binds GPC3; a chelator conjugated to the C-terminus of the single-domain monoclonal antibody; and a radiolabel that includes Al[18F] in complex with the chelator. In some aspects, the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1. In some examples, the CDR1, CDR2 and CDR3 sequences respectively include SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In other examples, the CDR1, CDR2 and CDR3 sequences respectively include SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some aspects, the amino acid sequence of the single-domain monoclonal antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, and includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1. In some examples, the amino acid sequence of the single-domain monoclonal antibody includes or consists of SEQ ID NO: 1. In some aspects, the chelator includes restrained complexing agent (RESCA). In other aspects, the chelator comprises DOTA, NOTA, DTPA, or MAG3. In some aspects, the chelator is conjugated to the single-domain monoclonal antibody via a peptide sequence that includes or consists of LPXTGGGK (SEQ ID NO: 9) or LPETGGGK (SEQ ID NO: 10). In some examples, the peptide sequence of SEQ ID NO: 9 or SEQ ID NO: 10 results from a sortase reaction between (1) the chelator (such as RESCA) conjugated to the glycine-containing amino acid sequence GGGK (SEQ ID NO: 13) and (2) the monoclonal antibody conjugated to the sortase recognition sequence LPXTG (SEQ ID NO: 11). In some aspects, the composition includes the single-domain monoclonal antibody specific for GPC3 conjugated to LPETGGGK((±)-Al[18F]F-RESCA). In specific examples, the composition includes SEQ ID NO: 1 conjugated to LPETGGGK((±)-Al[18F]F-RESCA). In some examples of the disclosed compositions, the composition further includes a pharmaceutically acceptable carrier, such as water or saline. In specific examples, the pharmaceutically acceptable carrier is suitable for intravenous, intramuscular, subcutaneous, intratumoral, or parenteral administration (e.g., by injection or infusion). Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known. Except insofar as any
4239-111519-02 conventional media or agent is incompatible with the active compound (e.g., the imaging agent), use thereof in the pharmaceutical compositions described herein is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some cases, the composition includes isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and/or gelatin. Sterile injectable solutions can be prepared by incorporating the 18F-labeled single-domain monoclonal antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients, such as those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation can include vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Thus, in some aspects the composition is lyophilized. In some aspects, the composition is frozen. In some aspects, the composition (e.g., HN3-LPXTG-RESCA) is provided in a sterile format that is subsequently radiolabeled with the Al[18F] in a GLP/GMP fashion for human administration. The amount of 18F-labeled single-domain monoclonal antibody that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of 18F-labeled single-domain monoclonal antibody that can be combined with a carrier material to produce a single dosage form will generally be an amount of the composition that produces a detectable signal (e.g., a diagnostically effective amount, as discussed below). V. Kits for Detection of GPC3-Positive Tumors Also provided herein are kits for the detection of GPC3-positive tumors, such as by PET imaging.
4239-111519-02 In some aspects, the kit includes a single-domain monoclonal antibody that specifically binds GPC3, wherein the single-domain monoclonal antibody includes a sortase recognition sequence at the C-terminus of the antibody; and a chelator conjugated to a glycine-containing amino acid sequence. In some examples, the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1. In specific examples, the CDR1, CDR2 and CDR3 sequences respectively include SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In other specific examples, the CDR1, CDR2 and CDR3 sequences respectively include SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some examples, the amino acid sequence of the single-domain monoclonal antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, and includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1. In specific examples, the amino acid sequence of the single-domain monoclonal antibody includes or consists of SEQ ID NO: 1. In some examples, the single-domain monoclonal antibody further includes a His tag at the C- terminus of the sortase recognition sequence, such as a 6-His tag. In some examples, the sortase recognition sequence includes or consists of LPXTG (SEQ ID NO: 11). In particular examples, the sortase recognition sequence includes or consists of LPETG (SEQ ID NO: 12). In some examples, the chelator includes restrained complexing agent (RESCA). In other aspects, the chelator comprises DOTA, NOTA, DTPA, or MAG3. In some examples, the glycine-containing amino acid sequence conjugated to the chelator (such as RESCA) includes or consists of the sequence GGGK (SEQ ID NO: 13). In some examples the kit further includes a sortase enzyme, such as sortase A. In particular examples, the sortase enzyme (such as sortase A) includes a His tag, such as 6-His. In some examples, the kit further includes Al[18F]F, nickel-nitrilotriacetic (NTA) beads or resin, buffer, instructional materials, or any combination thereof. In other aspects, the kit includes a single-domain monoclonal antibody that specifically binds GPC3; and a chelator conjugated to the C-terminus of the single-domain monoclonal antibody via a peptide sequence that includes LPETGGGK (SEQ ID NO: 10). In some examples, the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1. In specific examples, the CDR1, CDR2 and CDR3 sequences respectively include SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In other specific examples, the CDR1, CDR2 and CDR3 sequences respectively include SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some examples, the amino acid sequence of the single-domain monoclonal antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, and includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1. In specific examples, the amino acid sequence of the single-domain monoclonal antibody includes or consists of SEQ ID NO: 1.
