Imaging reagents and methods

Abstract

A method for in vivo imaging or detection of a cancer in a subject in need thereof, wherein the method comprises: - administering to the subject, an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject, - detecting the agent in the subject, wherein the cancer is not renal cell carcinoma.

Description

Imaging reagents and methodsField of the invention

[0001] The invention relates to agents for use in in vivo imaging and detection of cancers, and methods of use thereof.Related application

[0002] This application claims priority from Australian provisional application AU 2022903922, the contents of which are hereby incorporated by reference in their entirety.Background of the invention

[0003] Methods of medical imaging are often used to assist in the diagnosis and staging of progression of various cancers. Such methods may also be advantageous in removing or reducing the need for invasive techniques (such as obtaining a biopsy sample) for confirming diagnosis, which are not always necessary and can lead to complications.

[0004] However, not all cancers can be successfully detected using standard medical imaging. Moreover, many imaging techniques enable the detection of masses but cannot successfully distinguish between benign or malignant tissue.

[0005] The state of oncology management has advanced in recent decades in a dichotomous manner. While some types of cancer have benefitted significantly from research advances in diagnostic and therapeutic options, resulting in improved morbidity and mortality for their patient population, others have proven more elusive and continue to portend dismal prognoses for their patient populations.

[0006] Where this latter category of cancers has exhausted the limits of existing diagnostic and therapeutic modalities, there is a need for innovations based on new approaches to oncology management.

[0007] There is a need for improved methods and compositions for use in the in vivo detection and / or imaging of various cancers.

[0008] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0009] The present invention is based at least in part on the identification by the inventors of a subset of solid tumours expressing the antigen carbonic anhydrase IX (CAIX), and the finding that these tumours can be imaged in vivo using an imaging agent for binding CAIX.

[0010] The present invention therefore provides a method for in vivo imaging or detection of a cancer in a subject in need thereof,- administering to the subject, an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,- detecting the agent in the subject, wherein the cancer is selected from:• bladder cancer• breast cancer (including triple negative breast cancer, hormone receptor positive breast cancer (ER positive, PR positive, ER / PR positive), and HER2 positive breast cancer)• cervical cancer• colorectal cancer• esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma) gastric cancer (including gastric adenocarcinoma) glioblastoma multiforme• head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer and nasopharyngeal carcinoma)• liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)• lung cancer (including non-small cell and small cell carcinoma)• ovarian cancer (including epithelial ovarian carcinoma)• pancreatic cancer (including pancreatic ductal adenocarcinoma), and• soft tissue sarcoma whereby detection of said agent above a background or standard level indicates the presence of the cancer, thereby imaging or detecting the cancer in the subject.

[0011] The present invention also provides a method for the diagnosis of a cancer in a subject in need thereof, where the method comprises:- administering to the subject, an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,- determining the presence or absence of the agent in the subject, wherein the cancer is selected from:• bladder cancer• breast cancer (including triple negative breast cancer, hormone receptor positive breast cancer (ER positive, PR positive, ER / PR positive), and HER2 positive breast cancer)• cervical cancer• colorectal cancer• esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma)• gastric cancer (including gastric adenocarcinoma)• glioblastoma multiforme• head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal and nasopharyngeal carcinoma)• liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)• lung cancer (including non-small cell and small cell carcinoma)• ovarian cancer (including epithelial ovarian carcinoma)• pancreatic cancer (including pancreatic ductal adenocarcinoma), and• soft tissue sarcoma whereby determining the presence of said agent above a background or standard level indicates that the subject has said cancer, thereby diagnosing the cancer in the subject.

[0012] The present invention also provides a method for producing an image of a cancer, the method comprising:- administering to a subject suspected of having the cancer, an effective amount of an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,- detecting the agent in the subject wherein the cancer is selected from:• bladder cancer• breast cancer (including triple negative breast cancer, hormone receptor positive breast cancer (ER positive, PR positive, ER / PR positive), and HER2 positive breast cancer) cervical cancer• colorectal cancer• esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma)• gastric cancer (including gastric adenocarcinoma)• glioblastoma multiforme• head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal and nasopharyngeal carcinoma)• liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)• lung cancer (including non-small cell and small cell carcinoma)• ovarian cancer (including epithelial ovarian carcinoma)• pancreatic cancer (including pancreatic ductal adenocarcinoma) and• soft tissue sarcoma thereby producing an image of the cancer.

[0013] The present invention also provides a method for producing an image of a cancer, the method comprising:- infusing an effective amount of an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent,- detecting the agent, wherein the cancer is selected from:• bladder cancer breast cancer (including triple negative breast cancer, hormone receptor positive breast cancer (ER positive, PR positive, ER / PR positive), and HER2 positive breast cancer)• cervical cancer• colorectal cancer• esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma)• gastric cancer (including gastric adenocarcinoma)• glioblastoma multiforme• head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal and nasopharyngeal carcinoma)• liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)• lung cancer (including non-small cell and small cell carcinoma)• ovarian cancer (including epithelial ovarian carcinoma)• pancreatic cancer (including pancreatic ductal adenocarcinoma) and• soft tissue sarcoma thereby producing an image of the cancer. Optionally, the detectable moiety is a radioisotope and the detecting comprises detecting radiation such as Positron Emission Tomography (PET) imaging.

[0014] In any embodiment, the methods of the invention further may comprise allowing the agent to concentrate at sites and / or tissues in said subject where the CAIX antigen is found in the subject prior to detecting the agent.

[0015] Administration may be by any suitable means, preferably one that allows for systemic administration (eg intravenous infusion) of the agent such that the agent can accumulate at sites in the subject where CAIX is present on the cell surface. The mode of administration may be dictated by the nature of the agent for binding to CAIX. For example, in the case of an antibody for binding to CAIX, preferably the agent is administered by intravenous infusion. Labelled peptides or small molecules may be administered orally or by other means.

[0016] In any embodiment, a diagnostic method of the invention does not require additional in vitro diagnosis using a tissue biopsy or other biological sample obtained from the subject.

[0017] In any embodiment, the agent comprises a first moiety for binding to CAIX and a second moiety for enabling detection of the agent in vivo.

[0018] In any embodiment, the moiety for binding to CAIX may be a small molecule, peptide or polypeptide (such as an antibody or antigen binding fragment thereof).

[0019] In any embodiment, the agent for binding to CAIX is a small molecule, optionally selected from the group consisting of: SLC-01 1 1 , SLC-149, SLC-0121 , SLC-101 , PMI- 05, sulfamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781 , -MIP-1486, MIP- 1490, MIP-1504 (especially "mTc-HEHEHE-Z09781 ,99mTc-MIP-1486, "mTc-MIP-1490 or99mTc-MIP-1504 / 5) and PHC-102.

[0020] In any embodiment, the agent for binding to CAIX is a peptide, optionally selected from the group consisting of: 3B-301 , 3B-302 or CAIX-P1 .

[0021] In any embodiment, the agent for binding to CAIX is a polypeptide, including an antibody or antigen-binding fragment thereof.

[0022] In a particularly preferred embodiment, the agent comprises a first moiety for binding to CAIX, wherein the first moiety is in the form of an antibody or antigen binding fragment thereof.

[0023] In any embodiment, the agent is an antigen binding protein (antibody) such as girentuximab or a functional variant or fragment thereof that retains the ability to bind to CAIX. In some embodiments, the antigen binding protein that binds to or specifically binds to CAIX is G250. In some embodiments, the antigen binding protein that binds to or specifically binds to CAIX is a chimeric antibody or antigen binding fragment thereof. In some embodiments, the antigen binding protein that binds to or specifically binds to CAIX is a humanised antibody or antigen binding fragment thereof. Optionally, the antigen binding protein is humanised G250 (hG250).

[0024] In alternative embodiments, the antibody for binding to CAIX may comprise BCA-356, BAY-794620 or SLC-0131 .

[0025] The agent for use according to the methods of the present invention comprises a moiety for enabling detection thereof. Any suitable detectable moiety, for use in in vivo detection techniques, can be used and will be known to the skilled person.

[0026] The detectable moiety may be linked directly to the moiety for binding to CAIX, or may be conjugated via a chelator or other linking moiety. In certain embodiments, the aforementioned agents for binding to CAIX are detectable without the need for linking an additional detectable moiety thereto.

[0027] In any embodiment, the detectable moiety is a radioisotope. Examples of suitable isotopes include: gallium-67 and gallium-68 (67Ga and68Ga), indium-1 11 (111ln), iodine-123, iodine-124 or iodine 131 (123l,124l, or131I), lutetium-177 (177Lu), technetium- 99 (99mTc), yttrium-90 (90Y) and zirconium-89 (89Zr).

[0028] In any embodiment, the agent is an antibody for binding to CAIX and the detectable moiety is a radioisotope, optionally selected from: gallium-67 and gallium-68 (67Ga and68Ga), indium-1 11 (111ln), iodine-123, iodine-124 or iodine 131 (123l,124l, or131I), lutetium-177 (177Lu), technetium-99 (99mTc), yttrium-90 (90Y) and zirconium-89 (89Zr).

[0029] In any embodiment, the agent is a girentuximab antibody (including a chimeric or humanised version thereof), and the detectable moiety is a radioisotope, optionally selected from: gallium-67 and gallium-68 (67Ga and68Ga), indium-1 11 (111ln), iodine-123, iodine-124 or iodine 131 (123l,124l, or131I), lutetium-177 (177Lu), technetium-99 (99mTc), yttrium-90 (90Y) and zirconium-89 (89Zr).

[0030] In any embodiment, the agent is89Zr-,123l-,124l-,131I- or177Lu-girentuximab.

[0031] In preferred embodiments, the detectable moiety is a radioisotope and the detection of the agent comprises determination or detecting the presence or absence of radiation emitted by the radioisotope. In any embodiment, determination or detection of the presence of absence of radiation comprises Positron Emission Tomography (PET) imaging.

[0032] Other suitable detectable moieties include fluorescent labels and dyes. It will be appreciated that in any embodiment of the invention, more than one detectable moiety may be utilised in order to maximise imaging or detection of the agent and thereby the tumour or cancer expressing CAIX.

[0033] The invention also provides for an agent, or a composition comprising an agent for CAIX, as described herein, for use in a method of detection, imaging, for obtaining an image of a cancer as described herein, or for diagnosis of a cancer.

[0034] Further still, the invention provides for an agent, or a composition comprising an agent for CAIX, as described herein, when used in a method of detection, imaging or diagnosis of a cancer, or for obtaining an image of a cancer as described herein.

[0035] Further still, the invention provides a kit for use in accordance with any method described herein, wherein the kit comprises: and agent for binding to CAIX as described herein, and optionally, instructions for the use thereof for detecting, imaging or diagnosing a cancer.

[0036] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0037] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Description of the drawings

[0038] Figure 1 : In vitro binding of radiolabelled-DOTA-GmAb to various cell lines.

[0039] Figure 2: Representative images of mice bearing As-PC-1 (pancreatic cancer), FaDu (hypopharyngeal cancer) or HT-29 (colorectal cancer) tumour xenografts, and following injection with radiolabelled-DOTA-GmAb.

[0040] Figure 3: Quantification of the percentage injected dose at 24 hours and 72 hours following injection of radiolabelled-DOTA-GmAb. Closed circles = AsPC-1 radiolabelled-DOTA-GmAb; Closed boxes = FaDu radiolabelled-DOTA-GmAb; Closed triangles = HT-29 radiolabelled-DOTA-GmAb.

[0041] Figure 4: Quantification of biodistribution results. Ex vivo biodistribution (ie the distribution of radiolabelled-DOTA-GmAb observed in the organs of mice, following necropsy) was shown to correlate with in vivo biodistribution (ie the distribution of radiolabelled-DOTA-GmAb observed in mice following whole-body imaging). Closedcircles = AsPC-1 radiolabelled-DOTA-GmAb; Closed boxes = FaDu radiolabelled-DOTA-GmAb; Closed triangles = HT-29 radiolabelled-DOTA-GmAb.

[0042] Figure 5: Imaging of bladder cancer. Day 0: whole body89Zr-girentuximab scans: A: Coronal PET / CT fusion. B: maximum intensity projection (MIP) visualisation.

[0043] Figure 6: Imaging of bladder cancer. Day 2, A.89Zr-girentuximab pelvis PET / CT fusion images: (arrows) radiopharmaceutical uptake on different sides of bladder wall. B. 3D representation with superimposed image of bladder.Detailed description of the embodiments

[0044] The present invention relates to the imaging and diagnosis of cancers for which diagnosis is challenging due to the lack of robust oncology management options. To improve the morbidity and mortality of these oncologic indications, diagnostic and therapeutic innovation is needed.

