Imaging reagents and methods

Abstract

A method for performing in vivo imaging or detection of cancer in a subject in need thereof, the method comprising: - 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; and - detecting the agent in the subject, wherein the cancer is not renal cell carcinoma.

Description

[Technical Field]

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

[0002] Related Applications This application claims priority from Australian Provisional Application No. AU2022 / 903922, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Medical imaging methods are often used to aid in the diagnosis and staging of the progression of various cancers. Such methods can also be advantageous in eliminating or reducing the need for invasive procedures (such as obtaining biopsy samples) to confirm a diagnosis, which are not necessarily required but can cause complications.

[0004] However, not all cancers can be successfully detected using standard medical imaging. Furthermore, many imaging techniques allow for the detection of masses but do not successfully distinguish between benign and malignant tissue.

[0005] The state of oncology management has progressed in a dichotomous manner in recent decades: some types of cancer have benefited significantly from advances in developments in diagnostic and therapeutic options, resulting in improved morbidity and mortality in their patient populations, while others have been more elusive and continue to portend poor prognosis for their patient populations.

[0006] As this latter category of cancers has exhausted the limits of existing diagnostic and therapeutic modalities, there is a need for innovation based on novel 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] The reference herein to any prior art is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would be understood by, considered relevant, and / or could reasonably be expected to be taken in conjunction with other prior art by a person skilled in the art. Summary of the Invention

[0009] The present invention is based, at least in part, on the inventors' identification of a subset of solid tumors that express the antigen carbonic anhydrase IX (CAIX), and the discovery that these tumors can be imaged in vivo using imaging agents that bind to CAIX.

[0010] Accordingly, the present invention provides a method for in vivo imaging or detection of cancer in a subject in need thereof, comprising: administering to the subject an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety to allow in vivo detection of the agent in the subject; - detecting a drug in a subject; Cancer is 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) ● Stomach cancer (including gastric adenocarcinoma) Glioblastoma multiforme Head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer, and nasopharyngeal cancer) ● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) Lung cancer (including non-small cell and small cell lung cancer) ●Ovarian cancer (including ovarian epithelial cancer) Pancreatic cancer (including pancreatic ductal adenocarcinoma), and Selected from soft tissue sarcomas, Detection of the agent above background or standard levels indicates the presence of cancer, thereby imaging or detecting cancer in the subject.

[0011] The present invention also provides a method for diagnosing cancer in a subject in need thereof, the method comprising: administering to a subject an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety to allow in vivo detection of the agent in the subject; - determining the presence or absence of the drug in the subject; Cancer is 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) ● Stomach cancer (including gastric adenocarcinoma) Glioblastoma multiforme Head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer, and nasopharyngeal cancer) ● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) Lung cancer (including non-small cell and small cell lung cancer) ●Ovarian cancer (including ovarian epithelial cancer) Pancreatic cancer (including pancreatic ductal adenocarcinoma), and Selected from soft tissue sarcomas, Determining the presence of the agent above background or standard levels indicates that the subject has the cancer, thereby diagnosing cancer in the subject.

[0012] The present invention also provides a method for generating an image of cancer, the method comprising: administering to a subject suspected of having cancer an effective amount of an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety to allow in vivo detection of the agent in the subject; - detecting the agent in the subject; Cancer is 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) ● Stomach cancer (including gastric adenocarcinoma) Glioblastoma multiforme Head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer, and nasopharyngeal cancer) ● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) Lung cancer (including non-small cell and small cell lung cancer) ●Ovarian cancer (including ovarian epithelial cancer) Pancreatic cancer (including pancreatic ductal adenocarcinoma) and Selected from soft tissue sarcomas, This produces an image of the cancer.