4239-111519-02 In some examples, the chelator includes RESCA. In other examples, the chelator includes DOTA, NOTA, DTPA, or MAG3. In some examples, the kit further includes Al[18F]F, NTA beads or resin, buffer, instructional materials, or any combination thereof. VI. Methods for Detection of GPC3-Positive Tumors Also provided herein are methods of detecting a GPC3-positive tumor in a subject. In some aspects, the method includes administering to the subject a diagnostically effective amount of a composition disclosed herein, and performing positron emission tomography (PET) imaging (or another suitable imaging application) on the subject. In some examples, the subject is human. In some aspects, the subject has not been previously diagnosed with a GPC3-positive tumor. In other aspects, the subject has been previously diagnosed as having a GPC3-positive tumor. In some aspects, the methods include selecting a subject having a GPC3-positive tumor. In some aspects of the disclosed methods, the GPC3-positive tumor is a hepatocellular carcinoma (HCC), melanoma, ovarian clear-cell carcinoma, yolk sac tumor (YST), neuroblastoma, neuroendocrine prostate cancer, hepatoblastoma, Wilms' tumor, squamous cell carcinoma of the lung, liposarcoma, cervical intraepithelial neoplasia, adenoma of the adrenal gland, schwannoma or an embryonal tumor. In specific aspects, the GPC3-positive tumor is HCC. The term “administering” in the context of imaging agents refers to the physical introduction of a composition that includes the imaging agent (e.g., ssHN3-Al[18F]F-RESCA) to a subject, using any of the various methods and delivery systems known to a skilled person. Routes of administration for the imaging agents described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The term “parenteral administration” means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intratumoral, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, an imaging agent described herein (e.g., ssHN3-Al[18F]F-RESCA) can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), and/or over one or more extended periods (such as over 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year). The imaging agent (e.g., ssHN3-Al[18F]F-RESCA) is administered in a diagnostically effective amount, which refers to a dose sufficient to allow for a detectable signal by PET imaging or
4239-111519-02 another suitable imaging application. The detectable signal is distinguishable from other background signals that may be generated from the subject. In other words, there is a measurable and significant difference (e.g., a difference sufficient to distinguish among the detectable signal and the background, such as about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, or 40% or more difference between the detectable signal and the background) between the detectable signal and the background. Standards and/or calibration curves can be used to determine the relative intensity of the detectable signal and/or the background. In some examples, the imaging agent (e.g., ssHN3-Al[18F]F-RESCA) is administered a single time. In other examples, the imaging agent is administered multiple times, such as 2, 3, 4, or 5 times to achieve a detectable signal. The amount of imaging agent administered to a subject can depend upon several factors, such as the tumor type, the size of the tumor, the site to be examined, the degree of susceptibility of the subject, the age, gender, physical condition, and weight of the subject, idiosyncratic responses of the subject, and the like. Diagnostically effective amounts of imaging compositions can also vary according to the instrument and methodologies used. A skilled person can determine the diagnostically effective amount of imaging agent to administer to the subject based on the factors provided above and knowledge available in the art. In some aspects, the subject is administered a dose of the imaging agent (e.g., ssHN3- Al[18F]F-RESCA) that is between 0.005 μg/kg of body weight to 50 μg/kg of body weight, between 0.02 μg/kg of body weight to 10 μg/kg, between 0.1 μg/kg of body weight to 10 μg/kg of body weight, between 1 μg/kg of body weight to 