[0045] The detection of CAIX in the context of imaging and diagnosing renal cancers is known. However, prior to the present invention, it was not known whether the in vivo detection of CAIX could be used to successfully image and diagnose other solid cancers which may express CAIX.

[0046] Although CAIX is typically associated with advanced disease, it is not known whether detection and / or imaging of CAIX is possible during the early stages of certain cancers, or if the cancer can only be detected in late disease. Further, some cancer exhibit reduced expression as disease progresses and it is therefore also not clear whether CAIX imaging is a useful means for detecting these types of cancers.

[0047] Moreover, given the heterogeneity of many cancer, the simple presence of expression of CAIX (eg as determined by immunohistochemistry techniques), does not necessarily indicate that the cancer could be detected using whole-body or partial-body imaging methods.

[0048] For example, in the 2006 study by Henrickx et al., (Cancer Biotherapy & Radiopharmaceuticals, 21 :263-268), it was found that radiolabelled antibodies for binding to CAIX are not suitable for imaging of biliary cancer despite the fact that the biliary cancers were found to overexpress CAIX. Conversely, the same antibodies are known to be generally useful for imaging and detection of CAIX-overexpressing renal cellcarcinomas. Both biliary and renal carcinomas are malignancies of epithelial cells and are characterised by increased expression of CAIX. It is not understood why CAIX-binding antibodies are therefore useful for imaging and detection of renal cell carcinoma but not biliary cancer.

[0049] In another example, a recent study published by Huizing et al (2021 , Physics and Imaging in Radiation Oncology, 145-150), found that a111In-labelled F(ab’)2 form of the CAIX-binding antibody girentuxumab, was not able to discriminate between tumour and non-tumour cells and therefore was indicated not to be useful for quantification of changes in CAIX expression. This finding in particular, is in stark contrast to the findings of the inventors as reported herein, wherein89Zr-GmAb (in the form of a full IgG antibody) was shown to be useful for imaging and detection of the same cancer cell types in vivo.

[0050] The present invention is therefore based on the finding that a subset of cancers associated with increased CAIX expression can indeed by successfully detected and imaged using an agent for binding to CAIX and wherein the agent comprises a detectable moiety.General definitions

[0051] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects, and vice versa, unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0052] Those skilled in the art will appreciate that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0053] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0054] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0055] The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally- equivalent products, compositions and methods are clearly within the scope of the present invention.

[0056] Any example or embodiment of the present invention herein shall be taken to apply mutatis mutandis to any other example or embodiment of the invention unless specifically stated otherwise.

[0057] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in diagnostic technology, radioimaging, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0058] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.Carbonic anhydrase IX

[0059] As used herein, carbonic anhydrase is also known as: CA-IX, CA9, CAIX, Carbonate dehydratase IX, Carbonic anhydrase 9, Carbonic anhydrase IX, carbonic dehydratase, G250, Membrane antigen MN, P54 / 58N, pMW1 , RCC-associated antigen G250, RCC-associated protein G250, and Renal cell carcinoma-associated antigen G250.

[0060] Cancer cells primarily express the plasma-membrane-associated CA isoforms CAIX and CAXII, as well as intracellular CAs such as CAI and CAIL Amongst the cancer- related CAs, CAIX has gained most attention, since expression of this isoform in healthytissue is restricted to epithelial cells in the stomach and gut, but is strongly upregulated in renal cancers.

[0061] CAIX, the expression of which is under control of the hypoxia-inducible factor 1 (HIF-1 ), is predominantly located in chronically hypoxic tumour regions. However, CAIX can also be found in mild hypoxic or even normoxic regions, since the expression of CAIX can be activated by components of the mitogen-activated protein kinase (MAPK) pathway.

[0062] The agent for binding to CAIX and for use in accordance with the methods of the invention, may be any compound that specifically recognises or binds to CAIX, mediates its activity by binding to CAIX or a fragment or splice variant thereof; irreversibly binds at the entrance to the active site, and / or inhibits CAIX by coordinating to the zinc ion at the active site of CAIX.

[0063] Preferably the agent for binding to CAIX specifically interacts with a CAIX polypeptide. Specifically interacting with (e.g. recognising or binding to) means that the agent e.g. antibody, has a greater affinity for CAIX compared to other polypeptides. In one embodiment the agent interacts with (i.e. binds to or recognises) or modulates the activity of CAIX polypeptide and / or mediates an antibody dependent cell cytotoxicity (ADCC) and / or complement mediated cytotoxicity (CDC). Thus, according to one embodiment, the agent is a CAIX inhibitor. Said CAIX inhibitor may act on the protein level or the nucleic acid level. Examples for CAIX inhibitors e.g. acting on the protein level include but are not limited to peptides and anti-CAIX antibodies as well as functional fragments of those antibodies or small organic molecules, preferably having a molecular weight below 500 g / mol.

[0064] Examples of anti-CAIX antibodies or antibodies for binding to CAIX are described in EP 637 336, WO 93 / 18152, WO 95 / 34650, WO 00 / 24913, WO 02 / 063010, WO 04 / 025302, WO 05 / 037083, WO 201 1 / 139375, Murri-Plesko et al., Eur J Pharmacol 201 1 , 657: 173-183.

[0065] Examples of small organic molecules for binding to CAIX include but are not limited to sulphonamides, heteroaromatic sulphonamides, sulfamates, coumarins and thiocoumarins and BAY-79-4620. Examples for inhibitors acting on the nucleic acid level are siRNA molecules, ribozymes and / or antisense molecules.

[0066] As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that an agent for use according to the invention, reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a CAIX or cell expressing same than it does with alternative antigens or cells. For example, an antigen binding protein that binds to CAIX with materially greater affinity (e.g., 1 .5 fold or 2 fold or 5 fold or 10 fold or 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than it does to other antigens.

[0067] Methods for assessing binding to a protein (eg CAIX) are known in the art, e.g., as described in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such a method generally involves immobilizing the agent (eg antibody) and contacting it with labeled target (in the case of an antibody, the antigen). Following washing to remove non-specific bound protein, the amount of label and, as a consequence, bound antigen is detected. Of course, the antigen binding site can be labeled and the antigen immobilized. Panning-type assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.

[0068] Other standard methods for assessing binding of to a target, such as CAIX, are also known in the art.Small molecules

[0069] In any embodiment, the moiety for binding to CAIX is the small molecule SLC- 01 1 1 (CAS 178606-66-1 ), SLC-149 (as described in EP 3317255 B1 , incorporated herein by reference), SLC-0121 or SLC-101.

[0070] In any embodiment, the moiety for binding to CAIX is the small molecule / contrast dye PMI-05 (as described in US2019 / 0192699A1 , incorporated herein by reference).

[0071] In any embodiment, the moiety for binding to CAIX is the small molecule sulfamide-nitroimidazole (as described in Rami et al., (2013), J. Med. Chem, 56: 8512- 8520, incorporated herein by reference).

[0072] In any embodiment, the moiety for binding to CAIX is the small molecule JS-403 (as described in WO 2010 / 089752 A1 , incorporated herein by reference).

[0073] In any embodiment, the moiety for binding to CAIX is the small molecule UB- TT220 (as described in WO 2022 / 015955 A1 , incorporated herein by reference).

[0074] In any embodiment, the moiety for binding to CAIX is the small molecule99mTc- HEHEHE-Z09781 (Kim et al., (2017) Advanced Science, 4:1600471 ; Gebauer and Skerra (2009) Current Opin in Chem Biol, 13(3) :245-55; Schardt et al., (2017) Mol Pharmaceutics, 14(4):1047-56; Tolmachev et al., (2008) Bioconjugate Chem, 19(8) : 1579- 87; Liu et al., (2022) Analytical and Bioanalytical Chemistry, 414:1095-1 104; Grindel et al., (2022) ACS Chem Biol, 17(6): 1543-55, incorporated herein by reference),99mTc-MIP- 1486, "mTc-MIP-1490 (4-(2-bis((1 -(2-((1 ,5-dicarboxy-3-(2-carboxyethyl)pentan-3- yl)amino)-2-oxoethyl)-1 H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide where X= ethyl) or99mTc-MIP-1504 (4-(2-bis((1 -(2-((1 ,5-dicarboxy-3-(2-carboxyethyl)pentan-3- yl)amino)-2-oxoethyl)-1 H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide where X= n-butyloxy) (Hillier et al., (2012) Journal of Nuclear Medicine, 53(s1 ):217, incorporated herein by reference)

[0075] In any embodiment, the moiety for binding to CAIX is the small molecule PHC- 102 (as described in WO 2015 / 1 14171 A1 ; WO 2018154517 A1 ; US 2014 / 0357650 A1 ; WO 2015 / 114171 A1 , incorporated herein by reference).Peptides

[0076] In any embodiment, the moiety for binding to CAIX is a peptide. As used herein, a peptide will be understood to comprise a chain of more than 1 amino acid residues. Typically, a peptide may comprise from about 2 to 30 amino acids, e.g. from about 5 to 30, from about 10 to 30, from about 2 to 25, from about 5 to 25, from about 10 to 25, or from about 10 to 20 amino acids. A peptide may have a length of at least 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or 30 amino acids. Typically a peptide is no longer than about 40 amino acids, e.g. no longer than about 35, 30, 25, 20, 17, 15, 14, 13, 12, 1 1 or 10 amino acids.

[0077] In any embodiment, the moiety for binding to CAIX is the peptide 3B-301 (also known as Debio 0228; Queen et al., (2018) Int J of Biol Macromol, 106:840-850; Eldehna et al., (2019) Bioorganic Chem, 90:103102; Lavecchia et al., (201 1 ) Carbohydrate Res, 346(3):442-48; Krymov et al., (2022) Eur J of Medicinal Chem, 228:113997; Supuran (2008) BJI Int, 101 (s4):39-40; Koyuncu et al., (2019) J of Enzyme Inhibition and MedicinalChem, 34(1 ):703-1 1 ; Kumar et al., (2017) Eur J of Medicinal Chem, 136:52-62, incorporated herein by reference) or 3B-302.

[0078] In any embodiment, the moiety for binding to CAIX is the peptide CAIX-P1 , having the amino acid sequence YNTNHVPLSPKY (as described in Askoxylakis et al., (2010), PLoS One, 5(12): e15962), optionally wherein the peptide is labelled with125l or1311, for enabling detection thereof (although it will be appreciated that any suitable radiolabel or other detectable moiety may be used).Polypeptides and antibodies

[0079] According to especially preferred embodiments, the agent comprises a first moiety in the form of an anti-CAIX antibody and / or a functional fragment of such an antibody. The fragment of the anti-CAIX antibody may have essentially the same CAIX- binding and / or inhibiting activity as the full-length anti-CAIX antibody and / or is an epitopebinding fragment of the anti-CAIX antibody.

[0080] Reference herein to an antibody or antigen binding fragment thereof that “binds to” carbonic anhydrase IX (CAIX) provides literal support for an antibody or fragment thereof that “binds specifically to” or “specifically binds to” CAIX.

[0081] The antibody and / or the antibody fragment thereof may be selected from the group consisting of polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2, Fab', scFv, dsFv and chimerized, humanized and fully human variants thereof. The antibody may be multivalent, or multivalent and multispecific.

[0082] In particularly preferred embodiments, the antibody is a full antibody, comprising at least one antigen binding domain (Fab) of an antibody, and at least one Fc region of an antibody .The antibody may include human constant regions of IgG 1 , lgG2a, lgG3, or lgG4.

[0083] According to a further preferred embodiment, an anti-CAIX antibody or epitopebinding fragment thereof for use according to the invention, binds to the amino acid sequence LSTAFARV and / or ALGPGREYRAL.

[0084] In any embodiment, the moiety for binding to CAIX is in the form of the antibody BAY-794620, or an antigen binding fragment thereof (as described in WO 2003 / 100029 A2; WO 2003 / 033674 A2; Theiner et al., (2021 ) Tierarztl Prax Ausg G GrosstiereNutztiere, 49(6): 392-402; Kimani et al., (2011 ) Photochemistry and Photobiology, 88(1 ):175-87; NCT01065623 (v24, 30 September 2014); NCT01028755 (v30, 19 January 2015), incorporated herein by reference).

[0085] In any embodiment, the moiety for binding to CAIX is in the form of the antibody SLC-0131 , or an antigen binding fragment thereof.