[0013] The present invention also provides a method for generating an image of cancer, the method comprising: injecting an effective amount of an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety to allow in vivo detection of the agent; detecting the agent; Cancer is 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) ● Stomach cancer (including gastric adenocarcinoma) Glioblastoma multiforme Head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer, and nasopharyngeal cancer) ● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) Lung cancer (including non-small cell and small cell lung cancer) ●Ovarian cancer (including ovarian epithelial cancer) Pancreatic cancer (including pancreatic ductal adenocarcinoma) and Selected from soft tissue sarcomas, Thereby, an image of the cancer is produced. Optionally, the detectable moiety is a radioisotope and detecting comprises detecting radiation, such as positron emission tomography (PET) imaging.

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

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

[0016] In some embodiments, the diagnostic methods of the present invention do 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 allowing detection of the agent in vivo.

[0018] In any embodiment, the moiety for binding to CAIX can be a small molecule, peptide, or polypeptide (eg, 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-0111, SLC-149, SLC-0121, SLC-101, PMI-05, sulfamido-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, -MIP-1486, MIP-1490, MIP-1504 (particularly 99m Tc-HEHEHE-Z09781, 99m Tc-MIP-1486, 99m Tc-MIP-1490 or 99m Tc-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, the first moiety being 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 humanized antibody or antigen-binding fragment thereof. Optionally, the antigen binding protein is humanized G250 (hG250).

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

[0025] An agent for use in accordance with the methods of the present invention includes a moiety to enable its detection. For use in in vivo detection techniques, any suitable detectable moiety can be used and will be known to those of skill in the art.

[0026] The detectable moiety can be directly linked to the moiety for binding to CAIX or can 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 an additional detectable moiety to be linked thereto.

[0027] In any embodiment, the detectable moiety is a radioisotope. Examples of suitable isotopes include gallium-67 and gallium-68 ( 67 Ga and 68 Ga), Indium-111( 111 In), iodine-123, iodine-124, or iodine-131 ( 123 I, 124 I, or 131 I), lutetium-177( 177 Lu), technetium-99 ( 99m Tc), yttrium-90( 90Y), and zirconium-89 ( 89 Zr).

[0028] In any embodiment, the agent is an antibody for binding to CAIX and the detectable moiety is a radioisotope, optionally gallium-67 and gallium-68 ( 67 Ga and 68 Ga), Indium-111( 111 In), iodine-123, iodine-124, or iodine-131 ( 123 I, 124 I, or 131 I), lutetium-177( 177 Lu), technetium-99 ( 99m Tc), yttrium-90( 90 Y), and zirconium-89 ( 89 Zr).

[0029] In any embodiment, the agent is a girentuximab antibody (including chimeric or humanized versions thereof) and the detectable moiety is a radioisotope, optionally gallium-67 and gallium-68 ( 67 Ga and 68 Ga), Indium-111( 111 In), iodine-123, iodine-124, or iodine-131 ( 123 I, 124 I, or 131 I), lutetium-177( 177 Lu), technetium-99 ( 99m Tc), yttrium-90( 90 Y), and zirconium-89 ( 89 Zr).

[0030] In any embodiment, the agent is 89 Zr-, 123 I-, 124 I-, 131 I-, or 177 Lu-girentuximab.

[0031] In a preferred embodiment, the detectable moiety is a radioisotope and detecting the agent comprises determining the presence or absence of, or detecting, radiation emitted by the radioisotope. In any embodiment, determining the presence or detecting the absence of radiation comprises positron emission tomography (PET) imaging.

[0032] Other suitable detectable moieties include fluorescent labels and dyes. It should be understood that in any embodiment of the invention, one or more detectable moieties may be utilized to maximize imaging or detection of the agent, and thereby the tumor or cancer, that expresses CAIX.

[0033] The present invention also provides agents for CAIX, or compositions containing agents, as described herein, for use in the methods of detection, imaging, obtaining images of cancer, or diagnosing cancer, as described herein.

[0034] Still further, the present invention provides agents or compositions comprising agents for CAIX, as described herein, for use in methods for detecting, imaging, or diagnosing cancer, or for obtaining images of cancer as described herein.