10 μg/kg of body weight, between 2 μg/kg of body weight to 6 μg/kg of body weight, or between 4 μg/kg of body weight to 5 μg/kg of body weight, such as per day or per imaging event. In some aspects, the radioactivity of the imaging agent (e.g., ssHN3-Al[18F]F-RESCA) ranges from about 3.7 megabecquerels (MBq) to 3.7 gigabecquerels (GBq), from 18 MBq to 740 MBq, from 100 MBq to 500 MBq, from 100 MBq to 400 MBq, from 100 MBq to 333 MBq, from 100 MBq to 250 MBq, from 150 MBq to 250 MBq, from 200 MBq to 250 MBq or from 200 MBq to 225 MBq. Alternatively, the dosage may be measured by millicuries, for example. In some aspects, the amount of imaging agent (e.g., ssHN3-Al[18F]F-RESCA) administered for imaging studies is 1 to 10 mCi, 3 to 10 mCi, 5 to 10 mCi, 3 to 8 mCi, 4 to 7 mCi or 5 to 6 mCi. In some examples, the amount of imaging agent is about 5 to about 10 mCi, such as about 5, about 6, about 7, about 8, about 9 or about 10 mCi. In some examples, a diagnostically effective amount will be the amount of compound sufficient to produce emissions in the range of from 1 to 10 mCi, 3 to 10 mCi, 3 to 8 mCi, 4 to 7 mCi or 5 to 6 mCi. In certain examples, the imaging agent is administered to a human subject in an amount of 1 to 10 mCi, 3 to 10 mCi, 3 to 8 mCi, 4 to 7 mCi or 5 to 6 mCi. In some aspects, the subject is imaged using PET for an amount of time appropriate for the 18F radionuclide. The 18F-labelled single-domain antibody bound to GPC3-expressing cells is then detected by PET imaging. When using 18F, PET imaging is typically performed within 30 minutes, within 60 minutes, within 90 minutes or within 120 minutes of administration of the imaging agent
4239-111519-02 (e.g., ssHN3-Al[18F]F-RESCA). PET imaging can be used to qualitatively or quantitatively detect a target molecule (e.g., GPC3 expressed on tumor cells). Detection of GPC3-expressing tumor cells can be used to guide treatment decisions, such as by determining appropriate anti-cancer drugs capable of treating the cancer (e.g., immunotherapies targeting GPC3, chemotherapeutic drugs effective for treating liver cancer, radiation therapy, and the like). In some aspects, PET imaging is used to monitor the progression or regression of a GPC3- positive cancer, such as by imaging the subject periodically over time. In some examples, a subject is imaged every week, every 2 weeks, every 3 weeks, every 4 weeks, every 2 months, every 3 months, or every six months. PET imaging can also be used to determine the efficacy of a particular treatment regimen by imaging the subject prior to treatment, at various intervals during treatment and/or following treatment. In some aspects, PET images are obtained from only a portion of the subject, such as a portion that includes the subject’s liver. In other aspects, PET images are obtained from multiple locations of the subject’s body, such as the whole body. Exemplary PET imaging procedures are well-known and have been previously described (see, e.g., U.S. Patent No.11,806,40). In some aspects of the disclosed methods, the method further includes treating the subject with an effective amount of one or more appropriate anti-cancer drugs or treatments if the subject is diagnosed as having a GPC3-positive tumor. In some examples, the subject is administered an immunotherapy targeting GPC3 (e.g., a monoclonal antibody, an antibody-drug conjugate, a radioconjugate, an immunotoxin or chimeric antigen receptor (CAR)-expressing immune cells targeting GPC3). In other examples, the subject is administered a DNA damage repair inhibitor, such as a DNA-dependent protein kinase (DNA-PK) inhibitor, a poly(ADP ribose) polymerase (PARP) inhibitor, an ataxia telangiectasia mutated (ATM) kinase inhibitor, an ataxia telangiectasia and Rad3 related (ATR) kinase inhibitor. Other exemplary anti-cancer agents that can be administered include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Non-limiting examples of alkylating agents that can be administered include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine). Non-limiting examples of antimetabolites that can be administered include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.
