Methods of imaging and treating cancers having aberrant expression of EGFR

Abstract

Provided herein are radiolabeled antibody conjugates that are reactive to the 287-302 amino acid loop of domain II of epidermal growth factor receptor (EGFR). Provided herein are also methods of detecting and / or treating cancer using the provided radiolabeled antibody conjugates.

Description

METHODS OF IMAGING AND TREATING CANCERS HAVING ABERRANT EXPRESSION OF EGFR SEQUENCE LISTING

[0001] The present specification makes reference to a Sequence Listing, submitted electronically as an .xml file name “2026-02-27_DFCI 3595W01WO_sequence listing” on March 16, 2026. The .xml file was generated on February 27, 2026, and is 16 KB in size. The entire contents of the Sequence Listing are herein incorporated by reference.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit ofpriority under 35 U.S. C. § 119(e)toU.S. Provisional Application Nos: 63 / 773,052, filed March 17, 2025 and 63 / 820,954, filed on June 10, 2025, each of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under 5R00CA266921 awarded by the National Institute of Health (NIH). The government has certain rights to the invention.FIELD

[0004] Provided herein are radiolabeled antibody conjugates that are reactive to the 287-302 amino acid loop of domain II of epidermal growth factor receptor (EGFR). This loop structure is exposed in cancer cells where EGFR expression is very high (EGFR-hi) or where the EGFR variant III (EGFRvIII) is expressed. As such, the radiolabeled antibody conjugates target cells expressing EGFR-hi or EGFRvIII on their surface. Provided herein are also methods of detecting and / or treating cancer using the provided radiolabeled antibody conjugates.BACKGROUND

[0005] The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase. Overexpression of the receptor is present in at least 70% of human cancers, including skin, breast, lung, colorectal, prostate and glioblastoma (GBM). EGFR overexpression oftenresults from gene amplification, transcriptional activation, or activating mutations. One notable mutation, EGFR variant III (EGFRvIII), results from the deletion of exons 2-7, leading to ligand-independent, constitutive signaling that enhances cancer progression. This tumor-specific mutation creates unique solvent-exposed subdomains, such as the 287-302 amino acid loop, which is typically inaccessible in the wild-type receptor but fully exposed in EGFRvIII or is conditionally exposed when EGFR expression is very high (EGFR-hi).

[0006] Therapeutic strategies for targeting EGFR and its variants include small molecule inhibitors, monoclonal antibodies, antibody-drug conjugates, and CAR T-cell therapy. Small molecule inhibitors, such as gefitinib and erlotinib, can be used to inhibit EGFR in tumors with EGFR-hi, but are not known to target EGFRvIII. Several murine monoclonal antibodies, such as 40H3 and 806, target unique EGFRvIII-specific epitopes and have been conjugated with chemotherapeutic payloads or radiolabeled for diagnostic and therapeutic purposes. The chimeric (ch806) and humanized (ABT-806) forms of 806 have been radiolabeled and explored for cancer imaging and treatment.

[0007] Known small molecules cannot specifically target EGFRvIII so their use as dual detection / therapeutic agents (theranostic agents) is challenging. The ability of anti-EGFR or anti-EGFRvIII antibodies to selectively target cancers in vivo is variable and their use as theranostic agents cannot be generalized to entire classes. In particular, the ability of a labeled antibody to selectively target its target epitope in vitro, or target EGFRvIII or EGFR-hi in cellular models, cannot guarantee its activity in vivo. Likewise, the specificity of EGFR-targeting agents towards ex vivo tumors cannot predict tumor-specific uptake such that the label does not concentrate in off-target organs including the liver, kidneys, bladder, heart, muscle, lungs or bones. Therefore, there remains a need for EGFR-targeting agents that can be used as theranostic agents for cancer, such as epithelial-derived cancers or glioblastomas.SUMMARY

[0008] Provided are radiolabeled antibody conjugate that binds within the exposed 287-302 amino acid loop on EGFRvIII and EGFR-hi and their use in methods of detecting and / or treating cancer in vivo. Specifically, following in vivo administration of a radiolabeled antibody conjugate, EGFR-overexpressing tumors in which the 287-302 amino acid loop is exposed were clearly detectable by PET-CT imaging. The antibody conjugate bound EGFRvIII and EGFR-hi in vitro and the radiolabeled antibody conjugate showed selectivityfor EGFR-overexpressing tumors over control tumors in vivo. Selectivity for an EGFR-hi xenograft was also shown over another xenograft and organs such as the liver, kidneys, bladder, heart, muscle and lungs. The findings described herein indicate that radiolabeled antibody conjugates have utility in theranostic methods for detecting and treating EGFR-hi or EGFRvIII-expressing cancers, such as epithelial-derived cancers or glioblastomas.

[0009] Accordingly, in one aspect, there is provided a radiolabeled antibody conjugate comprising a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of64Cu,67Ga,89Zr,161Tb,177Lu,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human epidermal growth factor receptor (EGFR; SEQ ID NO: 1); optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine; and optionally wherein the radiolabeled antibody conjugate comprises a conjugate group comprising a chelator that forms a stable complex with the radioisotope with a thermodynamic stability constant (LogKML) of at least 18.

[0010] In another aspect, there is provided a method of detecting cancer cells in a subject having cancer, wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi), the method comprising: (a) administering an effective amount of a radiolabeled antibody conjugate to the subject; and (b) detecting any radiation retained within the subject; wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of64Cu,89Zr,161Tb,177Lu,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) and optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.

[0011] In yet another aspect, there is provided a method of treating cancer in a subj ect in need thereof, the method comprising administering a therapeutically effective amount of a radiolabeled antibody conjugate to the subject; wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi); wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of67Ga,177Lu,225Ac,211At,161Tb, and212Pb; wherein the monoclonal antibody specifically binds to anepitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) and optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.

[0012] In a further aspect, there is provided a theranostic method for detecting and treating cancer in a subject, wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpresses EGFR (EGFR-hi), the method comprising a combination of the methods of detecting and treating cancer described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Aspects will be described, by way of example, with reference to the following drawings.

[0014] FIG. 1 shows the binding of specified antibodies (rabbit anti-EGFR (Cell Signaling Technology; 2232S) or 40H3) or antibody conjugates (DOTA-40H3 or DFO*-40H3) to EGFR from specified tumor cell lines expressing EGFR (U87 cells) or overexpressing EGFR (A431 or MDA-MB-468). The prominent band between the highest two markers of the ladder on the immunoblots is EGFR (-180 kDa). The presence of this band confirms specific antibody binding between the antibody-cell pair. For example, 40H3, DOTA-40H3 and DFO*-40H3 display specific binding to cells overexpressing EGFR (A431 and MDA-MB-468) but not to cells lines EGFR normal levels of EGFR (U87 cells).

[0015] FIG. 2A shows representative PET / CT images of uptake of the radiolabeled EGFR antibody conjugate 89Zr-DFO*-40H3 in mice bearing A431 (EGFR-overexpressing) tumors. The images were taken 24-, 48-, and 72-hours post-administration of 89Zr-DFO*-40H3. The arrows indicate tumor location.

[0016] FIG. 2B shows the mean standard uptake values (SUV) of the radiolabeled EGFR antibody conjugate 89Zr-DFO*-40H3 in different tissues in mice bearing A431 (EGFR-overexpressing) and U87 MG (control) subcutaneous tumors. Mean SUV was calculated 24-, 48-, and 72-hours post-administration of 89Zr-DFO*-40H3 to the mice. Significant P values (<0.05) are displayed for tumor comparisons. A two-way ANOVA was used to calculate p values.

[0017] FIG. 3 is a scheme showing protein nucleophiles reacting with conjugation reagents. FIG. 3 A shows a generic nucleophile reacting with the reagent to form an antibody conjugate. FIG. 3B shows a nucleophilic conjugation with an isothiocyanate group. FIG. 3Cshows amine conjugation with a succinimide group. FIG. 3D shows thiol conjugation with a maleimide group.

[0018] FIG. 4 is a scheme showing generalized conjugation reagents based on DFO* (left) and DOTA (right). FIG. 4A shows the structures of DFO* (left) and DOTA (right). FIG.4B shows generalized reagents comprising a generalized reactive group and a conjugate group comprising a moiety capable of forming a stable reaction product with a radioisotope (i.e., DFO* (left) and DOTA (right)).

[0019] FIG. 5 is a scheme showing conjugation reagents based on DFO* (left) and DOTA (center and right). FIG. 5A exemplifies reagents DFO*-p-phenyl-NCS (left) and two examples of DOTA reagents (center and right). FIG. 5B depicts their conjugation products.DETAILED DESCRIPTION

[0020] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and as commonly used in the art to which this application belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. To facilitate ready understanding, certain terms used herein are first defined below.

[0021] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a nucleotide sequence” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0022] Throughout this specification and aspects, the words “have” and “comprise”, or variations such as “has”, “having”, “comprises”, or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0023] It is understood that wherever aspects are described herein with the language “comprising”, otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. In other words, if an aspect is described ascomprising A, B and C, aspects consisting essentially of A, B and C are also contemplated, as are aspects consisting of A, B and C.

[0024] The term “about” refers to an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term indicates a deviation from the indicated numerical value of ±10%. In some embodiments, the deviation is ±5% of the indicated numerical value. In certain embodiments, the deviation is ±1% of the indicated numerical value.

[0025] The term “antibody” refers to an antigen binding polypeptide that includes six complementarity determining regions (CDRs). Exemplary antigen-binding polypeptides comprised by the term “antibody” include, but are not limited to, IgA antibodies, IgG antibodies, IgE antibodies, IgM antibodies, bi- or multi- specific antibodies (including bispecific T cell engagers (BiTEs) and biparatopic antibodies), Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and single-chain variable fragments (scFvs). An antibody typically includes at least a heavy (H) chain variable region (abbreviated herein as VH), and at least a light (L) chain variable region (abbreviated herein as VL). An antibody may include two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., Fab, F(ab’)2, Fd, and Fv fragments), single chain antibodies (e.g., singlechain Fvs), as well as full-length antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). A full-length antibody typically includes a constant region. A full-length antibody may be an IgG, e.g., IgGl, IgG3, or IgG4. The light chains of the immunoglobulin can be of types kappa or lambda. An “antibody” may be an immunoglobulin molecule comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds, wherein the amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the CDRs contained therein. The CDRs are interspersed with regions that are well-known and generally conserved among and between species (e.g., mouse and human), which are termed framework regions (FRs).

[0026] The term “antigen-binding fragment” refers to a portion of an intact antibody that binds to an antigen (e.g., EGFR). An antigen-binding fragment can contain the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments. Antigen-binding fragments or regions also comprise single chain antibodies, such as single-chain Fvs (scFvs).

[0027] The term “cancer” refers to a pathological condition characterized by the abnormal and uncontrolled proliferation of cells resulting from genetic and / or epigenetic alterations. In some instances, the cancer may be derived from epithelial cells, e.g., the cancer may be carcinoma. In some instances, the cancer may be derived from glial cells and the cancer may be GBM. In some instances, the cancer may be selected from the group consisting of GBM, non-small cell lung carcinoma (NSCLC), colorectal cancer (e.g., metastatic colorectal cancer (mCRC)), breast cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, esophageal cancer, skin cancer, and head and neck cancer. When used herein, it is understood that the term “cancer” encompasses pre-cancerous conditions with the potential to develop into cancer, e.g., dysplasia. Similarly, the term “cancer cell” encompasses pre-cancerous cells with the potential for malignant transformation. Throughout this specification and aspects, the term “cancer” may be used interchangeably with the term “tumor”.

[0028] “ka” refers to an association rate of a particular binding means and a target to form a binding means / target complex.

[0029] “kd” refers to a dissociation rate of a particular binding means / target complex.

[0030] “KD” refers to a dissociation constant, which is obtained from the ratio of kd to ka(i.e., kd / ka) and is expressed as a molar concentration (M). KD values can be determined using methods well established in the art, e.g., by ELISA, as described in Example 4.

[0031] The term “radioisotope” is an elementally unstable form of a chemical element that releases radiation as it breaks down and becomes more stable. Radioisotopes suitable for in vivo detection / location / imaging and / or for therapy are known to those skilled in the art.