[0035] Still further, the present invention provides kits for use according to any of the methods described herein, the kits comprising an agent for binding to CAIX as described herein and, optionally, instructions for its use to detect, image, or diagnose cancer.

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

[0037] Further aspects of the 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, in which: [Brief explanation of the drawings]

[0038] [Figure 1] In vitro binding of radiolabeled DOTA-G mAb to various cell lines. [Figure 2] Representative images of mice bearing As-PC-1 (pancreatic cancer), FaDu (hypopharyngeal cancer), or HT-29 (colorectal cancer) tumor xenografts after injection with radiolabeled DOTA-GmAb. [Figure 3] Quantification of the percentage of injected dose 24 and 72 hours after injection of radiolabeled DOTA-GmAb. Filled circles = AsPC-1 radiolabeled DOTA-GmAb, filled squares = FaDu radiolabeled DOTA-GmAb, filled triangles = HT-29 radiolabeled DOTA-GmAb. [Figure 4] Quantification of biodistribution results. Ex vivo biodistribution (i.e., distribution of radiolabeled DOTA-GmAb observed in mouse organs after necropsy) was shown to correlate with in vivo biodistribution (i.e., distribution of radiolabeled DOTA-GmAb observed in mice after whole-body imaging). Filled circles = AsPC-1 radiolabeled DOTA-GmAb, filled squares = FaDu radiolabeled DOTA-GmAb, filled triangles = HT-29 radiolabeled DOTA-GmAb. [Figure 5] Bladder cancer imaging. Day 0: Whole-body 89Zr-girentuximab scan: A: Coronal PET / CT fusion. B: Maximum intensity projection (MIP) visualization. [Figure 6] Bladder cancer imaging. Day 2. A. 89Zr-girentuximab pelvic PET / CT fusion image: (arrows) radiopharmaceutical uptake on different sides of the bladder wall. B. 3D representation of overlaid images of the bladder. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention relates to the imaging and diagnosis of cancers, which are difficult to diagnose due to a lack of robust oncology management options. Diagnostic and therapeutic innovations are needed to improve morbidity and mortality in these oncology indications.

[0040] Detection of CAIX in the context of imaging and diagnosing kidney cancer is known. However, prior to the present invention, it was not known whether in vivo detection of CAIX could be used successfully to image and diagnose other solid tumors that may express CAIX.

[0041] Although CAIX is typically associated with aggressive disease, it is not known whether CAIX can be detected and / or imaged during the early stages of certain cancers, or whether the cancer can only be detected in later-stage disease. Furthermore, some cancers show decreased expression as the disease progresses, and therefore it is not clear whether CAIX imaging would be a useful tool for detecting these types of cancers.

[0042] Furthermore, given the heterogeneity of many cancers, the mere presence of CAIX expression (e.g., as determined by immunohistochemistry techniques) does not necessarily indicate that the cancer can be detected using whole-body or partial-body imaging methods.

[0043] For example, a 2006 study by Henrickx et al. (Cancer Biotherapy & Radiopharmaceuticals, 21:263-268) found that radiolabeled antibodies binding to CAIX were not suitable for imaging biliary tract cancer, despite the fact that biliary tract cancer was found to overexpress CAIX. Conversely, the same antibodies are known to be generally useful for imaging and detecting CAIX-overexpressing renal cell carcinoma. Both biliary tract cancer and kidney cancer are malignant tumors of epithelial cells and are characterized by increased expression of CAIX. Therefore, it is not understood why CAIX-binding antibodies are useful for imaging and detecting renal cell carcinoma but not biliary tract cancer.

[0044] In another example, a recent study published by Huizing et al. (2021, Physics and Imaging in Radiation Oncology, 145-150) 111 We observed that the In-labeled F(ab')2 form of the CAIX-binding antibody girentuximab cannot distinguish between tumor and non-tumor cells and is therefore not useful for quantifying changes in CAIX expression. 89 This is in stark contrast to our results reported herein, where Zr-GmAb (in the form of a whole IgG antibody) was shown to be useful for imaging and detecting the same cancer cell types in vivo.