[0086] According to further particularly preferred embodiments, the agent for binding to CAIX is the antibody anti-G250 and / or an antigen-binding fragment thereof. Anti-G250 antibodies are, e.g., described in EP-B-0 637 336. The antibody or fragment thereof may be chimeric or humanised G250 antibody. In some embodiments, the antigen binding protein that binds to or specifically binds to CAIX is as described in any of WO 2002 / 062972 A2 (US 2004 / 0219633 A1 ), WO 2004 / 002526 A1 (US 7,632,496 B2), WO 2006 / 002889 A2 (US 7,691 ,375 B2), WO 2009 / 056342 A1 (US 2014 / 0017252 A1 ), WO 201 1 / 032973 A1 (US 2012 / 0207672 A1 ), and WO 2014 / 128258 A1 (US 10,620,208 B2), or WO 2021 / 000017 A1 , the entire contents of each of these publications is incorporated herein by reference.

[0087] The antibodies for use in the present invention may be produced by any suitable method known in the art including but not limited by methods as described in PCT / EP02 / 01282 and PCT / EP02 / 01283, which are incorporated herein by reference.

[0088] An especially preferred antibody is cG250, preferably girentuximab (INN). Another especially preferred embodiment is the monoclonal antibody G250 produced by the hybridoma cell line DSM ACC 2526. The antibody cG250 is an IgG 1 kappa light chain chimeric version of an originally murine monoclonal antibody mG250.

[0089] Variants of the original chimeric G250 (cG250) antibody are known, including WX-G250 and WX-G250RIT (131 iodine) (Janssen Global Services LLC).

[0090] In a particularly preferred embodiment, the antibody is89Zr-girentuximab (ie89Zr-cG250),123l-,124l-, or131I- girentuximab, or177Lu-girentuximab.

[0091] In any embodiment, the antibody or antigen binding fragment thereof comprises:(a) a heavy chain variable domain (VH) comprising three complementarity determining regions (CDRs) of the amino acid sequence as set forth in SEQ ID NOs: 4, 20, 36, 52 or 68; and / or(b) a light chain variable domain (VL) comprising three complementarity determining regions (CDRs) of the amino acid sequence as set forth in SEQ ID NOs: 84, 100, 1 16, 132, 148 or 164.

[0092] In any embodiment, the antibody or antigen binding fragment thereof comprises an antigen binding domain that binds specifically to carbonic anhydrase IX (CAIX) and comprises:FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - linker - FR1 a - CDR1 a - FR2a - CDR2a - FR3a - CDR3a - FR4a wherein:FR1 , FR2, FR3 and FR4 are each framework regions;CDR1 , CDR2 and CDR3 are each complementarity determining regions;FR1 a, FR2a, FR3a and FR4a are each framework regions;CDR1 a, CDR2a and CDR3a are each complementarity determining regions; wherein the sequence of any of the complementarity determining regions have an amino acid sequence as described in Table 1 below. Preferably, the framework regions have an amino acid sequence also as described in Table 1 below, including amino acid variation at particular residues which can be determined by aligning the various framework regions derived from each antibody. The CDR1 , CDR2 and CDR3 may be sequences from the VH, CDR1 a, CDR2a and CDR3a may be sequences from VL, or the CDR1 , CDR2 and CDR3 may be sequences from the VL, CDR1 a, CDR2a and CDR3a may be sequences from VH.

[0093] In any embodiment, the antigen or antigen binding fragment thereof comprises:(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 1 , a CDR2 comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set in SEQ ID NO: 2, and a CDR3 comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 3;(ii) a VH comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in any of SEQ ID NO: 4, 20, 36, 52 or 68;(iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 81 , a CDR2 comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 82 and a CDR3 comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 83;(iv) a VL comprising a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 84, 100, 1 16, 132, 148 or 164;(v) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 1 , a CDR2 comprising a sequence set forth in SEQ ID NO: 2 and a CDR3 comprising a sequence set forth in SEQ ID NO: 3;(vi) a VH comprising a sequence set forth in any of SEQ ID NO: 4, 20, 36, 52 or 68;(vii) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 81 , a CDR2 comprising a sequence set forth in SEQ ID NO: 82, and a CDR3 comprising a sequence set forth in SEQ ID NO: 83;(viii) a VL comprising a sequence set forth in any of SEQ ID NO: 84, 100, 1 16, 132, 148 or 164;(ix) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 1 , a CDR2 comprising a sequence set forth between in SEQ ID NO: 2, and a CDR3 comprising a sequence set forth in SEQ ID NO: 3; and a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 81 , a CDR2 comprising a sequence set forth in SEQ ID NO: 82, and a CDR3 comprising a sequence set forth in SEQ ID NO: 83; or(x) a VH comprising a sequence set forth in any of SEQ ID NO: 4, 20, 36, 52 or 68 and a VL comprising a sequence set forth in any of SEQ ID NO: 84, 100, 116, 132, 148 or 164.

[0094] In a further embodiment, the antibody or antigen binding fragment thereof comprises:(i) a VH comprising a framework region (FR) 1 comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in any of SEQ ID Nos: 9, 25, 41 , 57 or 73; a FR2 comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in any of SEQ ID Nos: 10, 26, 42, 58 or 74; a FR3 comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in any of SEQ ID Nos: 1 1 , 27, 43, 59 or 75; a FR4 comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in any of SEQ ID Nos: 12, 28, 44, 60 or 76; and(ii) a VL comprising a framework region (FR) 1 comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in any of SEQ ID Nos: 89, 105, 121 , 137, 153 or 169; a FR2 comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in any of SEQ ID Nos: 90, 106, 122, 138, 154 or 170; a FR3 comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in any of SEQ ID Nos: 91 , 107, 123, 139, 155 or 171 ; a FR4 comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in any of SEQ ID Nos: 92, 108, 124, 140, 156 or 172.

[0095] In a further embodiment, the antibody or antigen binding fragment thereof comprises:(i) a VH comprising a framework region (FR) 1 comprising or consisting of a sequence as set forth in SEQ ID Nos: 9, 25, 41 , 57 or 73; a FR2 comprising or consistingof a sequence as set forth in SEQ ID Nos: 10, 26, 42, 58 or 74; a FR3 comprising or consisting of a sequence as set forth in SEQ ID Nos: 11 , 27, 43, 59 or 75; a FR4 comprising or consisting of a sequence as set forth in SEQ ID Nos: 12, 28, 44, 60 or 76, and(ii) a VL comprising a framework region (FR) 1 comprising or consisting of a sequence as set forth in SEQ ID Nos: 89, 105, 121 , 137, 153 or 169; a FR2 comprising or consisting of a sequence as set forth in SEQ ID Nos: 90, 106, 122, 138, 154 or 170; a FR3 comprising or consisting of a sequence as set forth in SEQ ID Nos: 91 , 107, 123, 139, 155 or 171 ; a FR4 comprising or consisting of a sequence as set forth in SEQ ID Nos: 92, 108, 124, 140, 156 or 172.

[0096] In any embodiment, the antibody or antigen binding fragment thereof that specifically binds to CAIX comprises an amino acid sequence that consists essentially of or consists of (in order of N to C terminus or C to N terminus) any one of SEQ ID NO: 4, 20, 36, 52 or 68 and / or any one of SEQ ID NO: 84, 100, 1 16, 132, 148, 164.

[0097] In any embodiment, the antibody or antigen binding fragment thereof comprises:(a) a heavy chain variable domain (VH) comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in SEQ ID NOs: 4, 20, 36, 52 or 68; and / or(b) a light chain variable domain (VL) comprising or consisting of a sequence at least about 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% identical to a sequence as set forth in SEQ ID NOs: 84, 100, 1 16, 132, 148 or 164.

[0098] In any embodiment, the antibody or antigen binding fragment thereof for binding to CAIX may be in the form of:(i) a single chain Fv fragment (scFv);(ii) a dimeric scFv (di-scFv); or(iii) one of (i) or (ii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

[0099] In any embodiment, the antibody or antigen binding fragment thereof for binding to CAIX may be in the form of:(i) a diabody;(ii) a triabody;(iii) a tetrabody;(iv) a Fab;(v) a F(ab’)2;(vi) a Fv; or(vii) one of (i) to (vi) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

[0100] In any embodiment, the antibody or antigen binding fragment thereof for use according to the invention may be a fusion protein comprising an antigen binding protein, immunoglobulin variable domain, antibody, dab (single domain antibody), di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule, or multispecific antibody as described herein.

[0101] An antigen binding fragment, immunoglobulin variable domain, antibody, dab, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein may be obtained by expressing a nucleic acid encoding the same.

[0102] An antibody or antigen binding fragment thereof as described herein may comprise a human constant region, e.g., an IgG constant region, such as an IgG 1 , lgG2, lgG3 or lgG4 constant region or mixtures thereof. In the case of an antibody or proteincomprising a VH and a VL, the VH can be linked to a heavy chain constant region and the VL can be linked to a light chain constant region.

[0103] In one example an antibody or antigen binding fragment thereof as described herein comprises a heavy chain constant region, comprising a stabilized heavy chain constant region, comprising a mixture of sequences fully or partially with or without the C-terminal lysine residue.

[0104] In one example, an antibody or antigen binding fragment thereof as described herein comprises a VH disclosed herein linked or fused to an lgG4 constant region or stabilized lgG4 constant region (e.g., as discussed above) and the VL is linked to or fused to a kappa light chain constant region.

[0105] The functional characteristics of an antigen binding fragment thereof as described herein will be taken to apply mutatis mutandis to an antibody as described herein.

[0106] An antibody or antigen binding fragment thereof for a use as described herein may be purified, substantially purified, isolated and / or recombinant.Table 1 : Summary of amino acid and nucleotide sequences of preferred CAIX-binding antibodies

[0107] In further embodiments, the moiety for binding to CAIX may comprise BCA-356, a bispecific antibody comprising affinity matured humanised anti-CAIX antibody linked to attenuated subunits of IL-12 fused to each of the heavy chains of the anti-CAIX antibody at the C-terminus via a linker into a “knobs in hole” format (as described in Nair et al., Journal for ImmunoTherapy of Cancer, 10:S2).

[0108] In particularly preferred embodiments of the methods and uses described herein, the agent for binding to CAIX is89Zr -Girentuximab,124l-Girentuximab,177Lu- Girentuximab, or111ln-Girentuximab-IRDye800CW (for example, as described in Stroet et aL, (2022), Cancers 14: 861 , incorporated herein by reference), G250RIT (when labelled with an appropriate detectable moiety), or9°Y-DOTA-cG250.

[0109] In an especially preferred embodiment, the agent for binding to CAIX is89Zr- Girentuximab.89Zr -Girentuximab is a chimeric monoclonal antibody (INN name: girentuximab (GTX), also known as cG250 and TLX250) with specificity for CAIX antigen,radiolabelled with the positron emitting radio-metal zirconium-89 via a NSuc-DFO-TFP- ester (DFO-TFP), linked to lysine residues of GTX to yield89Zr DFO-TFP-GTX.Constant regions

[0110] In preferred embodiments, any antibody and / or antigen binding fragment thereof as described herein for use in the present invention may comprise a constant region of an antibody. This includes antigen binding fragments of an antibody fused to an Fc.

[0111] Sequences of constant regions useful for producing the antibodies or antigen binding fragment thereof as described herein may be obtained from a number of different sources. In some examples, the constant region or portion thereof of the protein is derived from a human antibody. The constant region or portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including lgG1 , lgG2, lgG3 and lgG4. In one example, the constant region is human isotype lgG4 or a stabilized lgG4 constant region.

[0112] The neonatal Fc-receptor (FcRn) is important for the metabolic fate of antibodies of the IgG class in vivo. The FcRn functions to salvage IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. It is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (a-FcRn) and a 15 kDa p2-microglobulin (P2qi). FcRn binds with high affinity to the CH2-CH3 portion of the Fc-region of an antibody of the class IgG. The interaction between an antibody of the class IgG and the FcRn is pH dependent and occurs in a 1 :2 stoichiometry, i.e. one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc-region polypeptides (see e.g. Huber, A.H., et al, J. Mol. Biol. 230 (1993) 1077-1083).

[0113] Thus, an IgG’s in vitro FcRn binding properties / characteristics are indicative of its in vivo pharmacokinetic properties in the blood circulation. In the interaction between the FcRn and the Fc-region of an antibody of the IgG class different amino acid residues of the heavy chain CH2- and CH3 -domain are participating.

[0114] Different mutations that influence the FcRn binding and therewith the half-live in the blood circulation are known. Fc-region residues critical to the mouse Fc-region-mouse FcRn interaction have been identified by site-directed mutagenesis (see e.g. Dall'Acqua,W.F., et al. J. Immunol 169 (2002) 5171 -5180). Residues Ile253, H is310, His433, Asn434 and His435 (numbering according to EU index numbering system) are involved in the interaction (Medesan, C, et al., Eur. J. Immunol. 26 (1996) 2533-2536; Firan, M., et al, Int. Immunol. 13 (2001 ) 993-1002; Kim, J.K., et al, Eur. J. Immunol. 24 (1994) 542-548). (Using the Kabat system, the relevant residues are Ile266, His329, His464, Asn465 and His466). Residues Ile253, His310, and His435 were found to be critical for the interaction of human Fc-region with murine FcRn (Kim, J.K., et al, Eur. J. Immunol. 29 (1999) 2819- 2885).