4239-111519-02 Non-limiting examples of natural products that can be administered include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase). Non-limiting examples of miscellaneous agents that can be administered include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide). Non-limiting examples of hormones and antagonists that can be administered include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples of immunomodulators that can be administered that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM- CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman- LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech). Exemplary biologics that can be used in combination with the disclosed methods include one or more monoclonal antibodies (mAbs) used to treat cancer, such as mAbs specific for EGFR (e.g., cetuximab), VEGF (e.g., bevacizumab), PD-1 (e.g., nivolumab, JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001, dostarlimab (TSR- 042, WBP-285), INCMGA00012 (MGA012), AMP-224, or AMP-514), CD276 (e.g., atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (envafolimab), BMS-936559, BMS935559, MEDI- 4736, MPDL-3280A, or MEDI-4737), CD25 (e.g., daclizumab or basiliximab), CD20 (e.g., Tositumomab (Bexxar®); Rituximab (Rituxan, Mabthera); Ibritumomab tiuxetan (Zevalin, for example in combination with yttrium-90 or indium-111 therapy); Ofatumumab (Arzerra®), veltuzumab, obinutuzumab, ublituximab, ocaratuzumab), CD22 (e.g., narnatumab, inotuzumab ozogamicin, moxetumomab pasudotox) or CTLA4 (e.g., ipilimumab, tremelimumab). In some examples, the additional therapeutic agent administered is an anti-cancer monoclonal antibody, for
4239-111519-02 example one or more of: 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab , Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomab estafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan, Ofatumumab, Olaratumab, Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab, Zalutumumab, or combinations thereof In a specific example, the disclosed methods are used in combination with a therapeutic PD-1 mAb, such as one or more of nivolumab, JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001, dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, and AMP-514. In some examples, the methods further include surgical treatment, for example surgical resection of the cancer or a portion of it. In some examples, the methods further include administration of radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection. In some aspects of the disclosed methods, treating the subject reduces the size of a GPC3 expressing or overexpressing tumor, reduces the size of a GPC3 metastasis, and/or reduces the number of GPC3 metastases, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even complete eradication of the tumor (e.g., the tumor is no longer detectable), for example as compared to the tumor or metastases prior to treatment. VII. Methods of Producing an Antibody Site-Specifically Modified with a Radiolabel Described herein are two methods that can be used to site-specifically modify an antibody with a radiolabel. The methods discussed below are for modifying the C-terminus of an antibody, but
4239-111519-02 can be modified to allow for addition of a radiolabel at a different location of the antibody by changing the location of the sortase recognition sequence. In one aspect, the method includes (a) providing a chelator that includes restrained complexing agent (RESCA) conjugated to GGGK (SEQ ID NO: 13) (GGGK-RESCA); (b) providing a single-domain monoclonal antibody conjugated at the C-terminus to a sortase recognition sequence having the amino acid sequence LPXTG (SEQ ID NO: 11) (monoclonal antibody-LPXTG); (c) contacting the products of (a) and (b) with a sortase enzyme to generate the product monoclonal antibody-LPXTGGGK-RESCA; and (d) labeling the product of (c) with Al[18F]F to produce monoclonal antibody-LPXTGGGK-Al[18F]F-RESCA. In another aspect, the method includes (a) providing a chelator that includes RESCA conjugated to GGGK (SEQ ID NO: 13) (GGGK-RESCA); (b) labeling GGGK-RESCA with Al[18F]F to produce GGGK-Al[18F]F-RESCA; (c) providing a single-domain monoclonal antibody conjugated at the C-terminus to a sortase recognition sequence having the amino acid sequence LPXTG (SEQ ID NO: 11) (monoclonal antibody-LPXTG); and (d) contacting the products of (b) and (c) with a sortase enzyme to generate the product monoclonal antibody-LPXTGGGK-Al[18F]F-RESCA. In some examples of these methods, the monoclonal antibody-LPXTG conjugate further includes a histidine tag (such as 6-His) and the sortase enzyme includes a histidine tag (such as 6- His). In some examples, the methods further include (e) purifying the monoclonal antibody- LPXTGGGK-Al[18F]F-RESCA product using a nickel-NTA column. In some examples, the sortase enzyme is sortase A. In some examples, the monoclonal antibody specifically binds GPC3. In particular examples, the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1. In specific examples, the CDR1, CDR2 and CDR3 sequences respectively include SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In other specific examples, the CDR1, CDR2 and CDR3 sequences respectively include SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In particular examples, the amino acid sequence of the single-domain monoclonal antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, and includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1. In specific examples, the amino acid sequence of the single-domain monoclonal antibody includes or consists of SEQ ID NO: 1. EXAMPLES The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.