[0032] The term “radiolabeled antibody conjugate” is an antibody (or an antigen binding fragment thereof) to which one or more radioisotope has been attached (e.g., conjugated or chelated). The radioisotope is generally connected to the antibody via one or more amino acid sidechain of the antibody (e.g., one or more lysine or cysteine sidechain) or the N-terminal amine using bioconjugate chemistry known to those skilled in the art. The conjugate group attached to the antibody generally comprises a synthetic functional group (e.g., a chelator that forms a complex) attached to the radioisotope prior to administration to a subject. The radioisotope may be a metal (e.g., a detection isotope such as64Cu,89Zr,161Tb,177LU,212Pb and225Ac; or a therapeutic isotope such as67Ga,161Tb,177Lu,212Pb and225Ac)that may be chelated by the conjugated chelator to form the radiolabeled antibody conjugate. Alternatively, the radioisotope may be a halogen (e.g., a detection isotope such as18F,124I,125I or211At; or a therapeutic isotope such as131I or211At) that may be reacted with the synthetic functional group prior to conjugation or after the conjugate is attached to the antibody.

[0033] The term “EGFR variant III” or “EGFRvIII” means the expressed protein of a common mutation in the extracellular domain of EGFR that results in the deletion of exons 2-7 and fusion of exon 1 with exon 8. This results in the loss of 268 amino acids and creates solvent-exposed subdomains, such as the 287-302 amino acid loop, which is typically inaccessible in the wild-type receptor but fully exposed in EGFRvIII. The term “the cancer cells express EGFRvIII” means that some or all of the cancer cells (optionally within a tumor) express EGFRvIII to a level that is linked to cancer and can be detected using detection methods known to those skilled in the art. An example of a cancer that expresses EGFRvIII is glioblastoma.

[0034] The term “overexpresses EGFR” or “EGFR-hi” means that the cancer contains a high copy number of the EGFR gene. EGFR copy number can be classified into six categories (ParkH., etal. Mod. Pathol. 27, 2014, 1212-1222): disomy (<2 copies in >90% of cells); low trisomy (<2 copies in >40% of cells, three copies in 10-40% of cells, and >4 copies in <10% of cells); high trisomy (<2 copies in >40% of cells, three copies in >40% of cells, and >4 copies in <10% of cells); low polysomy (>4 copies in 10-40% of cells); high polysomy (>4 copies in >40% of cells); and gene amplification (presence of tight EGFR gene clusters and a ratio of the EGFR gene to chromosome 7 of >2, or >15 copies of EGFR per cell in >10% of cells). The term “a high copy number” includes high trisomy, low polysomy, high polysomy and gene amplification; preferably high polysomy and gene amplification; most preferably gene amplification. A high copy number is optionally associated with one or more mutations in the EGFR gene. The term “the cancer cells express EGFR-hi” means that some or all of the cancer cells (optionally within a tumor) overexpress EGFR to a level that is linked to cancer and can be detected using detection methods known to those skilled in the art. Overexpression may also be calculated at the protein level, for example using semi-quantitative immunoblotting (see Example 3) or immunohistochemistry (Park H., et al. Mod. Pathol. 27, 2014, 1212-1222) with overexpression of at least 2-fold (e.g., at least 3-fold) compared to non-EGFR-hi cells of a similar type (e.g., biopsied tissue from the same organ where the cancer is not present). Examples of cancer that overexpress EGFR includecarcinomas, and those selected from the group consisting of NSCLC, colorectal cancer (e.g., mCRC), breast cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, esophageal cancer, skin cancer, and head and neck cancer.

[0035] The term “administer” refers to the direct application of an agent to a subject, e.g., a radiolabeled antibody conjugate described herein. A compound (e.g., a radiolabeled antibody conjugate described herein) may be administered by any suitable route (e.g., by infusion, injection, or any other means), whether self-administered or administered by a clinician or other medical professional.

[0036] The term "detect" refers to a qualitative or quantitative identification of the presence, absence of an analyte to be detected. The analyte may be cancer cells where EGFR expression is very high or where EGFRvIII is expressed. Detection may be carried out by an attending physician or caregiver, prescribing a radiolabeled antibody conjugate described herein and thereby causing the application of the radiolabeled antibody conjugate to a subject, e.g., by infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified provider. Detection is generally achieved using an appropriate scanner, e.g., positron emission tomography (PET), optionally coupled with computed tomography (CT) (e.g., PET-CT imaging), or single-photon emission computed tomography (SPECT). The term “detect” may be used interchangeably with the verb “image” where the detection is processed to form an image. The term “detect” may be used interchangeably with the term “locate” where the analyte is clustered in one or more locations in the body, e.g., one or more tumors in a specific organ. Detection, imaging and / or location of radioisotopes retained within the body at the time of imaging (e.g., 1 to 14 days) may be used to confirm the presence or absence of tumors, the rate of disease progression and / or the efficacy of a treatment regimen.

[0037] The term "treat" refers to restraining, slowing, or reversing the progression or severity of an existing symptom, condition, or disorder, in a subject in need thereof. The treatment may target cancer cells where EGFR expression is very high or where EGFRvIII is expressed. Treatment may be carried out by an attending physician or caregiver, prescribing a radiolabeled antibody conjugate described herein and thereby causing the application of the radiolabeled antibody conjugate to a subject, e.g., by infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified provider.

[0038] The term "theranostics" or “theragnostics” is a technique used in personalized nuclear medicine, where one radioactive compound (e.g., a radiolabeled antibody conjugate comprising a detection isotope such as64Cu,89Zr,161Tb,177Lu,211At,212Pb and225Ac) is used to detect and a second radioactive compound (e.g., a radiolabeled antibody conjugate comprising a therapeutic isotope such as67Ga,161Tb,177Lu,211At,212Pb and225Ac) is used to treat cancerous tumors. Therefore, theranostics combines radiation detection and radiation therapy.

[0039] The phrase "effective amount" means an amount of a radiolabeled antibody conjugate that is sufficient to detect, inhibit, treat or kill cancer cells where EGFR expression is very high or where EGFRvIII is expressed. Such effective amounts may be achieved by the use of a consistent effective amount of radioisotope across all antibody molecules, or by combining highly labeled antibody with unlabeled antibody. One or more effective amounts may be administered to a subject to confirm the presence or absence of tumors, the rate of disease progression and / or the efficacy of a treatment regimen. One or more therapeutically effective amounts of a radiolabeled antibody conjugate may be administered during the treatment.

[0040] The term "subject" refers to a mammal, preferably a human to whom treatment is administered. The term “subject” may be used interchangeably with “patient” where the subject has cancer and requires treatment.

[0041] The term "co-administering" means the administration of a radiolabeled antibody conjugate and another compound described herein, separately, simultaneously, and / or sequentially over a period of time as determined by a drug label and attending physician or healthcare provider. Additionally, “co-administering” may include the administration of one or more additional therapies, for example, radiation, surgery, bone marrow transplantation, chemotherapy, immunotherapy, hormone therapy, or targeted therapy.

[0042] The term “epitope” is known in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions or residues of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope).

[0043] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein.Epidermal growth factor receptor (EGFR)

[0044] The epidermal growth factor receptor (EGFR or Erb-Bl) belongs to a family of receptor tyrosine kinases. EGFR is a cell-surface, membrane-bound protein with three main structural domains: the extracellular domain, the transmembrane domain, and the intracellular domain. The extracellular domain is responsible for binding ligands, such as epidermal growth factor (EGF), which triggers receptor activation. Upon ligand binding, the receptor undergoes a conformational change that facilitates its dimerization. The transmembrane domain is a single a-helix that spans the plasma membrane, anchoring the receptor in place. The intracellular domain, located inside the cell, contains a tyrosine kinase domain that, once activated, initiates phosphorylation events and activates various signaling pathways. These pathways play essential roles in regulating cellular processes like growth, differentiation, and survival.

[0045] The amino acid sequence of human EGFR is shown below. This sequence does not include the first 24 amino acids comprising the N-terminal signal peptide sequence. The residue numbering used herein begins with the first residue shown below, L. SEQ ID NO: 1 corresponds to residues 24 to 1210 of UniProtKB ID: P00533.LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLK TIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPM RNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPS CPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDC LVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTD HGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKH FKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDL HAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTIN WKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGP KIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAP NQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKAN KEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQ YLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEE KEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPAS EISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYL VIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLL SSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYIN QSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNST FDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEF IGA (SEQ ID NO: 1).EGFR in cancer

[0046] Dysregulation of EGFR has been implicated in various human cancers, with overexpression of the receptor present in at least 70% of human cancers (Seymour, L. K., Curr. Drug Targets 2, 2001, 117-133), including skin, breast, carcinomas (including head and neck), lung, prostate and colorectal cancer (da Silva Santos E., etal. Int. J. Pharm. 592, 2021, 120082). In particular, EGFR is highly expressed in up to 40% of cases of glioblastoma (GBM). EGFR-driven oncogenesis results from activating mutations, deletions, or receptor overexpression due to gene amplification or transcriptional activation and elevated ligand levels can also contribute to cancer progression (An, Z., et al. Oncogene 37, 2018,1561- 1575). Accordingly, EGFR represents a valuable therapeutic target.287-302 amino acid loop of EGFR

[0047] The extracellular region of EGFR comprises four main domains; domains I and III are responsible for ligand binding (Sun L., et al. Front. Oncol. 2020, 10, 2021, 586596), while domains II and IV contain cysteine-rich motifs involved in dimerization and proper receptor folding. A common mutation in the extracellular domain is the deletion of exons 2-7, resulting in the loss of 268 amino acids and formation of EGFR variant III (EGFRvIII), which fuses exon 1 with exon 8. This tumor-specific mutation creates unique solvent-exposed subdomains, such as the 287-302 amino acid loop which comprises the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 2), as shown in bold in SEQID NO: 1. This loop structure is typically inaccessible in the wild-type receptor but fully exposed in EGFRvIII or is conditionally exposed when EGFR expression is very high (EGFR-hi) (GanH., etal. Cancer Res. 72, 2012, 2924-2930; Jungbluth A., etal. PNAS 100, 2003, 639-644; Sugawa N., et al. PNAS 87, 1990, 8602-8606). EGFRvIII exhibits constitutive signaling activity independent of ligand binding, thus leading to enhanced cell proliferation, decreased apoptosis, increased angiogenesis, and accelerated invasiveness (Jungbluth A., etal. PNAS 100, 2003, 639-644).Antibodies that bind within the 287-302 amino acid loop

[0048] Various anti-EGFR / EGFRvIII antibodies, such as the monoclonal antibodies 40H3 (WO 2021 / 003297 Al; Ho et al., Antib Then 2019; 2(4):88-98), its humanized form A10 (WO 2025 / 014896 Al) and 806 (WO 2010 / 096434 A2; Jungbluth et al., PNAS 2003; 100(2): 639-644; Panousis etal., Br. J. Cancer 2005; 92(6): 1069-1077), and antigen-binding fragments, such as Fab 8709 (Miersch et al., ACS Chem Biol. 2017; 12(5): 1381-1389), have been developed that are capable of binding to epitopes within the 287-302 amino acid loop. Such antibodies and antigen-binding fragments are able to specifically target cancer cells that express EGFRvIII or EGFR-hi while avoiding healthy cells that express normal levels of wildtype EGFR.

[0049] Accordingly, the methods provided herein may use a radiolabeled antibody conjugate that specifically binds to an epitope (e.g., a non-contiguous epitope) within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1), optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.

[0050] The amino acid loop structure comprises the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 2). Accordingly, the epitope (e.g., the noncontiguous epitope) may comprise one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen) amino acids selected from C287, G288, A289, D290, S291, Y292, E293, M294, EX295, E296, D297, G298, V299, R300, K301 and C302 of human EGFR comprising the amino acid sequence of SEQ ID NO: 1.

[0051] Different anti-EGFR / EGFRvIII antibodies bind to different residues in the 297-302 loop. For example, in an alanine scan, 40H3 retained binding to D290Aand E293A,but not to R300A (Ho et al., Antib Then 2019; 2(4): 88-98). This indicates that the epitope bound by 40H3 includes the sidechain of residue R300.

[0052] Accordingly, the epitope may comprise an arginine residue at position 300 of human EGFR as recited in SEQ ID NO: 1. Preferably, the antibody epitope is such that the antibody does not bind to its epitope when R300 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (R300A; i.e., CGADSYEMEEDGVAKC (SEQ ID NO: 15)). Anti-EGFR / EGFRvIII antibodies that target the 287-302 loop structure are generally provided as isolated antibodies, in particular isolated monoclonal antibodies (“MAbs”). MAbs are antibodies derived from a single copy or clone including, for example, any eukaryotic, prokaryotic or phage clone.