[0115] More specifically, the antibody or antigen binding protein may comprise one or more amino acid substitutions that decrease the half-life of the protein. For example, the antibody or antigen binding fragment thereof may comprise a Fc region comprising one or more amino acid substitutions that decrease the affinity of the Fc region for the neonatal Fc region (FcRn).Preferred Modifications

[0116] In any embodiment, the antibody or antigen binding fragment thereof (for example a G250 antibody or variant thereof as described herein) is a modified IgG antibody or fragment thereof, comprising a heavy chain constant region having one or more amino acid substitutions compared to a wild-type antibody of the class IgG, wherein the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn), thereby reducing the serum half-life of the modified antibody compared to a wild-type antibody of class IgG.

[0117] In one embodiment, the one or more amino acid substitutions are selected from substitutions in the heavy chain constant region 2 (CH2) of the IgG molecule, reducing the affinity of the IgG molecule for FcRn. Alternatively, the one or more amino acid substitutions may be in the heavy chain constant region 3 (CH3) of the IgG molecule, thereby reducing the affinity of the IgG molecule for FcRn. Still further, the amino acid substitutions may include at least one substitution in the CH2 region, and at least one substitution in the CH3 region of the IgG molecule, whereby the substitutions reduce the affinity of the IgG for FcRn.

[0118] In certain preferred embodiments, the one or more amino acid substitutions may be at one or more of residues His310, His433, His435, His436, or Ile253 of IgG.Preferably, the amino acid substitutions comprise a substitution in the heavy chain constant region at positions His310 or at His435. More preferably, the amino acid substitutions that reduce the affinity of the antibody for FcRn are at both His310 and His435.

[0119] In other preferred embodiments, the antibody and / or antigen binding fragment thereof has a constant region substantially identical to a naturally occurring class IgG antibody constant region wherein at least one amino acid residue selected from the group consisting of residues His310, His435, and Ile253 is different from that present in the naturally occurring class IgG antibody, thereby altering FcRn binding affinity and / or serum half-life of said antibody relative to the naturally occurring antibody. In preferred embodiments, the naturally occurring class IgG antibody comprises a heavy chain constant region of a human IgG 1 , lgG2, lgG2M3, lgG3 or lgG4 molecule.

[0120] Also in preferred embodiments, amino acid residue 310 and / or residue 435 from the heavy chain constant region of the antibody having a constant region substantially identical to the naturally occurring class IgG antibody is any amino acid that is not histidine and which reduces the affinity of the constant region for FcRn. For example, the amino acid at residue 310 and / or 435 may be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine or glycine.

[0121] The amino acid substitutions may include substitution from a histidine residue to: alanine, glutamine, glutamic acid or aspartic acid. Preferably, the amino acid substitution at His310 is to alanine. Preferably the amino acid substitution at His435 is to glutamine. Preferably, the amino acid substitution at Ile253 is alanine.

[0122] In a preferred embodiment of the present invention, the binding affinity for FcRn and / or the serum half-life of the modified antibody is decreased by at least about 30%, 50%, 80%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold. In a preferred embodiment of the present invention, the binding affinity for FcRn and / or the serum half-life of said modified antibody is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0123] In addition, the antibodies or fragments thereof for use according to the present invention may be modified to comprise one or more mutations which modify the affinity of the antibodies for any one or more Fc gamma receptors. For example, the one or more amino acid modifications change the affinity of the antibody constant domain, Fc region, or Fc gamma receptor binding fragment, for any one or more Fc gamma receptors.

[0124] In certain embodiments, the modified antibody or antigen binding fragment thereof retains the ability to bind to one or more Fc-gamma receptors and accordingly, in certain embodiments the modified antibody retains the ability to stimulate effector responses (including ADCC). In one example, the Fc region of the constant region contains one or more amino acid substitutions that modulate effector function, including increasing effector function compared to a wild-type IgG.

[0125] In one example, the Fc region of the constant region has a reduced ability to induce effector function, e.g., compared to a native or wild-type human IgG 1 or lgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of an Fc region containing protein are known in the art and / or described herein.

[0126] In one example, the amino acid substitution that modifies that ability of the antibody to induce effector function is an amino acid substitution at residue Ile253 from the heavy chain constant region. In one example, the substitution is to any amino acid selected from be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine or glycine, wherein the substitution reduces the ability of the antibody to induce effector function. In preferred embodiments, the substitution from He at residue 253 is to arginine, proline, glutamic acid or aspartate, more preferably alanine.

[0127] In one example, the Fc region is an lgG4 Fc region (i.e., from an lgG4 constant region), e.g., a human lgG4 Fc region. Sequences of suitable lgG4 Fc regions will be apparent to the skilled person and / or available in publicly available databases (e.g., available from National Center for Biotechnology Information).

[0128] In one example, the constant region is a stabilized lgG4 constant region. The term “stabilized lgG4 constant region” will be understood to mean an lgG4 constant region that has been modified to reduce Fab arm exchange or the propensity to undergo Fab arm exchange or formation of a half-antibody or a propensity to form a half antibody. “Fab arm exchange" refers to a type of protein modification for human lgG4, in which an lgG4 heavy chain and attached light chain (half-molecule) is swapped for a heavy-light chain pair from another lgG4 molecule. Thus, lgG4 molecules may acquire two distinct Fab arms recognizing two distinct antigens (resulting in bispecific molecules). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. A “half antibody” forms when an lgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

[0129] In one example, a stabilized lgG4 constant region comprises a proline at position 241 of the hinge region according to the system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991 ). This position corresponds to position 228 of the hinge region according to the EU numbering system. In human lgG4, this residue is generally a serine. Following substitution of the serine for proline, the lgG4 hinge region comprises a sequence CPPC. In this regard, the skilled person will be aware that the “hinge region” is a proline-rich portion of an antibody heavy chain constant region that links the Fc and Fab regions that confers mobility on the two Fab arms of an antibody. The hinge region includes cysteine residues which are involved in inter-heavy chain disulfide bonds. It is generally defined as stretching from Glu226 to Pro243 of human IgG 1 according to the numbering system of Kabat (or Glu216 to Pro230 using the EU index). Hinge regions of other IgG isotypes may be aligned with the lgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain disulphide (S-S) bonds in the same positions (see for example WO2010 / 080538).

[0130] In alternative embodiments, the one or more amino acid modifications which reduce the affinity for the FcRn receptor also reduce the affinity for the Fc gamma receptors. The modified antibody or antigen binding fragment thereof may further comprise one or more amino acid substitutions compared a wild-type antibody of the class IgG, wherein the amino acid substitutions further reduce the affinity of the antibody for one or more Fc gamma receptors.

[0131] In a further embodiment, the modified antibody or antigen binding fragment thereof further comprises one or more amino acid substitutions compared a wild-type antibody of the class IgG, wherein the amino acid substitutions increase the stability of the CH1 -CH2 hinge region in the modified antibody compared to a wild-type antibody of the class IgG.

[0132] In any embodiment, the heavy chain constant region of the antibody or antigen binding protein comprises amino acid substitutions at both His310 and His435. The antibody may also comprise amino acid substitutions at residues equivalent to Ser228 and Leu235 of the constant heavy chain region.

[0133] In any embodiment, the antibody or antigen binding fragment thereof comprises mutations at Ser228, Leu235, His310 and His435. Preferably, the amino acid modifications are Ser228Pro, Leu235Glu, His310Ala and His435Gln.

[0134] Additional examples of stabilized lgG4 antibodies are antibodies in which arginine at position 409 in a heavy chain constant region of human lgG4 (according to the EU numbering system) is substituted with lysine, threonine, methionine, or leucine (e.g., as described in W02006 / 033386). The Fc region of the constant region may additionally or alternatively comprise a residue selected from the group consisting of: alanine, valine, glycine, isoleucine and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e., a CPPC sequence) (as described above).

[0135] In another example, the Fc region is a region modified to have reduced effector function, i.e., a “non-immunostimulatory Fc region”. For example, the Fc region is an IgG 1 Fc region comprising a substitution at one or more positions selected from the group consisting of 268, 309, 330 and 331. In another example, the Fc region is an lgG1 Fc region comprising one or more of the following changes E233P, L234V, L235A and deletion of G236 and / or one or more of the following changes A327G, A330S and P331 S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J Biol Chem. 276(9):6591 -604, 2001 ). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177 : 1129-1 138 2006; and / or Hezareh J Virol ;75: 12161 -12168, 2001 ).

[0136] In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one CH2 domain from an lgG4 antibody and at least one CH3 domain from an IgG 1 antibody, wherein the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0137] Preferably, the antibody or antigen binding fragment thereof comprises a heavy chain constant region comprising the sequence as set forth in any one of SEQ ID NOs: 177 to 180, preferably as set forth in SEQ ID NO: 178.

[0138] In a still further embodiment, the antibody or antigen binding fragment thereof preferably comprises a heavy chain comprising the sequence set forth in any one of SEQ ID NOs: 182 to 185, preferably as set forth in SEQ ID NO: 183.

[0139] In any embodiment, the antibody or antigen binding fragment thereof comprises a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 181. Preferably, the antibody or antigen binding protein comprises a light chain comprising the amino acid sequence as set forth in SEQ ID NO: 186.

[0140] In any embodiment, the antibody or antigen binding fragment thereof comprises the sequence set forth in SEQ ID NO: 183 and the sequence set forth in SEQ ID NO: 186.

[0141] In one embodiment, the antibody or antigen binding fragment thereof comprises: a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to, or comprises a sequence set forth in SEQ ID NO: 36 or 52 and the VL comprises a sequence at least about 95% or 96% or 97% or 98% or 99% identical to, or comprises a sequence set forth in SEQ ID NO: 1 16, 132 or 148.

[0142] Preferably, the VH comprises a sequence at least about 95% or 96% or 97% or 98% or 99% identical to, or comprises a sequence set forth in SEQ ID NO: 36 or 52 and a VL comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to, or comprises a sequence set forth in SEQ ID NO: 132 or 148.

[0143] More preferably, the VH comprises a sequence at least about 95% or 96% or 97% or 98% or 99% identical to, or comprises a sequence set forth in SEQ ID NO: 36and the VL comprises a sequence at least about 95% or 96% or 97% or 98% or 99% identical to, or comprises a sequence set forth in SEQ ID NO: 148.

[0144] Alternatively, the VH comprises a sequence at least about 95% or 96% or 97% or 98% or 99% identical to, or comprises a sequence set forth in SEQ ID NO: 52 and the VL comprises a sequence at least about 95% or 96% or 97% or 98% or 99% identical to, or comprises a sequence set forth in SEQ ID NO: 132 or 148, preferably the sequence set forth in SEQ ID NO: 148.Detectable moiety

[0145] The skilled person will be familiar with standard methods for conjugating a detectable moiety to an agent for binding to CAIX.

[0146] In any embodiment of the invention, a small molecule, a peptide, a protein or antibody for binding to CAIX and as described herein may be directly or indirectly linked to a detectable moiety, such as a radioisotope, dye or fluorescent moiety.

[0147] In any embodiment, the detectable moiety is a radioisotope. Examples of suitable isotopes include: gallium-67 and gallium-68 (67Ga and68Ga), indium-1 11 (111ln), iodine-123, iodine-124 or iodine 131 (123l,124l, or131I), technetium-99 (99mTc), and zirconium-89 (89Zr). As used herein, the term radionuclide may be used interchangeably with the term radioisotope.

[0148] It will be understood that the radioisotopes may be conjugated to a polypeptide (eg antibody) directly (via a chelating agent or prosthetic group or linker) or indirectly via binding to single or multiple amino acid residues in the protein (e.g. halogenation of tyrosine residues).