[0045] Thus, the present invention is based on the discovery that a subset of cancers associated with increased CAIX expression can indeed be successfully detected and imaged using an agent that binds to CAIX and that includes a detectable moiety.

[0046] General Definitions Throughout this specification, unless expressly stated otherwise or the context requires otherwise, a reference to a single step, composition of matter, group of steps, or composition of matter shall be construed to include one and the plurality (i.e., one or more) of that step, composition of matter, group of steps, or composition of matter. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the context clearly indicates otherwise. For example, a reference to "a" includes two or more and not just the singular, a reference to "an" includes two or more and not just the singular, a reference to "the" includes two or more and not just the singular, etc.

[0047] Those skilled in the art will understand that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention also includes all of the steps, features, compositions, and compounds, and any and all combinations, or any two or more of such steps or features, individually or collectively, referred to or indicated in this specification.

[0048] 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.

[0049] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0050] The present invention is not to be limited in scope by the specific examples described herein, which are for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention.

[0051] Any example or embodiment of the invention herein shall be construed to apply mutatis mutandis to any other example or embodiment of the invention unless expressly stated otherwise.

[0052] Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in diagnostic techniques, radiological imaging, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0053] 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 interpreted as providing explicit support for both meanings or either meaning.

[0054] Carbonic anhydrase IX As used herein, carbonic anhydrase is also known as CA-IX, CA9, CAIX, carbonic anhydrase IX, carbonic anhydrase 9, carbonic anhydrase IX, carbonic anhydrase, G250, membrane antigen MN, P54 / 58N, pMW1, RCC-associated antigen G250, RCC-associated protein G250, and renal cell carcinoma-associated antigen G250.

[0055] Cancer cells primarily express intracellular CAs, including the plasma membrane-associated CA isoforms CAIX and CAXII, as well as CAI and CAII. Among cancer-associated CAs, CAIX has attracted the most attention because its expression in healthy tissues is restricted to epithelial cells in the stomach and intestine, but is strongly upregulated in renal cancer.

[0056] CAIX, whose expression is under the control of hypoxia-inducible factor 1 (HIF-1), is primarily located in chronically hypoxic tumor regions. However, because CAIX expression can be activated by components of the mitogen-activated protein kinase (MAPK) pathway, CAIX can also be found in mildly hypoxic or even normoxic regions.

[0057] An agent for binding to CAIX and for use in the methods of the present invention may be any compound that specifically recognizes or binds to CAIX, mediates its activity by binding to CAIX or a fragment or splice variant thereof, binds irreversibly at the entrance to the active site, and / or inhibits CAIX by coordinating a zinc ion at the active site of CAIX.

[0058] Preferably, an agent for binding to CAIX specifically interacts with a CAIX polypeptide. Specifically interacting (e.g., recognizing or binding) means that the agent, e.g., an antibody, has a higher affinity for CAIX compared to other polypeptides. In one embodiment, the agent interacts with (i.e., binds to or recognizes) or modulates the activity of a CAIX polypeptide and / or mediates antibody-dependent cellular cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). Thus, according to one embodiment, the agent is a CAIX inhibitor. Such CAIX inhibitors may act at the protein level or nucleic acid level. For example, examples of CAIX inhibitors acting at the protein level include, but are not limited to, peptides and anti-CAIX antibodies, as well as functional fragments of these antibodies, or small organic molecules (preferably with a molecular weight of less than 500 g / mol).

[0059] Examples of anti-CAIX antibodies or antibodies for binding to CAIX are described in EP637336, WO93 / 18152, WO95 / 34650, WO00 / 24913, WO02 / 063010, WO04 / 025302, WO05 / 037083, WO2011 / 139375, Murri-Plesko et al., Eur J Pharmacol 2011, 657:173-183.