4239-111519-02 Example 1: Synthesis of ssHN3-Al[18F]F-RESCA The example describes the chemical and radiochemical syntheses of ssHN3-Al[18F]F- RESCA. Materials and Methods Boc-GGGK-NH2 and (±)-H3RESCA-TFP were purchased from Conju-Probe, LLC (San Diego, CA, USA) and CheMatech (Dijon, France), respectively, and used as received. Nickel NTA Agarose Bead was obtained from GOLDBIO (St Louis, MO, USA). Penta-mutant sortase A was purchased from GenScript (Piscataway, NJ, USA). All other chemicals and solvents were obtained from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Lyophilized whole human serum was obtained from MP Biomedicals, LLC (Solon, OH, USA) and dissolved in 2 mL saline. This serum was directly used without inactivation for the stability study. Fluorine-18 was obtained from the National Institutes of Health Cyclotron facility (Bethesda, MD, USA). Mass spectrometry (MS) was performed on a 6130 Quadrupole LC/MS, Agilent Technologies instrument equipped with a diode array detector.1H, 13C and 19F NMR spectra were recorded on a Varian spectrometer (400 MHz). Chemical shifts (ppm) are reported relative to the solvent residual peaks. High-performance liquid chromatography (HPLC) for purification and analytical analysis was performed on an Agilent 1200 Series instrument equipped with multi-wavelength detectors along with a flow count radio detector (Eckert & Ziegler, B-FC-3500 diode). Chemical Syntheses - Chelator H-GGGK((±)-H3RESCA)-NH2 A solution (2 mL phosphate buffer, pH 9.2) of (±)-H3RESCA-TFP (250 mg, 0.43 mmol) was added to a solution (2 mL phosphate buffer, pH 9.2) of BocGGGK-NH2 (175 mg, 0.42 mmol). The reaction mixture was stirred at 60°C for 3 hours followed by overnight at room temperature. The mixture was purified by HPLC using a semi-preparative column (condition: Agilent XDB 9.6 x 250 mm, 5 µm, 10-50% acetonitrile with 0.1% TFA in 0.1% TFA in water). The desired fraction was collected and lyophilized to obtain a white powder of intermediate Boc-protected chelator, BocGGGK((±)-H3RESCA)-NH2. To the dichloromethane solution (0.8 mL) of Boc-protected chelator, trifluoroacetic acid was added (0.2 mL) and stirred for 1 hour. The solvent was evaporated under a vacuum. Solid was redissolved in acetonitrile/water and lyophilized to obtain a white powder of H- GGGK((±)-H3RESCA)-NH2 (160 mg, 0.42 mmol, 52%). 1H NMR (400 MHz, D2O) δ 7.55 (d, J = 7.9 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 4.60 (d, J = 12.9 Hz, 1H), 4.30 – 4.17 (m, 3H), 4.03 (s, 2H), 3.99 (s, 1H), 3.94 (s, 2H), 3.89 (s, 2H), 3.59 (s, 2H), 3.52 – 3.42 (m, 1H), 3.38 – 3.22 (m, 2H), 3.17 (t, J = 6.8 Hz, 2H), 3.04 – 2.82 (m, 2H), 2.55 – 2.40 (m, 1H), 2.35 (d, J = 10.5 Hz, 1H), 1.99 (d, J = 9.7 Hz, 1H), 1.89 (d, J = 12.2 Hz, 1H), 1.85 – 1.62 (m, 3H), 1.50 (s, 3H), 1.42 – 1.02 (m, 5H).13C NMR (101 MHz, D2O) δ = 176.83, 174.03, 173.99, 173.80, 171.76, 171.30, 169.55, 167.86, 137.67, 131.37, 130.18, 128.43, 61.50, 59.23, 56.95, 53.52, 50.40,