[0149] In alternative embodiments, chelating agents or linkers may be used in order to conjugate the datable moiety to a peptide or protein for binding to CAIX. In one example, the peptide or protein (eg antibody) can be conjugated to a chelating moiety, selected from the group consisting of: TMT (6,6"-bis[N,N",N"'-tetra(carboxymethyl)aminomethyl)- 4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1 , 4,7,10- tetraazacyclododecane-NN',N"(N"'-tetraacetic acid, also known as tetraxetan), TCMC (the tetra-primary amide of DOTA), DO3A (1 ,4,7,10-Tetraazacyclododecane-1 ,4,7- tris(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1 ,4,7,10-tetraazabicyclo[5.5.2]tetradecan), NOTA (1 ,4,7-triazacyclononane-triacetic acid) Diamsar (3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1 ,8-diamine), DTPA (Pentetic acid or diethylenetriaminepentaacetic acid), CHX-A”-DTPA ([(R)-2-Amino-3-(4- isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1 ,2-diamine-pentaacetic acid), TETA (1 ,4,8,1 1 -tetraazacyclotetradecane-1 ,4,8), 11 -tetraacetic acid, Te2A (4,1 1 - bis(carboxymethyl)-1 ,4,8,11 -tetraazabicyclo[6.6.2]hexadecane), HBED, DFO (Desferrioxamine), DFOsq (DFO-squaramide) and HOPO (3,4,3-(LI-1 ,2-HOPO) or other chelating agent as described herein. Other known chelating moieties include 3p-C-NETA ({4-[2-(bis-carboxy-methylamino)-5-(4-nitrophenyl)pentyl]-7-carbo-xymethyl-[1 ,4,7]triazanonan-1 -yl} acetic acid), 5p-C-NETA (2-({1 -[4,7-b / s(carboxymethyl)-1 ,4,7- triazanonan-1 -yl]-7-(4-nitrophenyl)heptan-2-yl}(carbo-xymethyl) amino)acetic acid), NOTA (1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid) and NODA (1 ,4,7- triazacyclononane-1 ,4-diacetic acid).

[0150] In certain non-limiting embodiments discussed below, a chelating group may be used to attach18F or19F complexed with a metal, such as aluminum, to provide an alternative modality for imaging, detection and / or diagnosis. It is anticipated that fluorescent-labeled molecules may be of more use for intraoperative procedures, while18F-labeled molecules may be of greater use for pre- or post-operative imaging, detection and / or diagnosis of diseased tissues.

[0151] The agent may be modified to contain sulfhydryl groups for attaching maleimide- modified fluorescent probes. Alternatively, bis-functional cross-linking agents, or fluorescent dyes conjugated to other reactive species, may be used to attach the fluorescent probe to a different group on the agent for binding CAIX. For example, DYLIGHT® 488 and DYLIGHT® 800 are available as amine-reactive dyes derivatized with NHS ester for labeling primary amines (Product Nos. 46402 and 46421 , Thermo Electric, Rockford, HL). The skilled artisan will realize that the fluorescent probes of use are not limiting and other DYLIGHT® dyes, or alternative fluorescent probe molecules known in the art, may be used in the claimed methods and compositions.

[0152] In certain embodiments, a peptide or protein (eg antibody) for binding to CAIX may be conjugated to a fluorescent probe (to form an immunoconjugate). Methods for covalent conjugation of fluorescent probes and other functional groups are known in the art and any such known method may be utilized. For example, a fluorescent probes canbe attached at the hinge region of a reduced antibody component via disulfide bond formation or sulfhydryl-maleimide interaction. Alternatively, such agents can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et aL, Int. J. Cancer 56: 244 (1994). General techniques for such conjugation are well-known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991 ); Upeslacis et aL, “Modification of Antibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995).

[0153] Alternatively, the fluorescent probes can be conjugated via a carbohydrate moiety in the Fc region of the antibody. See, for example, Shih et aL, Int. J. Cancer 41 : 832 (1988); Shih et aL, Int. J. Cancer 46: 1101 (1990); and Shih et aL, U.S. Pat. No. 5,057,313, the Examples section of which is incorporated herein by reference. The general method involves reacting an antibody component having an oxidized carbohydrate portion with a fluorescent probes that has at least one free amine function. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0154] The Fc region may be absent if the antibody used as the antibody component of the immunoconjugate is an antibody fragment. However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et aL, J. Immunol. 154: 5919 (1995); U.S. Pat. Nos. 5,443,953 and 6,254,868, the Examples section of which is incorporated herein by reference. The engineered carbohydrate moiety is used to attach the functional group to the antibody fragment.

[0155] An alternative method for attaching fluorescent probes or other functional groups to a targeting molecule involves use of click chemistry reactions. The click chemistry approach was originally conceived as a method to rapidly generate complex substances by joining small subunits together in a modular fashion. (See, e.g., Kolb et aL, 2004, Angew Chem Int Ed 40:3004-31 ; Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistry reaction are known in the art, such as the Huisgen 1 ,3-dipolar cycloaddition copper catalyzed reaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), which is often referred to as the “click reaction.” Other alternatives include cycloaddition reactions such as the Diels-Alder, nucleophilic substitution reactions (especially to small strained rings like epoxy and aziridine compounds), carbonyl chemistry formation of urea compounds and reactions involving carbon-carbon double bonds, such as alkynes in thiol-yne reactions.

[0156] A copper-free click reaction has been proposed for covalent modification of biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc 126:15046-47.) The copper- free reaction uses ring strain in place of the copper catalyst to promote a [3+2] azidealkyne cycloaddition reaction. For example, cyclooctyne is a 8-carbon ring structure comprising an internal alkyne bond. The closed ring structure induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole. Thus, cyclooctyne derivatives may be used for copper-free click reactions.

[0157] Another type of copper-free click reaction was reported by Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involving strain-promoted alkyne-nitrone cycloaddition. To address the slow rate of the original cyclooctyne reaction, electron-withdrawing groups are attached adjacent to the triple bond. Examples of such substituted cyclooctynes include difluorinated cyclooctynes, 4-dibenzocyclooctynol and azacyclooctyne. An alternative copper-free reaction involved strain-promoted alkyne-nitrone cycloaddition to give N-alkylated isoxazolines. The reaction was reported to have exceptionally fast reaction kinetics and was used in a one-pot three-step protocol for site-specific modification of peptides and proteins. Nitrones were prepared by the condensation of appropriate aldehydes with N-methylhydroxylamine and the cycloaddition reaction took place in a mixture of acetonitrile and water. These and other known click chemistry reactions may be used to attach chelating moieties to antibodies or other CAIX-binding molecules in vitro.

[0158] In certain embodiments, the agent may comprise a peptide or protein (eg antibody) that is covalently coupled to the radioactive isotope124L This isotope is a positron emitter that can be attached to antibodies e.g. as described by Larsson et al. (J. Nucl. Med. 33 (1992), 2020-2023) or US 5,185,142, the content of which is herein incorporated by reference.

[0159] In any embodiment, radiolabelling of a protein or antibody is accomplished by covalent iodination, particularly with the lodogen Reagent (1 ,3,4,6-tetrachloro-3a,6a- diphenyl glycoluril). lodogen labeling is a solid phase oxidative method that is similar to the Chloramine-T method, but is generally considered to be milder, since the reaction takes place on the surface of the oxidant, minimizing exposure of the substrate (Salacinzki, P.R.P., et al., AnaLBiochem. 117:136 (1981 )).

[0160] Chelators with radiometals and other halogenated radioisotopes may be bound to proteins or antibodies via one or more amino acid residues or reactive moieties in the protein / antibody, including but not limited to one or more lysine residues, tyrosine residues or thiol moieties.

[0161] In another example, the protein or antibody may be conjugated to a bifunctional linker, for example, bromoacetyl, thiols, succinimide ester, TFP ester, a maleimide, or using any amine or thiol- modifying chemistry known in the art.

[0162] The skilled person will be familiar with standard methods for conjugating chelating agents to proteins, including antibodies and derivatives or fragments thereof. In addition, the skilled person will be familiar with approaches for selecting a relevant chelating agent for pairing with a radiometal, for example as described in Chem. Soc. Rev., 2014,43, 260, incorporated herein by reference.

[0163] In any embodiment, the datable moiety may be a fluorescent dye, such as but not limited to those described in US 20150086482, incorporated herein by reference.

[0164] In any embodiment, the fluorescent dye (which may also be referred to as a fluorescent probe) may be selected from: Alexa 350, Alexa 430, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein, 5-carboxy-2',4',5',7'- tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1 ,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, paraaminobenzoic acid, erythrosine, phthalocyanines, azomethines, cyanines, xanthines, succinylfluoresceins, rare earth metal cryptates, europium trisbipyridine diamine, aeuropium cryptate or chelate, diamine, dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin, phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), Tetramethylrhodamine, and Texas Red.Administration of agent

[0165] The skilled person will appreciate that dosage of the agent for use in accordance with the methods of the invention will depend on various factors including the age, sex, height and weight of the subject to whom the agent is administered, and depending on the agent.

[0166] Where the agent is an antibody for binding to CAIX, preferably the antibody is administered to a subject or infused at a dose from about 1 mg to about 50 mg, preferably in a dose from about 5 mg to about 20 mg, and more preferably in a dose of about 10 mg. The specific activity of the radiolabeled antibody is preferably about 15 to about 20 MBq / mg, more preferably about 18 to about 19 MBq / mg.

[0167] In certain embodiments, the agent for binding to CAIX is a radiolabelled girentuximab antibody, and the antibody is administered at a mass dose of about 10 mg of girentuximab by slow infusion.

[0168] The antibody is usually administered as a pharmaceutical composition with a pharmaceutically acceptable carrier, e.g. physiological saline solution, optionally comprising a protein stabilizer such as human serum albumin (HSA). The antibody is preferably administered by infusion.

[0169] The CAIX-binding agent, preferably girentuximab, or a humanised variant thereof, is preferably administered intravenously, preferably by infusion or intravenous injections. The administration of the antibody by infusion is preferably performed over a period of up to about 30 minutes, more preferably in about 15 minutes. Of course, the CAIX inhibitor can also be applied intraperitoneally or intramuscularly.Detection methods

[0170] It will be appreciated that the methods for detecting or imaging of the agents for use according to the invention will depend on the nature of the detectable moiety of the agent.

[0171] The step of detecting is preferably performed using PET, SPECT, fluorescence spectroscopy, or any other suitable method.

[0172] Examples of in vivo methods for determining the presence or expression of CAIX in a tumour include use of in v / vo / partial or whole body imaging techniques such as Positron Emission Tomography (PET) and single photon emission computed tomography (SPECT) imaging. Immuno-PET and immuno-SPECT imaging may include the use of CAIX-binding molecules that are conjugated to a radioisotope to enable non-invasive imaging of tissues and tumours expressing CAIX.

[0173] In the case where the detectable moiety is a radioisotope, the methods will therefore involve determining radiation for the subject who was administered the agent.

[0174] The in vivo detection step in the methods described above may be whole body imaging or local imaging at specific sites, such as but not limited to sites of anticipated or likely solid tumour growth.

[0175] In the case of SPECT, the agent for binding to CAIX typically comprises a detectable agent in the form of a gamma-emitting radioisotope (radionuclide), normally through injection into the blood stream. Typically gamma-emitting radioisotopes for use in SPECT include99mTc (technetium),123l or131I (iodine) and68Ga (gallium).

[0176] In any embodiment, where the agent comprises a radioisotope, the detection method may comprise Positron Emission Tomography (PET).

[0177] Optionally, the detection method comprises PET / CT imaging or PET / MRI scanning.

[0178] Following administration (preferably infusion) of the agent, it may be practical to wait for a period of time to allow for the agent to accumulate at the site of cancer cells expressing tumours. Typically, the period of time will be at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at leastabout 7 days, at least about 8 days, at least about 9 days, at least about 10 days. Preferably, the time between administration of the agent and the detection of the agent (eg by PET or other method described here), the period is typically no more than about 10 days, or no more than about 15 days or no more than about 20 days.

[0179] In the case where the imaging or detection of the cancer is using PET, the PET imaging may be performed preferably within 7 ± 2 days of infusion of the radiolabelled agent, in particular 5 ± 2 days after the infusion, in order to obtain the optimal imaging results including accumulation of the agent at sites where CAIX is present.

[0180] In the context where the detectable moiety is a fluorescent probe or dye, detection of the moiety can be using fluorescent imaging including during intraoperative, intravascular or endoscopic procedures, as described in U.S. Pat. Nos. 4,932,412; 6,096,289; 6,387,350; 7,201 ,890; the Examples section of each cited patent incorporated herein by reference. Such imaging methods may be of use, for example, to image the distribution of tumor tissue to facilitate its removal. Fluorescent imaging may also be of use for diagnostic purposes, for example to distinguish between malignant, benign and hyperplastic tissues.Cancers to be detected or diagnosed

[0181] The present invention provides methods for identifying or imaging cancers in vivo. Such methods are expected to be useful in the diagnosis of cancers which express CAIX, preferably without the need for additional, invasive techniques (such as biopsy collection and testing), for confirming diagnosis.

[0182] Accordingly in a preferred embodiment, the methods of the invention enable the diagnosis of any cancer recited herein, as the sole, principal or main mode of diagnosis of the cancer and preferably without the need for additional invasive methods of diagnosis including biopsy-related methods.

[0183] The methods of the invention are also expected to be useful for the staging of the progression of a cancer or the success of treatment of a cancer. Again, such methods provide the benefit of providing a non-invasive means for assessing the cancer in a subject.