[0060] Examples of small organic molecules for binding to CAIX include, but are not limited to, sulfonamides, heteroaromatic sulfonamides, sulfamates, coumarins and thiocoumarins, and BAY-79-4620. Examples of inhibitors that act at the nucleic acid level are siRNA molecules, ribozymes and / or antisense molecules.

[0061] As used herein, the terms "specifically binds" or "binds specifically" shall be taken to mean that an agent for use according to the invention reacts or associates with CAIX or cells expressing it more frequently, more rapidly, for a longer duration, and / or with higher affinity than with alternative antigens or cells, for example, an antigen-binding protein that binds to CAIX with substantially higher 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 to other antigens.

[0062] Methods for assessing binding to proteins (e.g., CAIX) are known in the art, as described, for example, in Scopes (Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve immobilizing an agent (e.g., an antibody) and contacting it with a labeled target (an antigen in the case of an antibody). After washing to remove non-specifically bound proteins, the amount of label, and consequently the 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.

[0063] Other standard methods for assessing binding to a target such as CAIX are known in the art.

[0064] small molecule In any embodiment, the moiety for binding to CAIX is the small molecule SLC-0111 (CAS178606-66-1), SLC-149 (described in EP3317255B1, incorporated herein by reference), SLC-0121, or SLC-101.

[0065] In any embodiment, the moiety for binding to CAIX is the small molecule / imaging agent PMI-05 (described in US2019 / 0192699A1, incorporated herein by reference).

[0066] In any embodiment, the moiety for binding to CAIX is the small molecule sulfamido-nitroimidazole (described in Rami et al., (2013), J. Med. Chem, 56:8512-8520, incorporated herein by reference).

[0067] In an optional embodiment, the moiety for binding to CAIX is the small molecule JS-403 (described in WO2010 / 089752A1, which is incorporated herein by reference).

[0068] In some embodiments, the moiety for binding to CAIX is the small molecule UB-TT220 (described in WO2022 / 015955A1, which is incorporated herein by reference).

[0069] In any embodiment, the moiety for binding to CAIX is a small molecule 99m Tc-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: 1095-1104; Grindel et al., (2022) ACS Chem Biol, 17(6): 1543-55 (incorporated herein by reference)), 99m Tc-MIP-1486, 99mTc-MIP-1490 (4-(2-bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide (wherein X=ethyl)), or 99m Tc-MIP-1504 (4-(2-bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2-oxoethyl)-1H-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).

[0070] In some embodiments, the moiety for binding to CAIX is the small molecule PHC-102 (described in WO2015 / 114171A1, WO2018 / 154517A1, US2014 / 0357650A1, WO2015 / 114171A1, which are incorporated herein by reference).

[0071] peptide In any embodiment, the moiety for binding to CAIX is a peptide. As used herein, a peptide is understood to include a chain of more than one amino acid residue. Typically, a peptide can include about 2 to 30 amino acids, e.g., about 5 to 30, about 10 to 30, about 2 to 25, about 5 to 25, about 10 to 25, or about 10 to 20 amino acids. A peptide can have a length of at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 amino acids. Typically, a peptide is about 40 amino acids or less, e.g., about 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids or less.

[0072] In any embodiment, the moiety for binding to CAIX is 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., (2011) 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 Medicinal Chem, 34(1):703-11; Kumar et al., (2017) Eur J of Medicinal Chem, 136:52-62 (hereby incorporated by reference), or 3B-302.

[0073] In any embodiment, the moiety for binding to CAIX is the peptide CAIX-P1 having the amino acid sequence YNTNHVPLSPKY (described in Askoxylakis et al., (2010), PLoS One, 5(12):e15962), and optionally the peptide is 125 I or 131 I (although it will be understood that any suitable radiolabel or other detectable moiety may be used).

[0074] Polypeptides and Antibodies According to particularly 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, which may have essentially the same CAIX-binding and / or inhibitory activity as the full-length anti-CAIX antibody and / or may be an epitope-binding fragment of the anti-CAIX antibody.