[0053] The amino acid sequences of the complementarity-determining regions (CDRs) of the heavy (HCR1-3) and light (LCDR1-3) chains of an exemplary MAb that specifically binds to an epitope in the 297-302 amino acid loop of human EGFR are shown in Table 1.Table 1 : Exemplary antibody amino acid CDR sequences

[0054] Accordingly, the methods provided herein may use a radiolabeled antibody conjugate wherein the MAb comprises a heavy chain complementarity determining region (HCDR) 1 comprising an amino acid sequence of GFSLTNYG (SEQ ID NO: 3), an HCDR2 comprising an amino acid sequence of MWRGGGT (SEQ ID NO: 4), an HCDR3 comprising an amino acid sequence of ARKGVGMGLGY (SEQ ID NO: 5); and a light chain complementarity determining region (LCDR) 1 comprising an amino acid sequence of QTIGTW (SEQ ID NO: 6), an LCDR2 comprising an amino acid sequence of GAT (SEQ ID NO: 7), and an LCDR3 comprising an amino acid sequence of QQLYSNPYT (SEQ ID NO:8). These CDRs are found in 40H3 (WO 2021 / 003297 Al) and its humanized forms, such as A10 (WO 2025 / 014896 Al).

[0055] The anti-EGFRvIII / EGFR-hi antibody may be human or humanized. The amino acid sequences of the heavy (VH) and light (VL) chain variable regions of an exemplary humanized MAb that specifically binds to an epitope in the 297-302 amino acid loop of human EGFR are shown in Table 2.Table 2, Exemplary humanized antibody variable region sequences

[0056] Accordingly, the methods provided herein may use a radiolabeled antibody conjugate wherein the MAb comprises an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence that is at least about 85% identical to, or identical to: QVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGG GTDYNAAFISRLTITKDTSKSQVVFTMTNMDPVDTATYYCARKGVGMGLGYWGQ GTLVTVSS (SEQ ID NO: 9), andan immunoglobulin light chain variable (VL) region comprising an amino acid sequence that is at least about 85% identical to, or identical to:DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIYGATNLAD GVPSRFSGSGSGTKFTLTISSLQPEDFATYYCQQLYSNPYTFGGGTKLEIK (SEQ ID NO: 10).

[0057] These variable regions are found in A10 (WO 2025 / 014896 Al).

[0058] The MAb antibody may have a dissociation constant (KD) for its epitope in the nanomolar range. For example, the antibody may have a KD of between about 0.06 nMto about 60 nM, preferably between about 0.6 nM to about 6 nM, for example as determined by a plate-based binding assay, such as an ELISA (e.g., as described in WO 2021 / 003297 Al; Ho E., et al. Antib. Then 2, 2019, 88-98; and / or WO 2025 / 014896 Al). Preferably, the antibody has a KD of between about 0.06 nM to about 60 nM, more preferably between about 0.6 nM to about 6 nM, for the peptide CGADSYEMEEDGVRKC (SEQ ID NO: 2).

[0059] The epitope may comprise the sidechain of arginine at residue 300 of SEQ ID NO: 1 (R300) such that when the arginine at residue 300 is substituted with alanine, the MAb does not specifically bind to this peptide (CGADSYEMEEDGVAKC (SEQ ID NO: 15)), for example as determined by a plate-based binding assay, such as an ELISA (e.g., as described in WO 2021 / 003297 Al; Ho E., et al. Antib. Then 2, 2019, 88-98; and / or WO 2025 / 014896 Al). Preferably, the antibody has a KD of about 600 nM or greater (e.g., about 600 nM to a value that is undetectably high, such as about 600 mM), more preferably about 6 mM or greater (e.g., about 6 mM to a value that is undetectably high, such as about 600 mM), for the peptide CGADSYEMEEDGVAKC (SEQ ID NO: 15).

[0060] The KD may be determined using a plate-based assay. The plate-based assay may be ELISA, as described in Example 4. The antigen used to determine the KD of the antibody for its epitope may be the peptide CGADSYEMEEDGVRKC (SEQ ID NO: 2), optionally comprising a C-terminally His-tag. Specific binding may be defined as a KD in the nanomolar or sub-nanomolar range, preferably between about 0.06 nM to about 60 nM, more preferably between about 0.6 nM to about 6 nM. Longer peptides comprising the peptide CGADSYEMEEDGVRKC (SEQ ID NO: 2) may be employed, such that the epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) is exposed and available to be recognized (e.g., EGFRvIII).

[0061] The MAb may be an IgG antibody, e.g., an IgGl antibody.

[0062] The MAb may be a bi- or multi-specific antibody, a single chain antibody, a nanobody, or a diabody.

[0063] An antigen-binding fragment is a portion of an intact antibody that binds to an antigen (e.g., EGFR). Antigen-binding fragments are generally smaller and more easily engineered than full-length antibodies, allowing for better tissue penetration, lower production costs, and greater flexibility in therapeutic design.

[0064] Accordingly, the MAb may comprise, or be formulated as, an antigen-binding fragment. For example, the antibody may comprise, or be, a Fab fragment, a Fab’ fragment,a F(ab’)2 fragment, an Fd’ fragment, an Fd fragment, and / or a single-chain variable fragment (scFv).

[0065] For example, the MAb may comprise, or be, an scFV Amino acid sequences of exemplary scFvs that specifically bind to an epitope in the 297-302 amino acid loop of human EGFR are shown in Table 3.Table 3, Exemplary scFV sequences

[0066] These scFV sequences are found in A10 (WO 2025 / 014896 Al).

[0067] Alternatively, the MAb may comprise, or be, a Fab fragment. Amino acid sequences of exemplary heavy and light chain Fab fragments that specifically bind to an epitope in the 297-302 amino acid loop of human EGFR are shown in Table 4.Table 4, Exemplary Fab fragment sequences

[0068] These Fab sequences are found in A10 (WO 2025 / 014896 Al).

[0069] An Fc region may provide one or more effector functions to an antibody. The effector functions may include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). Additionally or alternatively, an Fc region may provide an antibody extended half-life in circulation.

[0070] Accordingly, the MAb may comprise an Fc region.Conjugates of antibodies that bind within the 287-302 amino acid loop

[0071] The radiolabeled antibody conjugate requires a stable connection between the radioisotope and the antibody. Many conjugate systems are known to those skilled in the art.

[0072] It is possible to radiolabel proteins with a fluoride isotope. For example, 4-nitrophenyl-2-18F-fluoropropionate ([18F]NFP), N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), or 4-[18F]fluorobenzoic acid ([18F]FBA) may be used to conjugate18F via amine groups (e.g., lysine sidechains and / or N-terminal amines). For example, N-[6-(4-[18F]fluoro-benzylidene)aminooxyhexyl]maleimide ([18F]FBAM), N-[2-(4- [18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM), or [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO) may be used to conjugate18F via thiol groups (e.g., reduced cysteine sidechains).

[0073] It is possible to radiolabel proteins with an iodide isotope. For example, lodogen (l,3,4,6-tetrachloro-3a,6a-diphenylglycouril) may be used to conjugate iodineisotopes via phenyl groups (e.g., tyrosine sidechains). For example, an aromatic iodide with a lysine reactive group (e.g., an isothiocyanate or a succinimide such as radiolabeled succinimidyl iodobenzoate) or a cysteine reactive group (e.g., maleimide such as radiolabeled N-(p-iodophenethyl)maleimide) may be used to conjugate iodine isotopes.

[0074] In the case of211At, a conjugate may be formed with the astatine isotope covalently bound to the antibody (Vanermen et al. EJNMMI Radiopharm. Chem. 9, 2024, 69). For example, 2,3,5,6-tetrafluorophenyl l-(3-hydroxy-2-(hydroxymethyl)-2-(Astatom ethyl)propyl)- 1H- 1,2, 3 -triazol e-4- carboxylate orN-succinimidyl 5-[211At]astato-3-pyridinecarboxylate may be used to conjugate211At via amine groups (Tada et al., ChemMedChem 19, 2024, e202400369). For example, N-succinimidyl 3-211At-astato-4-guanidino-methylbenzoate (211At-SAGMB) may also be used to conjugate211At via amine groups (Feng et al., J. Nucl. Med. 64, 2023, 124-130). In the case of metallic radioisotope (and salts thereof), the conjugate group may be formed with a chelator covalently bound to the antibody, optionally via a spacer, wherein the chelator may chelate the metallic radioisotope or salt thereof to form a stable complex. The radioisotope:chelator complex may be formed before or after conjugation between the antibody and the chelator. Preferably, the complex is formed after conjugation between the antibody and the chelator. The complex preferably has a thermodynamic stability constant (LogKML) of at least 18, preferably at least 20, more preferably at least 25.Conjugation chemistry

[0075] The chemistry used to produce the antibody conjugate is not particularly limited (see Bioconjugation and crosslinking technical handbook, Thermo Scientific, 2022). Generally, a nucleophile within the protein (e.g., reactive amine or thiol groups) may be reacted, at a specific pH where the nucleophile is active and the antibody is stable (e.g., pH 6.0 to 7.5), with a reagent comprising a reactive group (e.g., an isothiocyanate, a succinimide or a maleimide) and a conjugate group comprising a radioisotope or a moiety capable of forming a stable reaction product with a radioisotope (e.g., a chelator capable of forming a complex), as shown in FIG. 3.

[0076] In FIG. 3A, the generic nucleophile (Nu:) reacts with the reagent (R-X) to form an antibody conjugate. X represents the reactive group, replaced by Y during the reaction. R represents the conjugate group (e.g., a synthetic functional group) comprising the radioisotope or a moiety capable of forming a stable reaction product with a radioisotope(e.g., a chelator), and optionally a spacer. Exemplary conjugation chemistries for conjugate groups (R groups) are shown in FIG. 3B to 3D. FIG. 3B shows a nucleophilic (e.g., amine or thiol depending on the pH and reactivity) conjugation with an isothiocyanate. Higher pH favors lysine labelling whereas lower pH favors cysteine (in the presence reduced cysteine residues) and N-terminal amine labelling. FIG. 3C shows amine conjugation with a succinimide group. FIG. 3D shows thiol conjugation with a maleimide group.

[0077] Exemplary amine reactive groups may be selected from the group consisting of isothiocyanates, succinimidyl esters, sulfosuccinimidyl esters, 2,4,5,6-tetrafluorophenyl (TFP) esters, sulfodicholorphenol (SDP) esters, carbonyl azides, sulfonyl chlorides and aldehydes, such as an isothiocyanate, a succinimidyl ester, or a sulfosuccinimidyl ester. Optionally, the amine reactive group is an isothiocyanate.

[0078] Exemplary thiol reactive groups may be selected from the group consisting of isothiocyanates, iodoacetamides, maleimides, benzylic halides and bromomethylketones, such as an isothiocyanates, an iodoacetamide or a maleimide.

[0079] It may be preferable that the conjugation reaction occurs at a pH between 7.0 and 7.5 where the conjugation occurs in a facile manner and the antibodies are generally stable. This favors cysteine (in the presence of reduced cysteine residues) and N-terminal amine labelling, as well as any specific acid lysine sidechains with lower pKa values.Conjugate groups

[0080] The conjugate group comprises the radioisotope or the moiety capable of forming a stable reaction product with the radioisotope (e.g., a chelator), and optionally a spacer.