[0184] As used herein, the term “cancer” refers to a malignant growth or tumour resulting from an uncontrolled division of cells. The term “cancer” includes primary tumours and metastatic tumours.

[0185] The methods of the present invention find particular utility in the imaging, detection and / or diagnosis of cancers which have not previously been identified using in vivo imaging techniques utilising an agent for binding to CAIX.

[0186] A subject for whom the diagnosis or detecting or imaging of the cancer described herein, may be suspected of having or be at risk of having the cancer. A subject suspected of having a cancer may exhibit one or more symptoms of the cancer, may have a family history of the cancer or may have one or more genetic markers indicating a risk or likelihood of developing the cancer. A subject considered at risk of having the cancer may exhibit one or more symptoms of the cancer, may have a family history of the cancer or may have one or more genetic markers indicating a risk or likelihood of developing the cancer.

[0187] In any embodiment, the cancer being detected, imaged or diagnosed is breast cancer. High levels of CAIX have previously been reported in breast cancers and CAIX expression has also been reported to be associated with resistance to chemotherapy or an indicator of treatment success. These observations date back over several decades and prior to now, diagnosis using a CAIX-binding imaging agent has not previously been reported for this patient group.

[0188] The breast cancer may be so-called “triple negative breast cancer” (TNBC), an aggressive, metastatic and drug-resistant form of breast cancer has limited therapeutic options, and which is negative for other biomarkers of breast cancer such as the Estrogen receptor (ER positive breast cancer), Progesterone receptor (PR positive breast cancer) and human epidermal growth factor receptor 2 (HER2 positive breast cancer).

[0189] In any embodiment, the breast cancer may be hormone receptor positive breast cancer, such as ER positive, PR positive, ER&PR positive. In any embodiment, the breast cancer may be positive for HER2 (including HER2 and hormone receptor positive breast cancer).In certain embodiments, the cancer being detected, imaged or diagnosed is not breast cancer.

[0190] In any embodiment, the cancer being detected, imaged or diagnosed is cervical cancer. The cervical cancer may be a squamous cell carcinoma, or an adenocarcinoma. In any embodiment, a subject for whom diagnosis or imaging of a cervical cancer may exhibit one or more symptoms of cervical cancer such as abnormal vaginal bleeding, including contact bleeding, or pelvic pain. A subject considered at risk of cervical cancer may have had a previous infection with HPV 16 or 18 strains, or have one or more genetic markers indicative of a risk of cervical cancer.

[0191] In any embodiment, the cancer being detected, imaged or diagnosed is colorectal cancer (including, for example, an epithelial colorectal adenocarcinoma). In any embodiment, a subject for whom diagnosis or imaging of a colorectal cancer may exhibit one or more symptoms of colorectal cancer such as persistent change in bowel habits, rectal bleeding or blood in stools, persistent abdominal discomfort, weakness or fatigue, and unexplained weight loss. A subject considered at risk of colorectal cancer may have a family history of the disease or have one or more genetic markers considered to be associated with increased risk of colorectal cancer or may previously have had intestinal polyps.

[0192] In any embodiment, the cancer being detected, imaged or diagnosed is esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma). A subject for whom diagnosis or imaging or detection of esophageal cancer may exhibit one or more symptoms selected from: dysphagia, unexplained weight loss, chest pain, pressure or burning, worsening indigestion or heartburn or coughing or hoarseness. A subject considered at risk of esophageal cancer may have a family history of the disease or have one or more genetic markers considered to be associated with increased risk of colorectal cancer, or may previously have been diagnosed with Barrett’s esophagus.

[0193] In any embodiment, the cancer being detected, imaged or diagnosed is gastric cancer (including gastric adenocarcinoma). A subject for whom diagnosis or imaging or detection of gastric cancer may exhibit one or more symptoms selected from: dysphagia, stomach pain, feeling bloated after small amounts of food, loss of appetite, indigestion, nausea and vomiting, fatigue, blackened stools. A subject considered at risk of gastric cancer may have a family history of the disease or have one or more genetic markers considered to be associated with increased risk of gastric cancer.

[0194] In any embodiment, the cancer being detected, imaged or diagnosed is glioblastoma multiforme. A subject for whom diagnosis or imaging or detection of glioblastoma may exhibit one or more symptoms including vision, hearing, balance, coordination, strength and reflex symptoms, nausea, vomiting, seizures or other neurological symptoms.

[0195] In any embodiment, the cancer being detected, imaged or diagnosed is head and neck cancer (including head and neck squamous cell carcinoma and nasopharyngeal and hypopharyngeal carcinoma). A subject for whom diagnosis or imaging or detection of head and neck cancer may exhibit one or more symptoms such as pain, swelling, hoarseness, sore throat, persistent cough, halitosis, unexplained weight loss. A subject considered at risk of head and neck cancer may have a family history of the disease, have one or more genetic markers considered to be associated with increased risk of head and neck cancer, may have had a previous infection with HPV or Epstein-Barr virus, a weakened immune system, poor oral hygiene including gum disease, smoking or chewing betel nut, areca nut, gutka or pan, or have an inherited condition such as Fanconi anaemia or Li-Fraumeni syndrome.

[0196] In any embodiment, the cancer being detected, imaged or diagnosed is liver cancer (including cholangiocarcinoma and hepatocellular carcinoma). As used herein, cholangiocarcinoma refers to biliary, or bile duct cancer. The cholangiocarcinoma may be intrahepatic, hilar of distal cholangiocarcinoma. The cancer may be gallbladder cancer or cancer of the ampulla of Vater. In any embodiment, a subject for whom diagnosis or imaging of a liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) may be a subject exhibiting one or more symptoms of liver cancer (including cholangiocarcinoma and hepatocellular carcinoma.

[0197] As used herein, one of more symptoms of cholangiocarcinoma include: abdominal pain, yellowish skin (jaundice), weight loss, generalised itching, fever, light coloured stool or dark urine. The skilled person will be familiar with various risk factors for cholangiocarcinoma include primary sclerosing cholangitis (an inflammatory disease of the bile ducts), ulcerative colitis, cirrhosis, hepatitis C, hepatitis B, infection with certain liver flukes, and some congenital liver malformations. However, most people have no identifiable risk factors.

[0198] In any embodiment, the cancer being detected, imaged or diagnosed is lung cancer (including epithelial non-small cell and small cell carcinoma). As used herein, the term "lung cancer" includes, but is not limited to all types of lung cancers at all stages of progression like lung carcinomas metastatic lung cancer, non-small cell lung carcinomas (NSCLC) such as lung adenocarcinoma, squamous cell carcinoma, or small cell lung carcinomas (SCLC). In some embodiments, the subject suffers from a non-small cell lung carcinomas (NSCLC).

[0199] In any embodiment, the cancer being detected, imaged or diagnosed is ovarian cancer (including epithelial ovarian carcinoma).

[0200] In any embodiment, the cancer being detected, imaged or diagnosed is pancreatic cancer (including pancreatic ductal adenocarcinoma).

[0201] In any embodiment, the cancer being detected, imaged or diagnosed is soft tissue sarcoma.

[0202] In any embodiment, the cancer being detected, imaged or diagnosed is bladder cancer. The bladder cancer may be non-muscle-invasive bladder cancer (NMIBC).

[0203] In particularly preferred embodiments, the cancer being detected or imaged or diagnosed is not a renal cancer (including clear cell renal cancer).

[0204] Imaging or diagnosis of the cancer will typically be assessed following administration of the agent, and detection thereof, by qualitatively assessing the detection of the agent compared with convention imaging. Quantitative assessment may be on a per lesion basis, including standardized uptake values (SUV) (SUVmax and SUVmean), SUV corrected for lean mass (SUL), metabolic tumor volume (MTV), and Tumour-to- background ratio (TBR)

[0205] The Tumor-to-Background Ratio (TBR) will typically be defined as the ratio of the lesion standardized uptake values (SUVmax) over the reference region SUV (liver, blood pool, etc). Comparison between the number, size and other characteristics of lesions detected by PET scan and standard imaging modalities including high-resolution CT / MRI and other potential imaging per patient (depending on the tumor type), type of lesion and indication will be performed.

[0206] Imaging Qualitative visual analysis (presence or absence of localized agent uptake associated with tumor, as seen on contrast-enhanced CT, MRI or FDG PET / CT), may be used to evaluate concordance of tumor lesion detection between agent-specific PET / CT and conventional imaging. RECIST 1.1 criteria for conventional imaging may be used as the main tool for concordance comparison vs PET.

[0207] In addition to the above, all visible tumoral lesions at conventional imaging may also be compared to PET imaging result.ExamplesExample 1 : Clinical trial protocol

[0208] The trial relates to the evaluation of CAIX expression in a subset of solid tumours using89Zr-labelled girentuximab deferoxamine PET / CT imaging.Primary objectives

[0209] To non-invasively evaluate89Zr-girentuximab PET / CT imaging of CAIX tumoral expression in different solid tumors. No formal imaging studies have been performed on the uptake of89Zr-girentuximab by these tumor types.

[0210] Primary endpoints: Qualitative (yes I no) and quantitative assessment of 89Zr- girentuximab uptake compared with the conventional imaging. Descriptive statistics are reported for each tumor type. Per lesion analysis include SUVmax, SUVmean, SUL (SUV corrected for lean mass), tumor-to-background ratio (TBR), and metabolic tumor volume (MTV).Secondary objectives

[0211] To evaluate tolerability and safety of89Zr-girentuximab administration in patients with different tumor types.

[0212] Secondary endpoints: Patient safety is evaluated based on the incidence and nature of adverse events (AEs) and serious adverse events (SAEs), and clinically significant changes in laboratory test values, vital signs, or findings on physical exam. Laboratory abnormalities are assessed according to the NCI CTCAE v.5.0 The patient is informed that in the event of abnormal physical signs occurring within 24 hours of theexamination, they must report to the principal investigator for registration. The NCI Common Toxicity Criteria, version 5.0 reference is used.Tertiary objectives

[0213] To evaluate the correlation between the standardized uptake values (SUV) of89Zrgirentuximab and CAIX histological expression in patients undergoing biopsy or surgery within 90 days from89Zr-girentuximab imaging.

[0214] T ertiary endpoints: If a biopsy or surgical sample is available within 90 days prior to dosing or 90 days after89Zr-girentuximab imaging (and for whom tissue sample is available), the correlation between the standardized uptake values (SUV) of89Zrgirentuximab and CAIX histological expression is assessed by comparing the 89Zrgirentuximab semi-quantitative data with the immunohistochemical results (IHC) of biopsied / resected tumors at that site.

[0215] All patients are followed for safety, up to EOS visit (Day 15-25).Overall study design

[0216] An open-label, non-randomized study is performed to evaluate expression of CAIX through89Zr-girentuximab PET / CT imaging in different tumor types and evaluate the feasibility of targeting CAIX for potential diagnostic and therapeutic applications.

[0217] A minimum of 5 subjects are enrolled for each of the tumor types, including but not limited to: cervical cancer, colorectal cancer, esophageal cancer (esophageal SCC and esophageal / esophagogastric junction adenocarcinoma), gastric cancer (gastric adenocarcinoma), glioblastoma multiforme, head and neck cancer (head and neck SCC and nasopharyngeal carcinoma), liver cancer (cholangiocarcinoma and hepatocellular carcinoma), lung cancer (non-small cell and small cell), ovarian cancer (epithelial ovarian carcinoma), pancreatic cancer (pancreatic adenocarcinoma) and soft tissue sarcoma.

[0218] The study involves a single administration of89Zr-girentuximab (37 MBq [1 mCi] ± 10%, containing a mass dose of 10 mg of girentuximab).

[0219] PET / CT imaging is conducted 5 ± 2 days post-administration. Image data analyses of the PET / CT imaging is performed by a nuclear medicine reader to assesstumor uptake of89Zr-girentuximab in a per lesion analysis, up to the 10 most active lesions and also according to RECIST 1.1 conventional imaging.

[0220] Qualitative visual analysis (presence or absence of localized89Zr-girentuximab uptake associated with tumor, as seen on contrast-enhanced CT, MRI or FDG PET / CT), is used to evaluate concordance of tumor lesion detection between 89Zr-girentuximab PET / CT and conventional imaging. Lesions demonstrated by 89Zr-girentuximab alone are described.

[0221] Tissue samples (from patient’s biopsy or surgery) are collected whenever possible and sent to a central lab for CAIX expression analysis.