[0075] Reference herein to an antibody or antigen-binding fragment thereof that "binds" to carbonic anhydrase IX (CAIX) provides literal support for the antibody or fragment thereof "binds specifically to" or "specifically binds to" CAIX.

[0076] The antibodies and / or antibody fragments 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 chimeric, humanized, and fully human variants thereof. The antibodies may be multivalent, multivalent, and multispecific.

[0077] In particularly preferred embodiments, the antibody is a whole antibody comprising at least one antigen-binding domain (Fab) of the antibody and at least one Fc region of the antibody. The antibody may comprise a human constant region of IgG1, IgG2a, IgG3, or IgG4.

[0078] According to a further preferred embodiment, the anti-CAIX antibody or epitope-binding fragment thereof for use according to the invention binds to the amino acid sequence LSTAFARV and / or ALGPGREYRAL.

[0079] In any embodiment, the moiety for binding to CAIX is in the form of the antibody BAY-794620, or an antigen-binding fragment thereof (described in WO2003 / 100029A2, WO2003 / 033674A2, Theiner et al., (2021) Tierarztl Prax Ausg G Grosstiere Nutztiere, 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)).

[0080] In one embodiment, the moiety for binding to CAIX is in the form of the antibody SLC-0131, or an antigen-binding fragment thereof.

[0081] According to a further particularly preferred embodiment, the agent for binding to CAIX is an antibody anti-G250 and / or an antigen-binding fragment thereof. Anti-G250 antibodies are described, for example, in EP-B-0637336. The antibody or fragment thereof may be a chimeric or humanized G250 antibody. In some embodiments, the antigen binding protein that binds or specifically binds to CAIX is as described in any of WO2002 / 062972A2 (US2004 / 0219633A1), WO2004 / 002526A1 (US7,632,496B2), WO2006 / 002889A2 (US7,691,375B2), WO2009 / 056342A1 (US2014 / 0017252A1), WO2011 / 032973A1 (US2012 / 0207672A1), and WO2014 / 128258A1 (US10,620,208B2) or WO2021 / 000017A1 (the entire contents of each of these publications are incorporated herein by reference).

[0082] Antibodies for use in the present invention may be produced by any suitable method known in the art, including, but not limited to, the methods described in PCT / EP02 / 01282 and PCT / EP02 / 01283, which are incorporated herein by reference.

[0083] A particularly preferred antibody is cG250, preferably girentuximab (INN). Another particularly preferred embodiment is the monoclonal antibody G250 produced by the hybridoma cell line DSM ACC 2526. The antibody cG250 is an IgG1 kappa light chain chimeric form of the original murine monoclonal antibody mG250.

[0084] Variants of the original chimeric G250 (cG250) antibody are known, including WX-G250 and WX-G250RIT (131 iodine) (Janssen Global Services LLC).

[0085] In a particularly preferred embodiment, the antibody 89 Zr-girentuximab (i.e. 89 Zr-cG250), 123 I-, 124 I-, or 131 I-Girentuximab, or 177 Lu-girentuximab.

[0086] In any embodiment, the antibody or antigen-binding fragment thereof (a) a heavy chain variable domain (VH) comprising three complementarity determining regions (CDRs) of the amino acid sequence set forth in SEQ ID NO: 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 set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0087] In any embodiment, the antibody or antigen-binding fragment thereof comprises an antigen-binding domain that specifically binds to carbonic anhydrase IX (CAIX); FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, During the ceremony, FR1, FR2, FR3, and FR4 are framework regions, CDR1, CDR2, and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a, and FR4a are each framework region; CDR1a, CDR2a, and CDR3a are each a complementarity determining region; The sequence of any of the complementarity determining regions has the amino acid sequence set forth in Table 1 below. Preferably, the framework regions have the amino acid sequence also set forth in Table 1 below, including amino acid variations at specific residues that can be determined by aligning various framework regions from each antibody. CDR1, CDR2, and CDR3 may be sequences from the VH and CDR1a, CDR2a, and CDR3a may be sequences from the VL, or CDR1, CDR2, and CDR3 may be sequences from the VL and CDR1a, CDR2a, and CDR3a may be sequences from the VH.