[0081] The moiety capable of forming a stable reaction product with a radioisotope optionally comprises a chelator. A suitable chelator for211At is closo-decaborate(2-) (CAS number 12356-13-7). Suitable chelators for chelating metal radioisotopes, or salts thereof, are known to those skilled in the art (Holik H. et al. Molecules 27, 2022, 3062). Exemplary chelators may be selected from the group consisting of DOTA (2,2',2",2"'-(l,4,7,10-tetraazacyclododecane-l,4,7,10-tetrayl)tetraacetic acid), DOTAM (2-[4,7,10-tris(2-amino-2-oxoethyl)-l,4,7,10-tetrazacyclododec-l-yl] acetamide), DOTAGA (2-(4,7,10-tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl)pentanedioic acid), ftmacropa (N,N’-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6; CAS Number 2146095-13-6), DTPA (diethylenetriamine pentaacetic acid), NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid), NODAGA (2-[l,4,7-triazacyclononan-l-yl-4,7-bis(tBu-ester)]-l,5-pentanedioic acid), NODASA (1,4,7-triazacyclononane-l-succinic acid-4, 7-diacetic acid), NETA ({4-[2-(bis-carboxy-methylamino)-5-(4-nitrophenyl)-entyl]-7-carboxymethyl-[l,4,7]tri-azonan-l-yl} acetic acid), TETA (triethylenetetramine), CB-TE2A (2,2-(l,4,8,ll-tetraazabicyclo[6.6.2]hexadecane-4,ll-diyl)diacetic acid), EUdedpa (l,2-bis[[(6-carboxypyridin-2-yl)methyl]amino]ethane), EUoctapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), EUCTZXdedpa (CHX= cyclohexyl / cyclohexane, EUdedpa = l,2-[[6-carboxy-pyri din-2 -yl]-methylamino]ethane), EUCffifoctapa (CHX= cyclohexyl / cyclohexane, EUoctapa = A,A'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-A,A'-diacetic acid), HYNIC (hydrazinonicotinamide), sarcophagine, HBED-CC (A,7\7-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-MN'-di acetic acid), PCTA (3,6,9, 15-tetraazabicyclo [9.3.1]pentadeca-l(15),ll,13-triene-3,6,9-triacetic acid), MANOTA (methyl amino triazacyclononane triacetic acid), THP (tris(hydroxypyridinone)), DFO (N'-[5-(acetyl-hydroxy-amino)pentyl]-N-[5-[3-(5-aminopentyl-hydroxy-carbamoyl) propanoylamino]pentyl]-N-hydroxy -butane diamide), DFO* (5,11,16,22-tetraazahexacosanediamide, Nl-[5-(acetylhydroxyamino)pentyl]-N26-(5-aminopentyl)-N26,5,16-trihydroxy-4,12,15,23-tetraoxo-), and DFOcyclo* (a DFO analogue elongated with a cyclic hydroxamate group having a different spacer length). Preferred chelators may be selected from the group consisting of DOTA (2, 2', 2", 2'"-(1 ,4,7, 10-tetraa5, 11,16,22-tetraazahexacosanediamide,zacyclododecane- 1,4,7,10-tetrayl)tetraacetic acid) and DFO* (WO 2015 / 140212 Al; Nl-[5-(acetylhydroxyamino) pentyl]-N26-(5-aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23-tetraoxo-; CAS number 1623757-38-9). FIG. 4A shows the structures of DFO* (left) and DOTA (right). FIG. 4B shows generalized reagents comprising a generalized reactive group (X; e.g., a isothiocyanates, a succinimide, an iodoacetamide or a maleimide) and a conjugate group comprising a moiety capable of forming a stable reaction product with a radioisotope (i.e., the chelators DFO* (left) and DOTA (right)).

[0082] Optionally, the antibody conjugate comprises DOTA when the radioisotope is (or is to be) selected from the group consisting of64Cu,67Ga,161Tb,177Lu,212Pb and225Ac. Optionally, the antibody conjugate comprises DFO* when the radioisotope is (or is to be)89Zr.Optional spacers

[0083] The conjugate group (R group) comprises the radioisotope or a moiety capable of forming a stable reaction product with a radioisotope, and optionally a spacer. It may be desirable to include a spacer between the radioisotope or a moiety capable of forming a stable reaction product with a radioisotope and the reactive group within the reagent. In some conjugate groups (e.g., DFO*), the moiety capable of forming a stable reaction product includes a portion that acts as a spacer (i.e., the 1-aminopentyl group connected to X). In some conjugate groups (e.g., DOTA), this section is shorter (i.e., -CH^CO)-) and an optional spacer may be appropriate. Chelators comprising DOTA and a spacer are known in the art.

[0084] FIG. 5A exemplifies reagents DFO*- / ?-phenyl-NCS (left; WO 2015 140212 Al) and two examples of DOTA reagents (S-2-(4-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane tetraacetic acid (center; used in the examples) and 2,2',2”-(I0-(I-carboxy-4-((4-isothiocyanatobenzyl)amino)-4-oxobutyl)-l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid (right; DOTA with a longer spacer). FIG. 5B depicts their conjugation products with an antibody where “Nu” represents a nitrogen atom in the form of -NH- (from a lysine sidechain or N-terminal amine) or a sulfur atom in the form of -S- (from a cysteine sidechain).

[0085] The spacer provides a covalent attachment between the radioisotope or a moiety capable of forming a stable reaction product with a radioisotope and the reactive group (e.g., a chelator). The structure of the spacer may not be critical, provided it does not substantially interfere with the reactivity of the reactive group or the structure or function of the antibody conjugate.

[0086] The spacer may be a Ci to C20 alkylene chain, optionally a C5 to C15 alkylene chain. The alkylene chain may be interrupted one or more -O-, -S-, -N(R')-, -C=C-, -C(O)-, — C(O)O— group, wherein R' is H, or Me. The alkylene chain interrupted one or more — O~ may be a polyethylene glycol (PEG) chain, optionally comprising 1-10 PEG units.

[0087] The spacer may be a pharmacokinetic-modifying spacer (PKM spacer), which may improve the “target-to-background” ratio by modifying the pharmacokinetic properties (distribution or excretion) of radiolabeled biomolecules. Exemplary PMK spacers may be selected from the group consisting of polyethylene glycols, aminohexanoic acid, amino acids and / or peptides (e.g., octreotide).

[0088] Optionally, the antibody conjugate is stable in human plasma for at least 24 hours, preferably at least 48 hours, more preferably at least 72 hours. A stable antibody conjugate means that there is a stable conjugate formed between the antibody and the conjugate group and that the conjugate group, and optionally spacer group, are stable in human plasma for the required time.Radiolabeled antibody conjugates

[0089] As described above, the radioisotope may form part of the conjugate group in the reagent, or be added after the conjugation reaction is complete.

[0090] For example,18F may be conjugated to amines in proteins using 4-nitrophenyl-2-18F-fluoropropionate ([18F]NFP), 4-[18F]fluorobenzoic acid ([18F]FBA) or N-succinimidyl-4-fluorobenzoate ([18F]SFB).18F may be conjugated to thiols in proteins using 7V-[6-(4-[18F]fluoro-benzylidene)aminooxyhexyl]mal eimide ([18F]FBAM), A-[2-(4-[18F]fluorobenz-amido)ethyl]maleimide ([18F]FBEM) or [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO). For example, an aromatic iodide may be conjugated via an amine reactive group (e.g., a succinimide such as radiolabeled succinimidyl iodobenzoate) or a cysteine reactive group (e.g., maleimide such as radiolabeled N-(p-iodophenethyl)maleimide) may be used to conjugate iodine isotopes. For example,211At may be conjugated to amines in antibodies using N-succinimidyl 3-[211At]astatobenzoate ([211At]SAB), N-succinimidyl-3-[211At]astato-4-guanidinomethylbenzoate ([211At]SAGMB) or isothiocyanatophenyl-closo-decaborate(2-). For example,211At may be conjugated to thiols in antibodies using maleimido-[211At]-closo-decaborate.

[0091] For example, the radioisotope may be selected from the group consisting of64Cu,67Ga,89Zr,161Tb,177Lu,211At,212Pb and225Ac; and may comprise a conjugate group comprising a chelator that forms a stable complex with the radioisotope with a thermodynamic stability constant (LogKML) of at least 18.

[0092] Based on the disclosure above, the radiolabeled antibody conjugate may comprise a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of64Cu,67Ga,89Zr,161Tb,177Lu,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human epidermal growth factor receptor (EGFR; SEQ ID NO: 1); optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine; and optionallywherein the radiolabeled antibody conjugate comprises a conjugate group comprising a chelator that forms a stable complex with the radioisotope with a thermodynamic stability constant (LogKML) of at least 18.

[0093] Optionally, the conjugate group includes a chelator. Preferably, the radioisotope is chelated to the chelator in the conjugate group after the conjugation reaction to the antibody is complete. This allows the antibody conjugate to be stored in a standard environment with the radioisotope stored separately and added prior to use.

[0094] Specific antibody-chelator-radioisotope combinations are contemplated below. For example, when the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of EGFR (SEQ ID NO: 1) the radioisotope may be89Zr in combination with the DFO* or DFOcyclo*. For example, when the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of EGFR (SEQ ID NO: 1) the radioisotope may be177Lu in combination with a chelator that forms a stable complex with the radioisotope with a thermodynamic stability constant (LogKML) of at least 18 (e.g., DOTA, DTPA, NETA, EUoctapa or PCTA).Radiolabeled antibody conjugates for diagnostic use

[0095] For diagnostic use, the radioisotope is preferably selected from the group consisting of64Cu,89Zr,161Tb,177Lu,211At,212Pb and225Ac, more preferably the group consisting of64Cu,89Zr,177Lu and212Pb, optionally89Zr.

[0096] Where the radioisotope is64Cu, the conjugate group preferably comprises a chelator, more preferably the conjugate group comprises a chelator selected from the group consisting of DOTA, NOTA, NOD AGA, NODASA, TETA, CB-TE2A and MANOTA.

[0097] Where the radioisotope is89Zr, the conjugate group preferably comprises a chelator, more preferably the conjugate group comprises a chelator selected from the group consisting of DFO, DFO* and DFOcyclo*, optionally the conjugate group comprises DFO*.

[0098] Where the radioisotope is161Tb, the conjugate group preferably comprises a chelator, optionally the conjugate group comprises DOTA.

[0099] Where the radioisotope is177Lu, the conjugate group preferably comprises a chelator, more preferably the conjugate group comprises a chelator selected from the group consisting of DOTA, DTPA, NETA, EUoctapa and PCTA, optionally the conjugate group comprises DOTA.

[0100] Where the radioisotope is211At, the conjugate group optionally comprises closo-decaborate(2-).

[0101] Where the radioisotope is212Pb, the conjugate group preferably comprises a chelator, more preferably a chelator comprising DOTA or DOTAM, optionally the conjugate group comprises DOTAM.

[0102] Where the radioisotope is225Ac, the conjugate group preferably comprises a chelator, more preferably the conjugate group comprises a chelator selected from the group consisting of DOTA and FFmacropa, optionally the conjugate group comprises DOTA.Radiolabeled antibody conjugates for therapeutic use

[0103] For therapeutic use, the radioisotope is preferably selected from the group consisting of67Ga,161Tb,177Lu,211At,212Pb and225Ac, more preferably the group consisting of161Tb,177LU,212Pb and225Ac, optionally177Lu.

[0104] Where the radioisotope is67Ga, the conjugate group preferably comprises a chelator, more preferably the conjugate group comprises a chelator selected from the group consisting of DOTA, NOTA, HztWAdedpa, THP and DFO.

[0105] Where the radioisotope is161Tb, the conjugate group preferably comprises a chelator, optionally the conjugate group comprises DOTA.

[0106] Where the radioisotope is177Lu, the conjugate group preferably comprises a chelator, more preferably the conjugate group comprises a chelator selected from the group consisting of DOTA, DTPA, NETA, FUoctapa and PCTA, optionally the conjugate group comprises DOTA.

[0107] Where the radioisotope is225Ac, the conjugate group preferably comprises a chelator, more preferably the conjugate group comprises a chelator selected from the group consisting of DOTA and FFmacropa, optionally the conjugate group comprises DOTA.

[0108] Where the radioisotope is211At, the conjugate group optionally comprises closo-decaborate(2-).

[0109] Where the radioisotope is212Pb, the conjugate group preferably comprises a chelator, more preferably a chelator comprising DOTA or DOTAM, optionally the conjugate group comprises DOTA.Radiolabeled antibody conjugates properties

[0110] The antibody conjugate may be conjugated with different ratios of the conjugate group comprising a radioisotope or a moiety capable of forming a stable reaction product with a radioisotope (e.g., a chelator forming a complex). For example, molar ratios of conjugate group to antibody of from about 0.1:1 to about 10:1 may be employed (e.g., to provide a molar ratio of radioisotope to antibody of from about 0.1:1 to about 10:1). Preferably, the ratio of conjugate group to antibody is from about 0.1:1 to about 3 : 1 (e.g., to provide a molar ratio of radioisotope to antibody of from about 0.1:1 to about 3:1), more preferably from about 0.5:1 to about 2:1 (e.g., to provide a molar ratio of radioisotope to antibody of from about 0.5:1 to about 2:1), even more preferably about 1:1 (e.g., to provide a molar ratio of radioisotope to antibody of about 1 : 1).[oni] Where the radiolabel in the radiolabeled antibody conjugate is attached via a chelator, the chelator preferably has an occupancy level of about 70-100%, more preferably about 80-100%, even more preferably about 90-100%. This ensures all antibody conjugate may be active and that the molar ratios of conjugate group to antibody may be equivalent to the molar ratio of radioisotope to antibody.