4239-111519-02 53.33, 47.84, 42.36, 42.26, 42.21, 40.45, 39.18, 30.50, 27.76, 23.73, 23.67, 23.61, 23.43, 22.37. MS (ESI) calculated mass for the parent C33H51N8O11735.37 [M+H], found 735.20. Radiochemical Syntheses - ssHN3-Al[18F]F-RESCA Fluorine-18 containing target water was passed through an anion-exchange cartridge (30-PS- HCO3). Fluorine-18 retained on the (30-PS-HCO3) cartridge was washed with water (3 mL) and eluted with saline (100 µL) in a reaction vial (~200 mCi). To this solution, 0.5 M sodium acetate solution (pH 5.2) of AlCl3 (80 µL, 0.2 mg/mL, 122 nmol) and H-GGGK( (±)-H3RESCA)-NH2 (60 µL, 4 mg/mL, 326 nmol) were added. The reaction mixture was incubated at room temperature for 10 minutes to prepare H3GGGK((±)-Al[18F]F-RESCA)-NH2. An aliquot of this solution (60 µL, ~50 mCi) was used for each radiolabeling reaction. Nanobody (ssHN3) was buffer exchanged into 50 mM tris buffer and concentrated to 5-10 mg/mL. ssHN3 (1 mg in 100-200 µL) was mixed with 30 µL of 1 M Tris buffer, 100 µL of sortase 10X buffer (500 mM Tris, 1500 mM NaCl, 100 mM CaCl2, pH 7.4), and 10 µL of sortase (35 mg/mL). To this mixture, fluorine-18 labeled chelator (60 µL, ~50 mCi) was added and incubated at 0°C for 30 minutes. Nickel NTA Agarose Bead (1.5 mL) was buffer exchanged with 50 mM tris buffer (0.5 mL). Nickel beads (200 µL) were added to the reaction mixture of ssHN3-Al[18F]F-RESCA and incubated for 5 minutes at room temperature. The final labeled nanobody was purified by size exclusion chromatography (column: PD10) using saline as an eluent to obtain ssHN3-Al[18F]F-RESCA in 1 mL saline (see FIG.1 for synthesis schematic and structure of ssHN3-Al[18F]F-RESCA). Shown in FIG.2 is a representative HPLC profile of ssHN3-Al[18F]F-RESCA. SE-HPLC conditions were as follows: eluent, 0.1 M sodium phosphate, 0.1 M sodium sulfate, 0.05% sodium azide, 10% iso-propyl alcohol (pH 6.8), flow rate 0.3 mL/min. In FIG.2, the dotted line is the UV detector, and the solid line is the radio detector. Example 2: Ex-vivo biodistribution in HepG2 subcutaneous and orthotopic xenografts This example describes two studies to evaluate ssHN3-Al[18F]F-RESCA in animal models of HCC. Nu/nu athymic mice (n = 4 at each timepoint) were implanted with HepG2 subcutaneous tumors. When tumors reached an average volume of 150 mm3, mice were injected with 10 µg (37 µCi) of ssHN3-Al[18F]F-RESCA, and organs were collected at 1, 2, and 3 hours post injection (p.i.). Radioactivity of collected tissues was measured with a gamma counter to assess percent of injected activity per mass of tissue (% IA/g). FIGS.3A-3B show PET/CT images of mice bearing GPC3+ (FIG.3A) and GPC3- (FIG.3B) subcutaneous tumors (circled in the figures). Biodistribution studies showed GPC3 specificity with statistically significantly higher uptake in GPC3+ (HepG2) tumors compared to GPC3- tumors (FIG.3C).