[0088] In any embodiment, the antigen or antigen-binding fragment thereof is (i) a complementarity determining region (CDR) 1 comprising a sequence that is 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 the sequence set forth in SEQ ID NO: 1; and 8. A VH comprising a CDR2 comprising a sequence that is 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 the 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 the sequence set forth in any of SEQ ID NOs: 4, 20, 36, 52, or 68; (iii) a CDR1 comprising a sequence that is 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 the sequence set forth in SEQ ID NO: 81; and a CDR1 that is 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 a VL comprising a CDR2 comprising a sequence that is 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 the sequence set forth in SEQ ID NO: 83, and a CDR3 comprising a sequence that is 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 the 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 the sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164; (v) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1, a CDR2 comprising the sequence shown in SEQ ID NO: 2, and a CDR3 comprising the sequence shown in SEQ ID NO: 3; (vi) a VH comprising a sequence set forth in any one of SEQ ID NOs: 4, 20, 36, 52, or 68; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 81, a CDR2 comprising the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising the sequence set forth in SEQ ID NO: 83; (viii) a VL comprising a sequence set forth in any one of SEQ ID NOs: 84, 100, 116, 132, 148, or 164; (ix) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1, a CDR2 comprising the sequence shown in SEQ ID NO: 2, and a CDR3 comprising the sequence shown in SEQ ID NO: 3, and a VL comprising a CDR1 comprising the sequence shown in SEQ ID NO: 81, a CDR2 comprising the sequence shown in SEQ ID NO: 82, and a CDR3 comprising the sequence shown in SEQ ID NO: 83; or (x) A VH comprising the sequence set forth in any of SEQ ID NOs: 4, 20, 36, 52, or 68, and a VL comprising the sequence set forth in any of SEQ ID NOs: 84, 100, 116, 132, 148, or 164.

[0089] In a further embodiment, the antibody or antigen-binding fragment thereof (i) a frame comprising or consisting of a sequence that is 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 any of the sequences set forth in SEQ ID NOs: 9, 25, 41, 57, or 73; Work region (FR) 1 and a FR comprising or consisting of a sequence that is 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 any of SEQ ID NOs: 10, 26, 42, 58, and 74. 2, 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%, or at least 99% identical to any of SEQ ID NOs: 11, 27, 43, 59, or 75, and , 28, 44, 60, or 76; and 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%, or at least 99% identical to a sequence set forth in any of (ii) a fragment 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%, or at least 99% identical to any of the sequences set forth in SEQ ID NOs: 89, 105, 121, 137, 153, or 169; and a framework region (FR) 1 comprising or consisting of a sequence that is 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 any of SEQ ID NOs: 90, 106, 122, 138, 154, or 170. and 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%, or at least 99% identical to any of SEQ ID NOs: 91, 107, 123, 139, 155, or 171. 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%, or at least 99% identical to any of the sequences set forth in SEQ ID NOs: 92, 108, 124, 140, 156, or 172;VL and

[0090] In a further embodiment, the antibody or antigen-binding fragment thereof (i) a VH comprising: a framework region (FR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 9, 25, 41, 57, or 73; a FR2 comprising or consisting of the sequence set forth in SEQ ID NO: 10, 26, 42, 58, or 74; a FR3 comprising or consisting of the sequence set forth in SEQ ID NO: 11, 27, 43, 59, or 75; and a FR4 comprising or consisting of the sequence set forth in SEQ ID NO: 12, 28, 44, 60, or 76; (ii) a VL comprising a framework region (FR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 89, 105, 121, 137, 153, or 169; FR2 comprising or consisting of the sequence set forth in SEQ ID NO: 90, 106, 122, 138, 154, or 170; FR3 comprising or consisting of the sequence set forth in SEQ ID NO: 91, 107, 123, 139, 155, or 171; and FR4 comprising or consisting of the sequence set forth in SEQ ID NO: 92, 108, 124, 140, 156, or 172.