[0112] To ensure the radioactivity targets the tumor in vivo, the radiolabeled antibody conjugate preferably has a high radiochemical purity (i.e., a high proportion of the total radioactivity in the sample may be present as the radiolabeled antibody conjugate rather than free in solution). More preferably, the radiolabeled antibody conjugate has a radiochemical purity of about 90-100%, even more preferably about 95-100%.Pharmaceutical compositions

[0113] A radiolabeled antibody conjugate as described herein may be in a pure form, or may be formulated into a pharmaceutical composition.

[0114] The pharmaceutical composition may comprise one or more of pharmaceutically acceptable carrier(s), diluent(s), and / or excipient(s).

[0115] The pharmaceutically acceptable carrier may be conventional (e.g., as described in Remington, The Science and Practice of Pharmacy, 22nd Edition, Loyd V, ed., Pharmaceutical Press, 2012). In general, the nature of the carrier depends on the mode of administration. For instance, parenteral formulations typically comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, such as water for injection,physiological saline, balanced salt solutions or the like as a vehicle. Pharmaceutical compositions can additionally include minor amounts of non-toxic auxiliary substances for stability (e.g., one or more buffering agent).

[0116] The carrier may be sterile and / or suspended or otherwise contained in a unit dosage form including one or more measured doses of the composition suitable for administration to a subject of an effective amount of a radiolabeled antibody conjugate described herein. Medications for use in therapy may also be included in such embodiments. The unit dosage form may be in a sealed vial that contains sterile contents or a syringe for injection into a subject, lyophilized for subsequent solubilization and administration. The pharmaceutical composition may be provided in a solid or controlled release dosage form.

[0117] A pharmaceutical composition typically contains an “effective” or “therapeutically effective” amount, as used interchangeably herein, of a radiolabeled antibody conjugate described herein. The dosages and dosage regimen to achieve the desired therapeutic result may depend on the means of administration. They may vary according to factors including, but not limited to, the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of a radiolabeled antibody conjugate described herein are outweighed by the therapeutically beneficial effects.Methods of detecting cancer cells

[0118] The described radiolabeled antibody conjugate can be used for the in vivo detection of cancer. For example, following administration of the radiolabeled antibody conjugate to a subject, the conjugate may specifically bind to any cancerous cells that express EGFRvIII or EGFR-hi via the antibody component. The radioisotope conjugated to the antibody component emits radiation. Detection of radiation within the subject following administration of the radiolabeled antibody conjugate (e.g., after 1 to 3 days) may indicate the presence of cancer cells.

[0119] Accordingly, in one aspect, there is provided a method of detecting cancer cells in a subject having cancer, wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi), the method comprising: (a) administering an effective amount of a radiolabeled antibody conjugate to the subject; and (b) detecting any radiation retained within the subject; wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope;wherein the radioisotope is selected from the group consisting of64Cu,89Zr,161Tb,177Lu,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) and optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.

[0120] The method may further comprise the steps of: (c) producing an image showing where the radiation is retained within the subject; (d) optionally diagnosing the subject as having cancer; and / or (e) optionally treating the subject if cancer cells are confirmed to be present.

[0121] Following administration, the radiolabeled antibody conjugate may initially distribute throughout the body, including to non-target tissues. Over time, unbound or non-specifically bound conjugates will clear from circulation. Additionally, the body's metabolism and excretion processes (e.g., via the kidneys, liver, or intestines) help to remove excess conjugate. Therefore, leaving a period of hours or days between administration and imaging helps to improve background noise, improve imaging contrast, and ensure that the signal comes predominantly from the targeted tissue rather than normal physiological distribution

[0122] Accordingly, the time period between administering the radiolabeled antibody conjugate to the subject and detecting any radiation retained within the subject may be 1 to 5 days. Preferably, the time period may be 2 to 3 days.

[0123] The cancer cells may be detected using positron emission tomography (PET). To further enhance detection of the pre-cancerous cells or cancer cells, PET may be coupled with computed tomography (CT). Alternatively, the cancer cells may be detected using single-photon emission computed tomography (SPECT).

[0124] EGFRvIII and EGFR-hi expression have been implicated in various cancers. In particular, EGFRvIII and EGFR-hi expression can occur in cancers of the epithelium and the central nervous system.

[0125] Accordingly, the cancer detected by the methods disclosed herein may be derived from epithelial cells. In particular, the cancer may be carcinoma. Alternatively, the cancer may be derived from glial cells and the cancer may be GBM.

[0126] The cancer may be selected from the group consisting of GBM, non-small cell lung carcinoma (NSCLC), colorectal cancer (e.g., mCRC), breast cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, esophageal cancer, skin cancer, and head and neck cancer. In particular, the cancer may be GBM.

[0127] Intravenous infusion allows the radiolabeled antibody conjugate to enter the bloodstream, ensuring rapid circulation and distribution to target cells. Accordingly, the radiolabeled antibody conjugate may be administered via intravenous infusion.

[0128] Those skilled in the art will be aware that the methods of detecting cancer cells described directly above may be combined with radiolabeled antibody conjugates described above. Furthermore, these methods may be claimed in alternative forms.

[0129] Therefore, in a related aspect, there is provided a radiolabeled antibody conjugate for use in a method of detecting cancer cells in a subject having cancer, wherein the cancer cells express EGFRvIII or overexpress EGFR, the use comprising: (a) administering an effective amount of a radiolabeled antibody conjugate to the subject; and (b) detecting any radiation retained within the subject; wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of64Cu,89Zr,161Tb,177Lu,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) and optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.

[0130] In another related aspect, there is provided use of a radiolabeled antibody conjugate in the manufacture of a medicament for detecting cancer cells in a subject having cancer; wherein the cancer cells express EGFRvIII or overexpress EGFR; wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of64Cu,89Zr,161Tb,177Lu,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) and optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.Methods of treating cancer

[0131] The described radiolabeled antibody conjugate can be used for the in vivo treatment of cancer. For example, following administration of the radiolabeled antibody conjugate to a subject, the conjugate may specifically bind to any cancer cells that express EGFRvIII or EGFR-hi via the antibody component. The radiation emitted by the radioisotope component may then be absorbed by the cancer cell to which the conjugate is bound, thereby killing the cell.

[0132] Accordingly, in another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of a radiolabeled antibody conjugate to the subject; wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi); wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of67Ga, 161Tb,177LU,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) and optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.

[0133] The method may further comprise the steps of: (a) repeating the administration of the therapeutically effective amount of the radiolabeled antibody conjugate to the subject one or more times; and (b) detecting any remaining cancer cells using the methods described herein.

[0134] The cancer may be derived from epithelial cells. In particular, the cancer may be carcinoma. Alternatively, the cancer may be derived from glia cells and the cancer may be GBM.

[0135] The cancer may be selected from the group consisting of GBM, NSCLC, colorectal cancer (e.g., mCRC), breast cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, esophageal cancer, skin cancer, and head and neck cancer. In particular, the cancer may be GBM. Non-metastatic colorectal cancer may be treated by surgery (i.e., removal of the localized tumor) but may be detected and / or treated using the described methods.

[0136] A therapeutically effective amount can be administered in a dosing regimen that may comprise multiple unit doses. A therapeutically effective amount (and / or anappropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, or combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and / or unit dose) for any particular subject may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific therapeutic molecule employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0137] It will be understood that the amount of the compound administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be detected, located or treated, the chosen route of administration, the selected compound or compounds administered, the age, weight, and response of the individual subject, and the severity of the subject’s symptoms. See, for example, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001.

[0138] Clinical efficacy of the therapeutic treatments described herein can be determined by measuring the clinical benefit rate (CBR) as in Schwartz Eur J Cancer. 2016 Jul; 62: 132-137. The clinical benefit rate is measured by determining the sum of the percentage of subjects who are in complete remission (CR), the number of subjects who are in partial remission (PR) and the number of subjects having stable disease (SD) at a time point at least 6 months out from the end of therapy. The CBR for a particular therapeutic regimen may be at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.

[0139] Intravenous infusion allows the radiolabeled antibody conjugate to enter the bloodstream, ensuring rapid circulation and distribution to target cells. Intratumor infusion involves the administration of the radiolabeled antibody conjugate directly into a tumor, enabling delivery of radiation directly to cancer cells. Accordingly, the radiolabeled antibody conjugate may be administered to the subject via intravenous or intratumor infusion.

[0140] Those skilled in the art will be aware that the methods of treating cancer described directly above may be combined with radiolabeled antibody conjugates described above. Furthermore, these methods may be claimed in alternative forms.

[0141] Therefore, in one related aspect, there is provided a radiolabeled antibody conjugate for use in a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the radiolabeled antibody conjugate to the subject; wherein the cancer cells express EGFRvIII or overexpress EGFR; wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of67Ga,161Tb,177LU,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) and optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.

[0142] In another related aspect, there is provided use of a radiolabeled antibody conjugate in the manufacture of a medicament for the treatment of cancer in a subject in need thereof; wherein a therapeutically effective amount of the radiolabeled antibody conjugate is administered to the subject; wherein the cancer cells express EGFRvIII or overexpress EGFR; wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope; wherein the radioisotope is selected from the group consisting of67Ga,161Tb,177LU,211At,212Pb and225Ac; wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1) and optionally wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.Combination therapy

[0143] It may be advantageous to combine treatment with a radiolabeled antibody conjugate with one or more additional therapies. For example, therapies including chemotherapy, conventional radiotherapy, antibody treatment and surgery have been shown to provide a therapeutic benefit in GBM.

[0144] Accordingly, the subject may be treated with one or more further therapies in addition to treatment with a radiolabeled antibody conjugate. These additional therapies may include chemotherapy, radiotherapy, antibody treatment and / or surgery. Alternatively, the subject may be treatment naive.

[0145] Treatments given in combination with a radiolabeled antibody conjugate may be given before, concurrently to or after treatment with a radiolabeled antibody conjugate,e.g., to increase or maintain a treatment response to the treatment with the radiolabeled antibody conjugate.Radiotheranostic methods

[0146] Radiothearnostics refers to the use of diagnostic radioisotopes (e.g.,64Cu,89Zr,161Tb,177LU,211At,212Pb and225Ac) to image biologic phenomena in vivo by assessing expression of specific biomarkers, followed by use of the same or a very similar targeting ligand to deliver therapeutic quantities of radiotherapeutic isotopes (e.g.,67Ga,161Tb,177Lu,211At,212Pb and225Ac) to target-expressing tissues. In the case of161Tb,177Lu,211At,212Pb and225Ac, the same radioisotope may be used for diagnosis and therapy, optionally in different amounts (e.g., a higher specific radioactivity for therapy compared to diagnosis).

[0147] A typical radiotheranostic has the structure of ligand-linker-receptor. The targeting ligand, e.g., antibody, serves as an anchor and acts to locally enrich the radioisotope (diagnostic or therapeutic) in, or near, the cancer cells. When antibodies are used as targeting ligands, the concept of radiotheranostics is similar to that of antibody-drug conjugates (ADCs) since both involve an antibody linked to a cytotoxic payload. However, in the case of radiotheranostics, the payload is a therapeutic radioisotope that cannot be administered systemically on its own.

[0148] Following visualization of target-expressing tissues, e.g. using PET or SPECT, the diagnostic radioisotope is replaced with a therapeutic radioisotope such as177Lu (which emits P" radiation) or225Ac (which emits a" radiation). When delivered via targeting ligands, these radiotherapeutic isotopes elicit highly specific cytotoxic radiation to target expressing tissues. For example, the mean penetration range of P-particles emitted by177Lu in soft tissue is 670 pm, enabling the delivery of cytotoxic radiation to small volumes and ensuring minimal damage to off-target, heathy surrounding tissues.

[0149] Accordingly, in another aspect, there is provided a theranostic method for detecting and treating cancer in a subject, wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpresses EGFR (EGFR-hi). The theranostic method comprises a combination of the methods of detecting and treating cancer described herein.

[0150] The cancer may be derived from epithelial cells. In particular, the cancer may be carcinoma. Alternatively, the cancer may be derived from glial cells and the cancer may be GBM.

[0151] The cancer may be selected from the group consisting of GBM, NSCLC, colorectal cancer (e.g., mCRC), breast cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, esophageal cancer, skin cancer, and head and neck cancer. In particular, the cancer may be GBM.