[0222] Study assessment is performed as displayed in the below table:1Can be obtained outside of the study within 28 days before Day 02Only if clinically indicated3Remote visit (phone follow-up)4Pre-study procedure, to be performed within 30 days of Day 05If the patient cannot undergo PET / CT, CT is contraindicated, or PET / MRI can be preferable option for the patient, PET / MRI can be performed instead of PET / CT.Dosing

[0223] The89Zr-girentuximab dose used here (37 MBq [1 mCi] ± 10%, containing a mass dose of 10 mg of girentuximab) is consistent with the dosing regimen of 89Zr- girentuximab in an ongoing Phase 3 clinical trial and was shown to allow PET imaging 4- 7 days after administration by Merkx et al, (2021 ).

[0224] 89Zr-girentuximab is a radiolabeled chimeric monoclonal antibody (INN name: Zirconium Zr89girentuximab deferoxamine. Girentuximab is a chimeric monocolonal antibody (INN: girentuximab, synonyms: cG250, TLX250) with specificity for the CAIX (carbonic anhydrase 9) antigen, radiolabelled with the positron emitting radio-metal zirconium-89 via a NSuc-DFO-TFP-ester (DFO-TFP), linked to lysine residues of girentuximab, to yield89Zr-DFO-girentuximab.

[0225] 89Zr-girentuximab is formulated as a solution for intravenous administration at the nominal dosage strength 37 MBq (±10%) (1 mCi±10%) containing a total of 10 mg of girentuximab for single intravenous use.89Zr-girentuximab solution for IV administration is supplied in either a glass vial or in a syringe (depending on the geographic region) in appropriate packaging (lead-shielded containers bearing a radioactive warning symbol in accordance with radioactive pharmaceutical requirements).

[0226] The compound is administered as a single dose administration of89Zr- girentuximab (37 Megabecquerel ± 10%, [1 mCi ± 0.1 mCi] containing a mass dose of 10 mg of girentuximab) by slow intravenous injection over 3 minutes via a single peripherally placed intravenous cannula. The injection volume is approx. 10 ml, depending upon administered activity.Inclusion criteria

[0227] All participants will meet the following criteria:1 . Written and voluntarily given Informed Consent.2. Male or female >18 years of age at time of consent.3. Have the capacity to understand the study and be willing and able to comply with all protocol requirements.4. Participants must have histologically or cytologically proven solid tumors of the following types, but not limited to:• cervical cancer• colorectal cancer• esophageal cancer (esophageal SCC and esophageal / esophagogastric junction adenocarcinoma)• gastric cancer (gastric adenocarcinoma)• glioblastoma multiforme• head and neck cancer (head and neck SCC and nasopharyngeal carcinoma)• liver cancer (cholangiocarcinoma and hepatocellular carcinoma)• lung cancer (non-small cell and small cell)• ovarian cancer (epithelial ovarian carcinoma)• pancreatic cancer (pancreatic ductal adenocarcinoma)• soft tissue sarcoma5. At least one non-CNS, measurable target lesion as per RECIST 1.1 documented at conventional imaging, performed within 30 days prior to Day 0.6. Participant agrees not to participate in another interventional study while participating in the present study, defined as signing the informed consent form (ICF) until completion of the last study visit.7. Negative serum pregnancy tests in female patients of childbearing potential at screening and confirmation of negative pregnancy test result from urine within 24 hoursprior to receiving investigational product. Female patients of non-childbearing potential must provide evidence by fulfilling one of the following criteria at screening:• Post-menopausal defined as aged more than 50 years and amenorrheic for at least 12 months following cessation of all exogenous hormonal treatments.• Women under 50 years old would be consider postmenopausal if they have been amenorrheic for 12 months or more following cessation of exogenous hormonal treatments and with luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels in the post-menopausal range for the institution.• Documentation of irreversible surgical sterilization by hysterectomy, bilateral oophorectomy, or bilateral salpingectomy but not tubal ligation8. For all participants, consent to practice double-barrier contraception until a minimum of 42 days after89Zr-girentuximab administration.Exclusion criteria

[0228] A patient is excluded from participation in the trial if one or more of the following criteria are met:1 . Exposure to murine or chimeric antibodies within the last 5 years.2. Previous administration of any radionuclide within 10 half-lives (of the radionuclide) prior to the intended administration of89Zr-girentuximab (i.e., within 10 half-lives of Day 0).3. Exposure to any CAIX targeting compound (diagnostic / therapeutic) in the last 3 months4. Serious non-malignant disease (e.g. psychiatric, infectious, autoimmune or metabolic) that may interfere with the objectives of the study or with the safety or compliance of the subject, as judged by the Investigator.5. Any clinically significant abnormalities detected during screening laboratory tests or physical exam that in the opinion of the Investigator would adversely affect the participants ability to participate in the study. Principal Investigator to assess patient suitability for inclusion based on pathology and tumor type.6. Mental impairment that may compromise the ability to give Informed Consent and comply with the requirements of the study.7. Exposure to any antineoplastic treatment within 14 days from the date of planned administration of89Zr-girentuximab (i.e. within 14 days of Day 0).8. Women who are pregnant or breastfeeding.9. Known allergy, hypersensitivity, or intolerance to girentuximab, DFO(desferrioxamine), or any of the components of the investigational agent.10. Renal insufficiency with glomerular filtration rate (GFR) < 45 millilitres / min / 1 ,73m21 1 . Vulnerable patients (e.g. being in detention).Efficacy assessment

[0229] Imaging is based on the ability of PET / CT imaging with89Zr-girentuximab to non-invasively evaluate CAIX tumoral expression in patients. After a single administration of89Zr-girentuximab on Day 0, whole body PET / CT scan is followed on Day 5 ± 2 days post-administration according to the table above, and the imaging manual. Patients with metastatic (suspected or confirmed) disease may undergo an optional additional wholebody PET / CT scan if clinically indicated (e.g. if the tumor-to-background ratio makes difficult identification of the tumoral lesion(s) and is expected to improve).

[0230] 89Zr-girentuximab tumor uptake will be qualitatively assessed (yes I no) compared with conventional imaging, up to the 10 most active lesions in individual patients. Quantitative assessment will be on a per lesion basis, including SUVmax, SUVmean, SUL, MTV, and TBR.

[0231] For patients who cannot undergo CT scan for any reason and / or for whom CT scan is contraindicated, PET / MRI can be performed instead of PET / CT, if PET / MRI is available at the study site. PET / MRI can also be performed in patients whom the disease condition can be better visualized by MRI (e.g., GBMR)

[0232] The Tumor-to-Background Ratio (TBR) will be defined as the ratio of the lesion SUVmax over the reference region SUV (liver, blood pool, etc). Comparison between the number, size and other characteristics of lesions detected by89Zr-girentuximab PET scanand standard imaging modalities including high-resolution CT / MRI and other potential imaging per patient (depending on the tumor type), type of lesion and indication will be performed.

[0233] Qualitative visual analysis (presence or absence of localized89Zr-girentuximab uptake associated with tumor, as seen on contrast-enhanced CT, MRI or FDG PET / CT), will be used to evaluate concordance of tumor lesion detection between89Zr-girentuximab PET / CT and conventional imaging. RECIST 1.1 criteria for conventional imaging must be used as the main tool for concordance comparison vs PET whenever possible. For radiological assessment of tumors with different recommendations as per Clinical Guidelines of Scientific Oncology Societies, these guidelines should also be followed. In addition to the above, all visible tumoral lesions at conventional imaging may also be compared to PET imaging result.

[0234] For each patient, the SUVmax, SUVmean, SUL, MTV, TBR and concordance rate with conventional imaging will be calculated locally by the nuclear medicine expert at each site and included in eCRF. Details will be included in the imaging manual.Example 2: Preparation of radiolabelled qirentuximab

[0235] Radiolabelled girentuximab was prepared as previously described (see for example, WO 2021 / 000017). Briefly, bioconjugated girentuximab was prepared using standard techniques to obtain DOTA-girentuximab or DFO-girentuximab), prior to being labelled with a radioisotope useful for imaging (eg89Zr).Example 3: In vitro and in vivo binding of radiolabelled qirentuximab to various cancers

[0236] Imaging studies using radiolabelled-DOTA-Girentuximab were conducted to assess the ability of the imaging reagent to detect non-RCC cancer types.

[0237] Firstly, the ability of radiolabelled-DOTA-Girentuximab to bind to various cell lines. These data, shown in Figure 1 , demonstrate the ability of the antibody to bind to various cell types which express CAIX, although the degree of in vitro binding is variable.

[0238] Subsequently, three groups of mice, each bearing different tumour xenografts, were tested. The groups were as follows:Group 1 : mice bearing an AsPc-1 cell xenograft (pancreatic cancer line); n= 4Group 2: mice bearing a FaDu cell xenograft (squamous carcinoma pharynx / hypopharyngeal cancer cell line); n = 4Group 3: mice bearing an HT-29 xenograft (colorectal cancer cell line); n = 4

[0239] The radiolabelled girentuximab was administered intravenously and imaging was conducted at 24 and 72 hour time points. Biodistribution was assessed at 72 hours following administration. Radioactivity and dose of the administered antibody is summarised in the below table:

[0240] Representative images for mice from each of the three groups are shown in Figure 2.

[0241] Figure 3 shows quantification of the percentage injected dose (at 24 hours and 72 hours following injection) in the tumours. The results confirm the observations made using flow cytometry to demonstrate the ability of the radiolabelled antibody to bind to each respective cancer cell lines. In other words, the results demonstrate (other than forthe FaDu cells - see further comments below) that the antibody is able to bind to target tumour cells in an in vivo context with similar affinity to in vitro.

[0242] The results also show that at 72 hours following administration, the radiolabelled antibody is still detectable in circulation and in the spleen.

[0243] Ex vivo biodistribution of radiolabelled-DOTA-GmAb was compared to the in vivo biodistribution. Briefly, ex vivo biodistribution corresponds to the distribution of radiolabelled-DOTA-GmAb in mouse organs, as assessed following necropsy. In vivo biodistribution corresponds to the biodistribution seen in whole-mouse imaging experiments (eg as shown in Figure 1 ).

[0244] Figure 4 shows that there was a high degree of correlation between in vivo and ex vivo quantification of the signal in the tumour.

[0245] The results show that positive in vitro binding results (eg with good binding to HT-29 and AsPc-1 cells) were recapitulated with positive binding observed in vivo.

[0246] Interestingly, the inventors observed that although there was very poor binding of the radiolabelled antibody to FaDu cells in vitro, the antibody was able to bind to tumour cells in vivo. These results indicate that a negative finding with in vitro binding may not be predictive of binding in vivo and therefore the potential utility of radiolabelled-DOTA- GmAb for imaging of particular cancer types.Example 4: Imaging of alternative cancer types

[0247] Similar experiments to those conducted in Example 2 are performed using89Zr- DFO-GmAb to detect the presence of tumour xenografts in mice as follows:Group 1 : mice bearing an A-549 cell line xenograft (lung cancer)Group 2: mice bearing a MDA-MB-468 cell line xenograft (triple negative breast cancer)Group 3: mice bearing a HeLa cell line xenograft (cervical cancer)Group 4: mice bearing a AGS cell line xenograft (gastric cancer)Group 5: mice bearing a HepG2 cell line xenograft (liver cancer)Group 6: mice bearing a A2780 cell line xenograft (ovarian cancer)Group 7: mice bearing a SK-LMS1 cell line xenograft (soft tissue sarcoma - vulvar leiomyosarcoma)

[0248] At 24 hours and 72 hours following intravenous injection of89Zr-DFO-GmAb into the xenograft-bearing mice, the mice are subjected to PET / CT scanning to determine the ability of the radiolabelled to detect the cancer cells in vivo.

[0249] The results will show that the radiolabelled antibody is able to bind to the tumour xenografts. In other words, the results will demonstrate that the antibody is able to bind to target tumour cells in an in vivo context and with similar affinity to in vitro.

[0250] These results will indicate that radiolabelled-GmAb is suitable for use in producing an image of various cancer types in vivo, and is therefore useful as a non- invasive diagnostic reagent for diagnosis and detection of cancers other than renal cell carcinoma.Example 4: Imaging of triple negative breast cancer

[0251] Triple Negative Breast Cancer (TNBC) is an aggressive, metastatic and drugresistant cancer with limited therapeutic options.

[0252] The inventors believe that CAIX, a hypoxia-mediated breast tumour growth regulator, may be important for the maintenance of breast cancer stem cells within hypoxic region. The inventors therefore assessed imaging of TNBC using PET / CT imaging with89Zr-labeled girentuximab in 12 metastatic TNBC patients.