4239-111519-02 ssHN3-Al[18F]F-RESCA was also tested in an orthotopic HepG2 tumor model. PET/CT images of Al18F-HN3 in animals bearing orthotopic HepG2 tumors showed high uptake in tumors, as well as signal in kidney and bladder (clearance organs) (FIG.3D). Biodistribution studies show persistence of tumor signal and an excellent tumor:liver ratio (FIG.3E). Example 3: PET imaging of HepG2 orthotopic model in nude athymic mice This example describes a study to evaluate use of ssHN3-Al[18F]F-RESCA for PET imaging in an animal model of HCC. Four nu/nu athymic mice were implanted with HepG2 orthotopic tumors, injected with 50 µCi of ssHN3-Al[18F]F-RESCA (~15 µg) and imaged 1, 2, and 3 hours post injection. A representative image taken 1 hour post-infection is shown in FIG.4. Terminal biodistribution was measured after the 3-hour imaging time point. As shown in FIG.5, ssHN3-Al[18F]F-RESCA was detected primarily in HepG2 tumors and the kidney. Example 4: Functional imaging utilizing ssHN3-Al[18F]F-RESCA This example evaluates the use of ssHN3-Al[18F]F-RESCA following radiofrequency ablation (RFA) treatment. Nu/nu athymic mice were implanted with HepG2 orthotopic tumors, injected with ~10 µg of ssHN3-Al[18F]F-RESCA, and imaged 1 hour post-injection. Representative image are shown in FIG. 6A (top). The following day, tumors were fully ablated using RFA (4 mm probe inserted into the tumor, ablated at 90°C for 90 seconds), a common treatment for patients with HCC tumors. The following week, post-treatment imaging was done to assess treatment response. PET images from the same mice as in FIG.6A (top) showed complete tumor ablation (FIG.6A, bottom). A similar study was conducted using partial tumor ablation. Nu/nu athymic mice were implanted with HepG2 orthotopic tumors, injected with ~10 µg of ssHN3-Al[18F]F-RESCA, and imaged 1 hour post-injection. Representative images are shown in FIG.6B (top). The following day, tumors were partially ablated (a 4 mm probe was inserted into the tumor, ablated at 90°C for 30 seconds) using radiofrequency ablation. The following week, post-treatment imaging was done to assess treatment response. PET images from the same mice as in FIG.6B (top) showed partial tumor ablation (FIG.6B, bottom). Immunohistochemical staining from mice subjected to total or partial ablation shows a decrease in antigen staining for GPC3 and Ki-67 (a marker for dividing cells), with total ablation resulting in the greatest decrease (FIG.6C). Example 5: Al18F-HN3 demonstrates binding corresponding to GPC3 expression This example describes a study to test uptake of Al18F-HN3 in animals bearing HepG2 (GPC3-positive), Hep3B (GPC3-positive) or Huh7 (GPC3-negative) xenograft tumors. PET/CT of
4239-111519-02 Al18F-HN3 showed uptake in xenograft tumors of liver cancer cell lines corresponding to expression levels of GPC3 where HepG2 >Hep3B > Huh7 (FIG.7A). The results of biodistribution studies are shown in FIG.7B. The greatest uptake was observed in the tumor and kidney. The liver:tumor ratio was the highest in mice bearing HepG2 tumors. FIG.7C shows immunohistochemical staining for GPC3 and Ki-67 (a marker for dividing cells), which is quantified in FIG.7D. The immunohistochemistry results confirm the in vivo imaging results showing highest expression of GPC3 in HepG2 tumors. As expected, Ki-67 expression is detected in all tumor types. It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.