[0152] The radiolabeled antibody conjugate may be administered via intravenous infusion. Where the cancer is a solid tumor, the radiolabeled antibody conjugate may be administered via intratumoral injection. Where the tumor is located within the central nervous system, such as GBM, the radiolabeled antibody conjugate may be administered via intrathecal infusion or intracerebroventricular infusion.EXAMPLES

[0153] Although methods and materials similar or equivalent to those described herein can be used, suitable methods and materials are described below. The following examples are for illustrative purposes only and are not intended to be limiting.Example 1. Production of the radiolabeled antibody conjugate89Zr-DFO*-40H3

[0154] This example illustrates that an EGFR antibody described herein (e.g., 40H3) can be conjugated with a reagent (e.g., DFO*-p-phenyl-NCS) comprising a reactive group (e.g., an isothiocyanate) and a conjugate group comprising a moiety capable of forming a stable reaction product with a radioisotope (e.g., the DFO* chelator) to form an antibody conjugate. This antibody conjugate (e.g., DFO*-40H3) can then chelate a radioisotope (e.g.,89Zr) to form a stable and active radiolabeled antibody conjugate (e.g.,89Zr-DFO*-40H3).

[0155] Conjugate production: 1 pL of DFO*-p -phenyl -NCS (Advanced Biochemical Compounds GmbH; 5, l l,l6,22-Tetraazahexacosanediamide, / ' '-[5-(acetylhydroxyamino)pentyl]-7V26,5,16-trihydroxy-7V26-[5-[[[(4-isothiocyanatophenyl)amino]thioxomethyl]amino]pentyl]-4, 12, 15,23-tetraoxo-; WO 2015 / 140212 Al; CAS number 1810009-29-0) in DMSO (14.7 mg / mL, 2.1 molar excess) was added to 1 mg of 40H3 antibody (1.092 mg / mL; National Cancer Institute; Ho E., et al. Antib. Then 2, 2019, 88-98) in PBS (930 pL). The reaction mixture pH measured -7-7.5 using litmus paper, and the mixture was incubated overnight at 27 °C. The conjugated protein was purified on a pre-conditioned PD-10 column, using lx PBS as buffer. The protein was eluted in three 1 mL fractions and one 0.5 mL fraction of lx PBS. Following analysis on the Nanodrop, fractions with the highest amount of conjugated protein were combined andredistributed among four 1.5 mL Eppendorf tubes and a small aliquot was removed for analysis on size exclusion HPLC. Volumes in each Eppendorf were 500, 500, 500 and 450 pL of 0.42 mg / mLDFO*-40H3. The conjugated protein was 91.41% pure via HPLC analysis at 280 nm, with a single impurity peak present in the starting material, suggestive of an aggregate. The ratio of conjugate: antibody was about 1:1. The product remained stable in terms of aggregation state and radiochemical purity when stored overnight at ambient temperature. The sample was stored at -80 °C until use. Conjugation of 40H3 at higher pH values of 8.0 to 8.5 resulted in higher levels of conjugation (2- to 5-fold higher conjugate: antibody ratios) that increased when the molar excess of the conjugate was raised from ~2 to ~4. However, some antibody degradation was observed by HPLC after 72 hours storage for these samples suggesting that stability may be less optimal at higher pH and / or higher molar excess of conjugate.

[0156] Radiolabeling of the antibody conjugate: Zr-89 (0.864 mCi, 7.5 pL) was transferred to a 1.5 mL microcentrifuge tube, and the pH was raised to 7 with the addition of 100 pL of 0.5 M HEPES buffer (pH 7.2). DFO*-40H3 protein (190 pg in 450 pL of PBS) was added and the reaction mixture was incubated at 30°C for 1 hour on a thermomixer. A small aliquot was removed for analysis of the crude reaction mixture, and the bulk was purified on a pre-conditioned PD MiniTrap column. The reaction mixture was loaded, and the radiolabeled antibody conjugate was eluted in two 0.5 mL fractions using lx PBS. The radiolabeled antibody conjugate eluted in the first fraction, and measured 203 pCi (23.4% radiochemical yield, 82.16% radiochemical purity with the remainder being aggregated antibody present in the starting material). Final QC confirmed purity of 91% with a small amount of aggregated antibody.

[0157] Radio-TLC Method: iTLC-SA chromatography paper (Agilent, A120B12) was used. Mobile phase (0.1 M citrate solution) was prepared by dissolving 1.94 g of sodium citrate tribasic dehydrate, 0.655 g of citric acid (monohydrate) and 0.09 g of sodium chloride in 100 mL of HyClone water. The iTLC-SA paper was spotted at 10 mm from the bottom with 1.0 pL of the radiolabeled product, and the TLC was developed by letting the mobile phase travel 70 mm from the origin. Radiolabeled product remained at the origin; free Zr-89 radiometal traveled with the mobile phase. The developed TLC papers were scanned using AR-2000 radio-TLC scanner (Eckert & Ziegler).

[0158] Analytical HPLC Method and Calibration Curve: A serial dilution was performed on the original 40H3 antibody with IxPBS, and the dilution series were injected on the analytical HPLC to generate the calibration curve.Analytical HPLC method:Instrument: Waters Acquity ArcPath: 2Wash / Purge solvent: 50% Acetonitrile in WaterInjection Volume: lO uLColumn: Waters XBridge Premier Protein SEC 250A 2.5 um 4.6 x 150 mm - S / N186009959Mobile Phase: lx PBS (isocratic)Flow rate: 0.25 mL / min

[0159] Quality control and stability testing: Specific activity was determined by integrating the HPLC UV peak (280 nm) of the radiolabeled product and comparing this against a calibration curve. Total protein content was 0.938 mg / mL for89Zr-DFO*-40H3 with 0.99 mCi / mL. Based on this, the specific activity was calculated to be 1.06 mCi / mg. The pH was determined as 7.0 (Hydrion strip, pH 4.5 - 7.5). The radiochemical purity was 95.8% (TLC) or 91% (HPLC with the major impurity being aggregated antibody). The product was stored at ambient temperature overnight, and an aliquot was analyzed via TLC and HPLC 24 hours post synthesis. The radiochemical purity remained constant.

[0160] Variation in the procedure: The conjugation conditions were varied such that 2 to 4 equivalents of DFO*-p-phenyl-NCS were added to 40H3 at a pH of 7 to 8.5.Example 2. Production of the radiolabeled antibody conjugate177Lu-DOTA-40H3

[0161] This example illustrates that an EGFR antibody described herein (e.g., 40H3) can be conjugated with a reagent (e.g., DOTA-NCS) comprising a reactive group (e.g., an isothiocyanate) and a conjugate group comprising a moiety capable of forming a stable reaction product with a radioisotope (e.g., the DOTA chelator) to form an antibody conjugate. This antibody conjugate (e.g., DOTA-40H3) can then chelate a radioisotope (e.g.,177Lu) to form a stable and active radiolabeled antibody conjugate (e.g.,177Lu-DOTA-40H3).

[0162] Conjugate production: 3.4 pL of DOTA-NCS (CAS number 1020407-41-3; S-2-(4-Isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane tetraacetic acid) in DMSO (4mg / mL, 4 molar excess) was added to 0.678 mg of 40H3 antibody (1.195 mg / mL; National Cancer Institute; Ho E., et al. Antib. Then 2, 2019, 88-98) in PBS (567 pL). The reaction mixture pH measured -7-7.5 using litmus paper, and the mixture was incubated overnight at 27°C. The conjugated protein was purified on a pre-conditioned PD-10 column, using lx PBS as buffer. The protein was eluted in three 1 mL fractions and one 0.5 mL fraction of lx PBS. Following analysis on the Nanodrop, fractions with the highest amount of conjugated protein were combined and concentrated on a 50 kDaMWCO centrifugal filter. The resulting protein solution measured 1.758 mg / mL, 280 pL. A small aliquot (4 pL) was removed for HPLC analysis. The sample was stored at -80 °C until use.

[0163] Radiolabeling of the antibody conjugate: In a 1.5 mL Eppendorf tube containing 200 pg of protein (114 pL in PBS), 100 pL of 0.1 M NaOAc and 97 pL of 0.04 M HC1 was added, to bring the pH down to 5.0. To this solution, 2.79 mCi of Lu-177 (3 pL in 0.04M HC1) was added. The reaction mixture was incubated at 38 °C for 1 hour. A small aliquot was removed for analysis of the crude reaction mixture, and the bulk was purified on a pre-conditioned PD-10 column. The reaction mixture was loaded, and the radiolabeled product was eluted in six 0.5 mL fractions using lx PBS. Product eluted in fractions 2 and 3, and measured 624 pCi in 1 mL. Residual impurities were observed during both TLC and HPLC analyses, and additional purification was performed using 5 kDa MWCO filters. The product was transferred to the filtration column, diluted with 4 mL of lx PBS and centrifuged at 6000 rpm for 30 minutes. After centrifugation, the remaining sample measured 372 pCi in 800 pL.

[0164] Quality control and stability testing: Quality control was performed as described for89Zr-DFO*-40H3. Specific activity was determined by integrating the HPLC UV peak (280 nm) of the radiolabeled product and comparing this against a calibration curve. Total protein content was 0.088 mg / mL for177Lu-DOTA-40H3 with 0.465 mCi / mL. Based on this, the specific activity was calculated to be 5.3 mCi / mg. The pH was determined as 7.0 (Hydrion strip, pH 4.5 - 7.5). The radiochemical purity was 72% (TLC with the major impurity being free metal) or 94.6% (HPLC).

[0165] This example illustrates that an EGFR antibody that specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (e.g., 40H3; first disclosed in WO 2021 / 003297 Al) retains EGFR binding to tumor cells overexpressingEGFR (e.g., A431, and MDA-MB-468 cells) when conjugated to a chelator (e.g., DFO* or DOTA) to form an antibody conjugate (e.g., DFO*-40H3 or DOTA-40H3).

[0166] EGFR binding assays: A431 (ATCC CRL-1555), MDA-MB-468 cells (ATCC HTB-132) and U87 MG cells (ATCC HTB-14) were purchased from ATCC. A431 cells overexpress EGFR (Lin C., et al. Science 224, 1984, 843-848) as do MDA-MB-468 cells (Filmus J., et al. Biochem. Biophys. Res. Commun. 128, 1985, 898-905).

[0167] DFO*-40H3 and 40H3-DOTA were prepared using the procedure described above with a 2.1 molar excess of the reagent DFO*-p-phenyl-NCS or DOTA-NCS.

[0168] Cells grown in 2D culture were washed with PBS and lysed with RIPA lysis buffer (Boston Bioproducts; BP-1155) mixed with lx phosphatase inhibitor cocktail 2 (Sigma; P5726), lx phosphatase inhibitor cocktail 3 (Sigma; P0044), and EDTA-free protease inhibitor (Roche; 11836170). The lysates were centrifuged at 10,000 rpm for 15 minutes at 4 °C, and the supernatant containing total protein was extracted and stored at -80°C. Protein concentration was quantified using the Pierce BCA protein assay kit (Life Technologies; 23225), and 100 ng / mL of protein was extracted for each cell line. Protein samples were mixed with lx Tris-Glycine SDS sample buffer and heat-shocked at 95 °C for 2 minutes (Thermo Fisher Scientific; LC2676). The denatured protein samples were loaded onto an 8-16% Tris-Glycine mini protein gel (Thermo Fisher Scientific; XP08162) and run with lx Tris-Glycine-SDS buffer (Bio-Rad; 1610732). The gels were transferred to nitrocellulose membranes (Bio-Rad) using the Trans-Blot Turbo RTA Mini 0.2 pm nitrocellulose transfer kit (Bio-Rad; 1704270). The membranes were blocked with Intercept TBS blocking buffer (Thermo Fisher Scientific; NC1660550) and incubated overnight with primary antibodies: rabbit anti-epidermal growth factor receptor (EGFR) (1:1000, Cell Signaling Technology; 2232S), 40H3, DFO*-40H3 and 40H3-DOTA. After incubation, the membranes were washed three times with lx Tris-buffered saline (Boston BioProducts; BM301) and Tween-20 (Sigma; P1379). The membranes were then incubated with secondary antibodies: goat anti-rabbit IgG (heavy- and light-chain) conjugated with AlexaFluor Plus 800 (Life Technologies; A32735) and goat anti-mouse IgG (heavy- and light-chain) conjugated with AlexaFluor Plus 680 (Life Technologies; A21058). Using an Odyssey infrared imaging system, the membranes were imaged and analyzed for respective bands using Image Studio.

[0169] FIG. 1 EGFR demonstrates that 2232S binds to EGFR from U87 MG cells that do not overexpress EGFR as well as A431 and MDA-MB-468 cells that do overexpress EGFR. The relative expression of the EGFR protein can be assessed by the intensity of the bands. FIG. 1 40H3 demonstrates that 40H3 selectively binds to A431 and MDA-MB-468 cells that overexpress EGFR. FIG. 1 40H3-DOTA and FIG. 1 40H3-DFO* demonstrate that conjugation of the antibody did not impact either the binding to cellular EGFR or the selectivity for cells overexpressing EGFR.