[0253] Patients underwent imaging with Fludeoxyglucose F18 (FDG) and89Zr- girentuximab PET-CT and CT. Patients received a single slow intravenous administration of 37±10% MBq89Zr-girentuximab (10 mg). At day 3 post administration, a skull to midthigh PET / CT was acquired with 10 minute acquisition time per bed position. The gold standard was determined by FDG PET / CT, CT and follow-up; lesion detected at least by 2 modalities was considered as true positive. T umour SUV[max, mean], total lesion glycolysis (TLG) and metabolic tumour volume (MTV) were measured. Immunohistochemistry (IHC) was performed with Bond RX fully automated research staining with anti-CAIX antibody (Leica, clone TH22). Staining was evaluated with semi-quantitative analysis (percentage and intensity of tumour cells expression) and SUV values compared to the degree of CAIX expression evaluated by IHC.

[0254] Preliminary results from 4 patients were examined and include data derived from a total of 49 lesions (lymph nodes, bone, lung and breast) which were detected in these patients (41 by89Zr-girentuximab, 42 by CT and 49 by FDG PET / CT). Forty-four lesions were confirmed by gold standard: 24, 5, 4, 2, 9 in nodes, lung, bone, skin and breast respectively.

[0255] Overall sensitivity of89Zr-girentuximab PET / CT was 93.2%, with 100% sensitivity for bone, lung, breast, skin and 87.5% for nodes. Overall sensitivity for both CT and FDG-PET / CT was 82.7%. Median tumor SUVmax were 3.45 [IQ: 2.03-4.69] and 4.68 [IQ: 3.27-10.71 ] for89Zr-girentuximab and89Zr-girentuximab FDG, respectively. IHC showed two CAIX-high-expression lesions [100%, 20%] for two patients whereas two patients presented respective low profiles [3%, 0%]. IHC CAIX cell status and89Zr- girentuximab SUVmean presented a weak correlation (rho=0.80; p=0.20). No89Zr- girentuximab safety issues were reported.

[0256] The results demonstrate that89Zr-girentuximab is useful for PET / CT imaging and diagnosis of TNBC in patients and provides a superior result to biopsy IHC.Example 6: Imaging of bladder cancer

[0257] Patients with non-muscle-invasive bladder carcinoma (NMIBC) are usually treated with cystectomy. Therefore, new treatment options which enable preservation of the urinary bladder are needed.

[0258] CAIX is expressed on the luminal surface of papillary structures in direct contact with vesical cavity. The inventors conducted a pilot prospective study aimed at ensuring intravesical radioactivity confinement and tumor targeting after intravesical instillation of89Zr-girentuximab.

[0259] Patients had one intravesical instillation of 37±10% MBq of89Zr-girentuximab (10 mg) and retained urine for 2 hours. Then 4 PET / CT scans were performed, 3 with one step on pelvis (H+2, Day1 and D2) and one from skull to mid-thigh at H+4 to observe intravesical radioactivity evolution over time.

[0260] A blood sample was taken at D1 to quantify a possible vascular passage of radioactivity. For all acquisitions, 10 minutes acquisition time per bed position was used. The gold standard was determined by a second look cystoscopy and trans urethralresection bladder (TURB) on89Zr-girentuximab PET / CT positive sites. Immunochemistry (IHC) was performed with an anti-CAIX antibody (Leica, clone TH22). Staining was evaluated with semi-quantitative analysis (percentage and intensity of tumour cells expression) and89Zr-girentuximab PET / CT bladder pattern compared to the degree of CAIX expression evaluated by IHC.

[0261] Results from 4 / 6 patients were obtained. Despite previous multiple previous intravesical instillations with bacillus Calmette-Guerin (BCG - a commonly used intravesical immunotherapy to treat bladder cancer), recurrent pTaG3 was identified in each patient.

[0262] 89Zr-girentuximab PET / CT showed no extra-vesical leakage. In 2 / 4 patients with positive IHC, uptake spots on the bladder wall was confirmed by TURB for one patient with corresponding recurrence foci and inflammatory scarring reaction for the second one. For the two other patients no uptake was observed in line with negative IHC. No adverse radiation contamination was observed during the process and no particular worker exposure was observed.

[0263] The results (as shown in Figure 5 and 6) indicate that intravesical instillation of89Zr-girentuximab showed radioactivity confinement into the bladder and, in patients with positive IHC, indicating that89Zr-girentuximab is useful for detecting and imaging tumours in this patient group.

[0264] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

CLAIMS1 . A method for in vivo imaging or detection of a cancer in a subject in need thereof, wherein the method comprises:- administering to the subject, an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,- detecting the agent in the subject, wherein the cancer is selected from:• bladder cancer• breast cancer (including triple negative breast cancer, hormone receptor positive breast cancer (ER positive, PR positive, ER / PR positive), and HER2 positive breast cancer)• cervical cancer• colorectal cancer• esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma)• gastric cancer (including gastric adenocarcinoma)• glioblastoma multiforme• head and neck cancer (including head and neck squamous cell carcinoma and nasopharyngeal carcinoma)• liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)• lung cancer (including non-small cell and small cell carcinoma)• ovarian cancer (including epithelial ovarian carcinoma) pancreatic cancer (including pancreatic ductal adenocarcinoma) and• soft tissue sarcoma whereby detection of said agent above a background or standard level indicates the presence of the cancer, thereby imaging or detecting the cancer in the subject.

2. A method for the diagnosis of a cancer in a subject in need thereof, where the method comprises:- administering to the subject, an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,- determining the presence or absence of the agent in the subject, wherein the cancer is selected from: bladder cancer breast cancer (including triple negative breast cancer, hormone receptor positive breast cancer (ER positive, PR positive, ER / PR positive), and HER2 positive breast cancer) cervical cancer colorectal cancer esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma) gastric cancer (including gastric adenocarcinoma) glioblastoma multiforme head and neck cancer (including head and neck squamous cell carcinoma, hypopharangral cancer, and nasopharyngeal carcinoma) liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) lung cancer (including non-small cell and small cell carcinoma)• ovarian cancer (including epithelial ovarian carcinoma)• pancreatic cancer (including pancreatic ductal adenocarcinoma) and• soft tissue sarcoma whereby detection of said agent above a background or standard level indicates that the subject has said cancer, thereby diagnosing the cancer in the subject.

3. A method for producing an image of a cancer, the method comprising:- administering to a subject suspected of having the cancer, an effective amount of an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,- detecting the agent in the subject wherein the cancer is selected from: bladder cancer breast cancer (including triple negative breast cancer, hormone receptor positive breast cancer (ER positive, PR positive, ER / PR positive), and HER2 positive breast cancer) cervical cancer colorectal cancer esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma) gastric cancer (including gastric adenocarcinoma) glioblastoma multiforme head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer and nasopharyngeal carcinoma)liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)• lung cancer (including non-small cell and small cell carcinoma)• ovarian cancer (including epithelial ovarian carcinoma)• pancreatic cancer (including pancreatic ductal adenocarcinoma) and soft tissue sarcoma thereby producing an image of the cancer.4 The method of any one of claims 1 to 3, wherein the method further comprises allowing the agent to concentrate at sites and / or tissues in said subject where the CAIX antigen is found in the subject prior to detecting the agent or determining the presence or absence of the agent in the subject.5 The method of any one of claims 1 to 4, wherein the method does not require additional in vitro methods for the detection, diagnosis or imaging of the cancer.6 The method of any one of claims 1 to 4, wherein the method is the sole method require to enable detection, diagnosis or imaging of the cancer.7 The method of any one of claims 1 to 6, wherein the agent for binding to CAIX is a small molecule, peptide or a polypeptide (such as an antibody or antigen binding fragment thereof).8 The method of any one of claims 1 to 7, wherein the agent for binding to CAIX is a small molecule, optionally selected from the group consisting of: SLC-01 1 1 , SLC- 149, SLC-0121 , SLC-101 , PMI-05, sulfamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781 , -MIP-1486, MIP-1490, MIP-1504 (especially "mTc-HEHEHE-Z09781 ,99mTc-MIP-1486, "mTc-MIP-1490 or99mTc-MIP-1504 / 5) and PHC-102.9 The method of any one of claims 1 to 7, wherein the agent for binding to CAIX is a peptide, optionally selected from the group consisting of: 3B-301 , 3B-302 or CAIX- P110 The method of any one of claims 1 to 7, wherein the agent for binding to CAIX is a polypeptide.1 1 . The method of any one of claims 1 to 7, wherein the agent for binding to CAIX is an antibody, or antigen binding fragment thereof.

12. The method of claim 11 , wherein the antibody or antigen binding fragment thereof is girentuximab, including a chimeric or humanised variant thereof.

13. The method of claim 11 , wherein the antibody or antigen binding fragment thereof is BCA-356, BAY-794620 or SLC-0131 .

14. The method of any one of the preceding claims, wherein the detectable moiety of the agent is conjugated to the agent directly, or via a chelator or linker.

15. The method of any one of claims 1 to 14, wherein the detectable moiety is a fluorescent labels or dye.

16. The method of any one of claims 1 to 14, wherein the detectable moiety is a radioisotope.

17. The method of claim 16, wherein the radioisotope is selected from: gallium-67 and gallium-68 (67Ga and68Ga), indium-11 1 (111ln), iodine-123, iodine-124 or iodine 131 (123l,124l, or131I), lutetium-177 (177Lu), technetium-99 (99mTc), yttrium-90 (90Y) and zirconium-89 (89Zr).

18. The method of claims 1 to 14, 16 or 17, wherein the detectable moiety is a radioisotope and the detection of the agent or detecting the presence or absence of the agent, comprises determination or detecting the presence or absence of radiation emitted by the radioisotope.

19. The method of claim 18, wherein the determination or detection of the presence of absence of radiation comprises Positron Emission Tomography (PET) imaging.

20. The method of any one of claims 1 to 14, wherein the agent is selected from:89Zr-girentuximab,123l-,124l-, or131I- girentuximab.21 . The method of any one of claims 1 to 14, wherein the agent is89Zr-girentuximab.

22. The method of any one of claims 1 to 6 wherein the cancer is breast cancer (including triple negative breast cancer, hormone receptor positive breast cancer (ER positive, PR positive, ER / PR positive), and HER2 positive breast cancer).

23. The method of any one of claims 1 to 6 wherein the cancer is cervical cancer.

24. The method of any one of claims 1 to 6 wherein the cancer is colorectal cancer.

25. The method of any one of claims 1 to 6 wherein the cancer is esophageal cancer.

26. The method of any one of claims 1 to 6 wherein the cancer is gastric cancer.

27. The method of any one of claims 1 to 6 wherein the cancer is glioblastoma multiforme.

28. The method of any one of claims 1 to 6 wherein the cancer is head and neck cancer (such as hypopharyngeal cancer, pharyngeal cancer).

29. The method of any one of claims 1 to 6 wherein the cancer is liver cancer.

30. The method of any one of claims 1 to 6 wherein the cancer is lung cancer.

31. The method of any one of claims 1 to 6 wherein the cancer is ovarian cancer32. The method of any one of claims 1 to 6 wherein the cancer is pancreatic cancer.

33. The method of any one of claims 1 to 6 wherein the cancer is soft tissue sarcoma.

34. The method of any one of claims 1 to 6 wherein the cancer is bladder cancer.

35. The method of claim 22 wherein the agent is a radiolabelled girentuximab antibody.

36. The method of claim 23 wherein the agent is a radiolabelled girentuximab antibody.

37. The method of claim 24 wherein the agent is a radiolabelled girentuximab antibody.

38. The method of claim 25 wherein the agent is a radiolabelled girentuximab antibody.

39. The method of claim 26 wherein the agent is a radiolabelled girentuximab antibody.

40. The method of claim 27 wherein the agent is a radiolabelled girentuximab antibody.41 . The method of claim 28 wherein the agent is a radiolabelled girentuximab antibody.

42. The method of claim 29 wherein the agent is a radiolabelled girentuximab antibody.

43. The method of claim 30 wherein the agent is a radiolabelled girentuximab antibody.

44. The method of claim 31 wherein the agent is a radiolabelled girentuximab antibody.

45. The method of claim 32 wherein the agent is a radiolabelled girentuximab antibody.

46. The method of claim 33 wherein the agent is a radiolabelled girentuximab antibody.

47. The method of claim 34 wherein the agent is a radiolabelled girentuximab antibody.

48. The method of any one of claims 1 to 47, wherein the agent is radiolabelled girentuximab that comprises one or more amino acid substitutions in the Fc region of the antibody which reduce the serum half-life of the antibody.

49. The method of any one of claims 35 to 48, wherein the radiolabelled girentuximab comprises girentxuximab conjugated to a radioisotope selected from: gallium-67 and gallium-68 (67Ga and68Ga), indium-11 1 (111ln), iodine-123, iodine-124 or iodine 131 (123l,124l, or131I), lutetium-177 (177Lu), technetium-99 (99mTc), yttrium-90 (90Y) and zirconium-89 (89Zr).

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