[0170] It is known from WO 2025 / 014896 that 40H3 and its humanized version A10 bind to cells that overexpressed EGFR or cells with transfected EGFRvIII. Given that the 287-302 amino acid loop is typically inaccessible in the wild-type receptor, conditionally exposed when EGFR is overexpressed and fully exposed in EGFRvIII, the data described above suggest that 40H3-DOTA and 40H3-DFO* may display similar selectivity for EGFRvIII-expressing cells.Example 4. Methods for detecting EGFR binding activity of DFO*-40H3 in ELISA and cellular models

[0171] Published methods may be used to confirm that an EGFR antibody conjugate (e.g., DFO*-40H3) retains EGFR binding activity to both the EGFR 287-302 loop peptide and EGFRvIII. They may be used to illustrate that antibody conjugation at lower pH values (e.g., 7.5) and / or lower conjugate:antibody ratios (e.g., 1:1) retain binding to the epitope in plate-based assays and / or to MDA-MB-468 cells that overexpress EGFR. Alternatively, binding may be confirmed as described in Example 3 and / or Example 5.

[0172] EGFR binding assays: Plate binding assays may be performed as described in WO 2021 / 003297 Al and / or WO 2025 / 014896 Al. For example, binding of 40H3 or the DFO*-40H3 antibody conjugate to the 287-302 peptide (CGADSYEMEEDGVRKC; SEQ ID NO: 2) and EGFRvIII (SEQ ID NO: 1 with the deletion of exons 2-7) may be performed as described previously (WO 2021 / 003297 Al; Ho E., el al. Antib. Then 2, 2019, 88-98; WO 2025 / 014896 Al). Briefly, ELISA binding may be detected for C-terminally His-tagged EGFRvIII or EGFR287-302 peptide bound to nickel-coated 96-well plates. Antibodies may be included (e.g., 1 to 300 ng / mL or from 0.01 nM to saturated binding) for about 1 hour before detection with secondary antibody (e.g., peroxidase-conjugated donkey anti-mouse IgG (HG+L) (Cat# 715-035-150; Jackson ImmunoResearch, ME, USA) and may be developed with a 3,3’,5,5’-tetramethylbenzidine substrate solution (Thermo Fisher Scientific).

[0173] MDA-MB-468 cell binding: Cell binding assays may be performed as described in WO 2021 / 003297 Al; Ho E., et al. Antib. Then 2, 2019, 88-98; and / or WO 2025 / 014896 Al. For example, MDA-MB-468 cells may be cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, NY, USA), 2 mM GlutaMAX (Thermo Fisher Scientific), Minimum Essential Media (MEM) non-essential amino acid (Thermo Fisher Scientific) under sterile conditions (e.g., 100 U / ml Penicillin and 100 pg / ml streptomycin).

[0174] Flow cytometry may be performed as described previously (WO 2021 / 003297 Al; Ho E., et al. Antib. Then 2, 2019, 88-98; WO 2025 / 014896 Al). Briefly, 40H3 or the DFO*-40H3 antibody conjugate (e.g., concentrations ranging from 10 pg / ml to 0.1 pg / ml) may be incubated with suspended cells (e.g., 1 to 2.5 xlO cells per tube) in FACS buffer at 4 °C for 1 hour. Bound antibodies may be detected with R-Phycoerythrin AffiniPure F(ab') Fragment Goat Anti -Human IgG (H+L) (Cat# 109116088; Jackson ImmunoResearch, ME, USA). Antibody binding may be characterized with the S A3800 Spectral Analyzer (Sony Biotechnology, San Jose, CA, USA) and the data may be analyzed.

[0175] Antibody conjugates that may be tested: Different conjugation chemistries (pH and molar equivalents of DFO*) were explored in order to label 40H3 with DFO* in a manner that did not interfere with the epitope and retained EGFR binding. It was demonstrated that conjugation of DFO* to 40H3 at either two or four molar equivalents at a lower pH (pH 7.5) ensured that DFO*-40H3 retained EGFR binding (consistent with the immunoblotting data in Example 3 and the in vivo imaging data in Example 5). These samples displayed conjugate: antibody ratios of about 1:1 and were stable in solution. In contrast, higher pH values of 8.0 or 8.5 resulted in higher conjugate: antibody ratios and minor aggregation in solution. Without wishing to be bound by theory, it appears that conjugation to the 40H3 antibody at pH values 8.0 or higher results in higher conjugate: antibody ratios on lysine residues and / or results in destabilization of the antibody. For this reason, it may be preferred that the conjugation reaction is performed at a pH between 7.0 and 7.5 where the conjugation occurs in a facile manner at relatively low conjugate: antibody ratios (e.g., 1:1) and the 40H3 antibody and antibody conjugates are confirmed to retain activity (e.g., immunoblotting colocalization with EGFR and / or in vivo imaging). The binding assays described above may be used to qualify binding or quantify the dissociation constants of these antibody conjugates, with or without a radiolabel attached, to predict binding in vivo.Example 5. In vivo detection of EGFR-over expressing tumors

[0176] This example illustrates that a radiolabeled EGFR antibody conjugate (e.g.,89Zr-DFO*-40H3) is capable of selectively binding to EGFR-overexpressing tumors in vivo.

[0177] A431 and U87 MG cells were cultured in DMEM and EMEM (Coming). Both media were supplemented with 10% fetal bovine serum (Gibco). Cells were grown at 37°C with 5% CO2. Five million A431 or U87 MG cells were suspended in a 1:1 (v / v) mixture of media and Matrigel (Corning).

[0178] Female Crl:NU(NCr)-Foxnlnu mice were purchased from Charles River. Xenograft mouse models were generated via subcutaneous injection of 50 pL of the A431 or U87 MG cell suspension into the right shoulder of the mice (seven per group). Tumor volumes and weights were measured weekly. Tumor volumes were calculated using the equation 0.5 x (L x W2), where L = tumor length and W = tumor width. PET imaging was performed once tumors reached approximately 80 mm3.

[0179] 89Zr-DFO*-40H3 PET imaging was carried out on a dedicated small animal PET / CT scanner (Siemens Multimodality Inveon, Siemens Medical Solutions USA) at the Lurie Family Imaging Center (DFCI). The mice were housed five to a cage with ad libitum access to food and water in 20°C ambient temperature, 40-50% humidity and a 12h lightdark cycle. The mice received a bolus intravenous injection (via the lateral tail vein) of89Zr-DFO*-40H3 (~3.7MBq / ~100 pCi) under anesthesia (~3% isoflurane / medical air inhalation). Warming was used to maintain a healthy core body temperature of the mice during periods of unconsciousness. The mice were rehoused prior to PET / CT imaging time points at 24h, 48h and 72h post-injection. The mice were anesthetized using ~3% sevoflurane / medical air inhalation prior to and throughout the duration of the scans.

[0180] A static PET emission scan was acquired in list mode format over 10 minutes, followed by a low-dose CT acquisition (80kVp, 0.5 mA) for anatomical reference and to provide guidance for the delineation of selected tissues volume of interest (VOI). The acquired PET data were then sorted into 0.5 mm sinogram bins and one time frame for image reconstruction using FORE / 3D-OSEM-MAP (16 subsets, two iterations). The reconstructed PET / CT images were analyzed with the Siemens Inveon Research Workplace software (IRW v.4.2, Siemens Medical Solutions USA). The radioactivity retention within the selected tissue was obtained from mean voxel intensity values within the VOI and then converted to MBq ml-1using the calibration factor determined for the Inveon PET system. These valueswere then divided by the administered activity in MBq and animal body weight to obtain an image VOI-derived standard uptake value (SUV). The mean and maximum SUV values (SUVmean and SUVmax, respectively) within a VOI were used as quantitative imaging metrics.

[0181] As can be seen in FIG. 2A, the A341 tumors were clearly detectable by PET / CT imaging. Excellent selectivity of89Zr-DFO*-40H3 for the target A341 tumors, compared to control U87 MG tumors, was observed from 24 hours through to at least 72 hours (FIG. 2B). By 72 hours, there was clear selectivity for the target tumor over other tissues, including the liver, kidneys, bladder, heart, muscle and lungs (FIG. 2B).

[0182] This example demonstrates that radiolabeled EGFR antibody conjugates are capable of selectively binding to EGFR-overexpressing tumors in vivo. This confirms the utility of radiolabeled EGFR antibody conjugates as in vivo probes for detecting and potentially treating EGFR-hi or EGFRvIII expressing cancers in which the 287-302 amino acid loop region is exposed.

[0183] Based on the properties of89Zr-DFO*-40H3 and177Lu-DOTA-40H3, it is predicted that radiolabeled antibody conjugates for detecting cancer cells may be produced using further radioisotopes such as64Cu,161Tb,211At,212Pb and225Ac. Furthermore, it is predicted that radiolabeled antibody conjugates for treating cancer may be produced using further radioisotopes such as67Ga,161Tb,211At,212Pb and225Ac. Furthermore, it is predicted that such radiolabeled antibody conjugates may be used in combination in theranostic methods for detecting and treating cancer.

[0184] It should be understood that the particular embodiments described herein are given by way of illustration only, not limitation. Other features, objects, and advantages are apparent from the above detailed description, drawings and examples. Various changes and modifications will be apparent to those skilled in the art.

[0185] All patents, patent publications, and non-patent publications referenced herein are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.ASPECTSA method of detecting cancer cells in a subject having cancer, wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi), the method comprising:a) administering an effective amount of a radiolabeled antibody conjugate to the subject; andb) detecting any radiation retained within the subject;wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope;wherein the radioisotope is selected from the group consisting of64Cu,89Zr,161Tb,177LU,211At,212Pb and225Ac;wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1); and wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.The method of aspect 1, further comprising the steps of:c) producing an image showing where the radiation is detected within the subject;d) optionally diagnosing the subject as having cancer; ande) optionally treating the subject if cancer cells are confirmed to be present.The method of aspect 1 or aspect 2, wherein the radioisotope is selected from the group consisting of64Cu,89Zr,177Lu and212Pb, optionally wherein the radioisotope is89Zr.The method of any one of the preceding aspects, wherein the time period between administering the radiolabeled antibody conjugate to the subject and detecting any radiation retained within the subject is 1 to 5 days, preferably 2 to 3 days.The method of any one of the preceding aspects, wherein the cancer cells are detected using single-photon emission computed tomography (SPECT) or positronemission tomography (PET), optionally wherein PET is coupled with computed tomography (CT).A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of a radiolabeled antibody conjugate to the subject;wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi);wherein the radiolabeled antibody conjugate comprises a monoclonal antibody and a radioisotope;wherein the radioisotope is selected from the group consisting of67Ga,161Tb,177LU,211At,212Pb and225Ac;wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human EGFR (SEQ ID NO: 1); and wherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.The method of aspect 6, wherein the radioisotope is selected from the group consisting of161Tb,177Lu,212Pb and225Ac, optionally wherein the radioisotope is177LU.The method of aspect 6 or aspect 7, further comprising:a) repeating the administration of the therapeutically effective amount of the radiolabeled antibody conjugate to the subject one or more times;b) detecting any remaining cancer cells using the method of any one of aspects 1-5.A theranostic method for detecting and treating cancer in a subject, wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi), the method comprising the methods of aspect 1 and aspect 6 in any order.The method of any one of the preceding aspects, wherein the radiolabeled antibody conjugate comprises a molar ratio of the radioisotope to antibody of from about0.1:1 to about 3:1, preferably from about 0.5:1 to about 2:1, more preferably about 1:1.The method of any one of the preceding aspects, wherein the monoclonal antibody comprises:a) a heavy chain complementarity determining region (HCDR) 1 comprising an amino acid sequence of GFSLTNYG (SEQ ID NO: 3), an HCDR2 comprising an amino acid sequence of MWRGGGT (SEQ ID NO: 4), an HCDR3 comprising an amino acid sequence of ARKGVGMGLGY (SEQ ID NO: 5); andb) a light chain complementarity determining region (LCDR) 1 comprising an amino acid sequence of QTIGTW (SEQ ID NO: 6), an LCDR2 comprising an amino acid sequence of GAT (SEQ ID NO: 7), and an LCDR3 comprising an amino acid sequence of QQLYSNPYT (SEQ ID NO: 8).The method of any one of the preceding aspects, wherein the monoclonal antibody comprises:a) an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence that is at least about 85% identical to, or identical to, QVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLG MMWRGGGTDYNAAFISRLTITKDTSKSQVVFTMTNMDPVDTATYYCA RKGVGMGLGYWGQGTLVTVSS (SEQ ID NO: 9); andb) an immunoglobulin light chain variable (VL) region comprising an amino acid sequence that is at least about 85% identical to, or identical to, DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIY GATNLADGVPSRFSGSGSGTKFTLTISSLQPEDFATYYCQQLYSNPYTFG GGTKLEIK (SEQ ID NO: 10).The method of any one of the preceding aspects, wherein the monoclonal antibody specifically binds to a peptide comprising residues 287-302 of human EGFR (SEQ ID NO: 1) with a dissociation constant (KD) of between about 0.06 nM and about 60 nM, preferably between about 0.6 nM to about 6 nM, as determined a plate-based binding assay, such as an ELISA.The method of any one of the preceding aspects, wherein the monoclonal antibody is human or humanized.The method of any one of the preceding aspects, wherein the monoclonal antibody is selected from:a) an IgG or an IgM antibody, e.g., an IgGl antibody;b) a bi- or multi-specific antibody;c) a single chain antibody;d) a nanobody; ore) a diabody.The method of any one of the preceding aspects, wherein the monoclonal antibody comprises or is a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, an Fd’ fragment, an Fd fragment, and / or a single-chain variable fragment (scFv).The method of any one of the preceding aspects, wherein the monoclonal antibody comprises an Fc region.The method of any one of the preceding aspects, wherein the radioisotope is a metallic radioisotope or salt thereof, and wherein the radiolabeled antibody conjugate comprises a conjugate group comprising a chelator that forms a stable complex with the radioisotope with a thermodynamic stability constant (LogKML) of at least 18.The method of aspect 18, wherein the radiolabeled antibody conjugate comprises a chelator selected from the group consisting of closo-decaborate(2-), DOTA (2,2',2'',2'"-(l,4,7,10-tetraazacyclododecane-l,4,7,10-tetrayl)tetraacetic acid), DOTAM (2-[4,7,10-tris(2-amino-2-oxoethyl)-l,4,7,10-tetrazacyclododec-l-yl] acetamide), DOTAGA (2-(4,7,10-tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl)pentanedioic acid), Fhmacropa (N,N’-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6), DTPA (diethylenetriamine pentaacetic acid), NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid), NODAGA (2-[l,4,7-triazacyclononan-l-yl-4,7-bis(tBu-ester)]-l,5-pentanedioic acid), NODASA (1,4,7-triazacyclononane-1 -succinic acid-4, 7-diacetic acid), NETA ({4-[2-(bis-carboxy-methylamino)-5-(4-nitrophenyl)-entyl]-7 -carboxymethyl-[ 1 ,4,7]tri-azonan- 1 -yl } acetic acid), TETA (triethylenetetramine), CB-TE2A (2,2-(l ,4,8, 11 -tetraazabicyclo[6.6.2]hexadecane-4,ll-diyl)diacetic acid), Fbdedpa (l,2-bis[[(6-carboxypyridin-2-yl)methyl]amino]ethane), FUoctapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), FhCffiYdedpa (CHX= cyclohexyl / cyclohexane, Fbdedpa = l,2-[[6-carboxy-pyridin-2-yl]-methylamino]ethane), HA WAoctapa (CHX= cyclohexyl / cyclohexane, FUoctapa = A,M-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-7V,7V'-diacetic acid), HYNIC (hydrazinonicotinamide), Sarcophagine, HBED-CC (N, A'-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-A,A-diacetic acid), PCTA (3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-l(15),ll,13-triene-3,6,9-triacetic acid), MANOTA (methyl amino tri azacyclononane triacetic acid), THP (tris(hydroxypyridinone)), DFO (N'-[5-(acetyl-hydroxy-amino)pentyl]-N-[5-[3-(5-aminopentyl-hydroxy-carbamoyl) propanoylamino]pentyl]-N-hydroxy-butane diamide), and DFO* (5,11, 16,22-tetraazahexacosanediamide, Nl-[5-(acetylhydroxyamino)pentyl]-N26-(5-aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23-tetraoxo-), and DFOcyclo*; optionally the radiolabeled antibody conjugate comprises DOTA or DFO*.The method of any one of the preceding aspects, wherein the radiolabeled antibody conjugate is formulated as a pharmaceutical composition, optionally wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipient(s).The method of any one of the preceding aspects, wherein the cancer is selected from the group consisting of glioblastoma (GBM), non-small cell lung carcinoma (NSCLC), colorectal cancer, breast cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, esophageal cancer, skin cancer, and head and neck cancer, optionally wherein the cancer is GBM.The method of any one of the preceding aspects, wherein the radiolabeled antibody conjugate is administered to the subject via intravenous infusion, intratumor injection, intrathecal infusion or intracerebroventricular infusion.

Claims

CLAIMS1. A radiolabeled antibody conjugate comprising a monoclonal antibody and a radioisotope;wherein the radioisotope is selected from the group consisting of64Cu,67Ga,89Zr,161Tb,177LU,211At,212Pb and225Ac;wherein the monoclonal antibody specifically binds to an epitope within an amino acid loop region at residues 287-302 of human epidermal growth factor receptor (EGFR; SEQ ID NO: 1); andwherein the monoclonal antibody does not specifically bind to a corresponding loop region in which the arginine at residue 300 is substituted with alanine.

2. The radiolabeled antibody conjugate according to claim 1, wherein the radiolabeled antibody conjugate comprises a molar ratio of the radioisotope to antibody of from about 0.1:1 to about 3:1, preferably from about 0.5:1 to about 2:1, more preferably about 1:1.

3. The radiolabeled antibody conjugate according to claim 1 or claim 2, wherein the monoclonal antibody comprises:a) a heavy chain complementarity determining region (HCDR) 1 comprising an amino acid sequence of GFSLTNYG (SEQ ID NO: 3), an HCDR2 comprising an amino acid sequence of MWRGGGT (SEQ ID NO: 4), an HCDR3 comprising an amino acid sequence of ARKGVGMGLGY (SEQ ID NO: 5); andb) a light chain complementarity determining region (LCDR) 1 comprising an amino acid sequence of QTIGTW (SEQ ID NO: 6), an LCDR2 comprising an amino acid sequence of GAT (SEQ ID NO: 7), and an LCDR3 comprising an amino acid sequence of QQLYSNPYT (SEQ ID NO: 8).

4. The radiolabeled antibody conjugate of any one of the preceding claims, wherein the monoclonal antibody comprises:a) an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence that is at least about 85% identical to, or identical to,QVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGGGTDYNAAFISRLTITKDTSKSQVVFTMTNMDPVDTATYYCA RKGVGMGLGYWGQGTLVTVSS (SEQ ID NO: 9); andb) an immunoglobulin light chain variable (VL) region comprising an amino acid sequence that is at least about 85% identical to, or identical to, DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIY GATNLADGVPSRFSGSGSGTKFTLTISSLQPEDFATYYCQQLYSNPYTFG GGTKLEIK (SEQ ID NO: 10).

5. The radiolabeled antibody conjugate of any one of the preceding claims, wherein the monoclonal antibody specifically binds to a peptide comprising residues 287- 302 of human EGFR (SEQ ID NO: 1) with a dissociation constant (KD) of between about 0.06 nM and about 60 nM, preferably between about 0.6 nM to about 6 nM, as determined a plate-based binding assay, such as an ELISA.

6. The radiolabeled antibody conjugate of any one of the preceding claims, wherein the monoclonal antibody is human or humanized.

7. The radiolabeled antibody conjugate of any one of the preceding claims, wherein the monoclonal antibody is selected from:a) an IgG or an IgM antibody, e.g., an IgGl antibody;b) a bi- or multi-specific antibody;c) a single chain antibody;d) a nanobody; ore) a diabody.

8. The radiolabeled antibody conjugate of any one of the preceding claims, wherein the monoclonal antibody comprises or is a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, an Fd’ fragment, an Fd fragment, and / or a single-chain variable fragment (scFv).

9. The radiolabeled antibody conjugate of any one of the preceding claims, wherein the monoclonal antibody comprises an Fc region.

10. The radiolabeled antibody conjugate of any one of the preceding claims, wherein the radioisotope is a metallic radioisotope or salt thereof, and wherein the radiolabeled antibody conjugate comprises a conjugate group comprising a chelator that forms a stable complex with the radioisotope with a thermodynamic stability constant (LogKML) of at least 18.

11. The radiolabeled antibody conjugate of claim 10, wherein the radiolabeled antibody conjugate comprises a chelator selected from the group consisting of closo- decaborate(2-), DOTA (2,2',2'',2'"-(l,4,7,10-tetraazacyclododecane-l,4,7,10- tetrayljtetraacetic acid), DOTAM (2-[4,7,10-tris(2-amino-2-oxoethyl)-l,4,7,10- tetrazacyclododec-l-yl] acetamide), DOTAGA (2-(4,7,10-tris(carboxymethyl)- l,4,7,10-tetraazacyclododecan-l-yl)pentanedioic acid), EUmacropa (N,N’-bis[(6- carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6), DTPA (di ethylenetriamine pentaacetic acid), NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid), NOD AGA (2-[l,4,7-triazacyclononan-l-yl-4,7-bis(tBu-ester)]-l,5-pentanedioic acid), NODASA (1, 4, 7-triazacy cl ononane-1 -succinic acid-4, 7-diacetic acid), NETA ({4- [2-(bis-carboxy-methylamino)-5-(4-nitrophenyl)-entyl]-7-carboxymethyl-[l,4,7]tri- azonan-l-yl} acetic acid), TETA (triethylenetetramine), CB-TE2A (2,2-(l,4,8, 11- tetraazabicyclo[6.6.2]hexadecane-4,ll-diyl)diacetic acid), EUdedpa (l,2-bis[[(6- carboxypyridin-2-yl)methyl]amino]ethane), EUoctapa (N,N'-bis(6-carboxy-2- pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), EECfflfdedpa (CHX= cyclohexyl / cyclohexane, EUdedpa = l,2-[[6-carboxy-pyridin-2-yl]- methylamino]ethane), EUCffifoctapa (CHX= cyclohexyl / cyclohexane, EUoctapa = AAf'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-AAf'-diacetic acid), HYNIC (hydrazinonicotinamide), Sarcophagine, HBED-CC (N, A'-bis-[2-hydroxy- 5-(carboxyethyl)benzyl]ethylenediamine-A,A-diacetic acid), PCTA (3,6,9,15- tetraazabicyclo [9.3.1]pentadeca-l(15),ll,13-triene-3,6,9-triacetic acid), MANOTA (methyl amino tri azacyclononane triacetic acid), THP (tris(hydroxypyridinone)), DFO (N'-[5-(acetyl-hydroxy-amino)pentyl]-N-[5-[3-(5-aminopentyl-hydroxy- carbamoyl) propanoylamino]pentyl]-N-hydroxy-butane diamide), and DFO* (5,11, 16,22-tetraazahexacosanediamide, Nl-[5-(acetylhydroxyamino)pentyl]-N26-(5- aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23-tetraoxo-), and DFOcyclo*; optionally the radiolabeled antibody conjugate comprises DOTA or DFO*.

12. The radiolabeled antibody conjugate of any one of the preceding claims, wherein the radiolabeled antibody conjugate is formulated as a pharmaceutical composition, optionally wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipient(s).

13. A method of detecting cancer cells in a subject having cancer, wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi), the method comprising:a) administering an effective amount of the radiolabeled antibody conjugate according to any one of claims 1-12 to the subject; andb) detecting any radiation retained within the subject;wherein the radioisotope is selected from the group consisting of64Cu,89Zr,161Tb,177LU,211At,212Pb and225Ac.

14. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of a radiolabeled antibody conjugate according to any one of claims 1-12 to the subject;wherein the cancer cells express epidermal growth factor receptor (EGFR) variant III (EGFRvIII) or overexpress EGFR (EGFR-hi);wherein the radioisotope is selected from the group consisting of67Ga,161Tb,177Lu,211At,212Pb and225Ac.

15. The method of any one of claims 13-14, wherein the cancer is selected from the group consisting of glioblastoma (GBM), non-small cell lung carcinoma (NSCLC), colorectal cancer, breast cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, esophageal cancer, skin cancer, and head and neck cancer, optionally wherein the cancer is GBM.

16. The method of any one of claims 13-15, wherein the radiolabeled antibody conjugate is administered to the subject via intravenous infusion, intratumor injection, intrathecal infusion or intracerebroventricular infusion.

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