[0091] In any embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CAIX comprises an amino acid sequence (in N- to C-terminal or C- to N-terminal order) that consists essentially of or consists of any one of SEQ ID NOs: 4, 20, 36, 52, or 68, and / or any one of SEQ ID NOs: 84, 100, 116, 132, 148, or 164.

[0092] In any embodiment, the antibody or antigen-binding fragment thereof (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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 4, 20, 36, 52, or 68; and / or (b) comprises 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%, or at least 99% identical to the sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0093] In any embodiment, the antibody or antigen-binding fragment thereof for binding to CAIX is (i) single chain Fv fragment (scFv), (ii) dimeric scFv (di-scFv), or (iii) It may be in the form of one of (i) or (ii) linked to the constant region of an antibody, Fc, or heavy chain constant domain (CH)2 and / or CH3.

[0094] In any embodiment, the antibody or antigen-binding fragment thereof for binding to CAIX is (i) diabodies, (ii) triabodies, (iii) tetrabodies, (iv) Fab, (v) F(ab')2, (vi) Fv, or (vii) It may be in the form of one of (i) to (vi) linked to the constant region of an antibody, Fc, or heavy chain constant domain (CH)2 and / or CH3.

[0095] In any embodiment, the antibody or antigen-binding fragment thereof for use according to the invention may be an antigen-binding protein described herein, an immunoglobulin variable domain, an antibody, a dab (single domain antibody), a dis-scFv, an scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single-chain antibody molecule, or a fusion protein comprising a multispecific antibody.

[0096] Antigen-binding fragments, immunoglobulin variable domains, antibodies, dabs, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies, fusion proteins or conjugates described herein can be obtained by expressing nucleic acids encoding them.

[0097] The antibodies or antigen-binding fragments thereof described herein can comprise a human constant region, e.g., an IgG constant region, such as an IgG1, IgG2, IgG3, or IgG4 constant region, or a mixture thereof. In the case of antibodies or proteins comprising 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.

[0098] In one example, the antibodies or antigen-binding fragments thereof described herein comprise heavy chain constant regions, including stabilized heavy chain constant regions, that comprise, in whole or in part, a mixture of sequences, with or without a C-terminal lysine residue.

[0099] In one example, an antibody or antigen-binding fragment thereof described herein comprises a VH disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as discussed above), and the VL is linked or fused to a kappa light chain constant region.

[0100] The functional characteristics of the antigen-binding fragments thereof described herein are used mutatis mutandis for the antibodies described herein.

[0101] Antibodies or antigen-binding fragments thereof for use as described herein may be purified, substantially purified, isolated, and / or recombinant.

[0102] [Table 1-1]

[0103] [Table 1-2]

[0104] [Table 1-3]

[0105] [Table 1-4]

[0106] [Table 1-5]

[0107] [Table 1-6]

[0108] [Table 1-7]

[0109] [Table 1-8]

[0110] [Table 1-9]

[0111] [Table 1-10]

[0112] [Table 1-11]

[0113] [Table 1-12]

[0114] [Table 1-13]

[0115] [Table 1-14]

[0116] [Table 1-15]

[0117] [Table 1-16]

[0118] In a further embodiment, the moiety for binding to CAIX may comprise BCA-356, a bispecific antibody comprising an affinity-matured humanized anti-CAIX antibody linked to an attenuated subunit of IL-12 fused at the C-terminus to each of the heavy chains of the anti-CAIX antibody via a linker in a "knobs-in-holes" format (Nair et al., Journal for Immuno Therapy of Cancer, 10:S2).

[0119] In particularly preferred embodiments of the methods and uses described herein, the agent for binding to CAIX is 89 Zr-girentuximab, 124 I-girentuximab, 177 Lu-girentuximab, or 111 In-girentuximab-IRDye800CW (e.g., as described in Stroet et al., (2022), Cancers 14:861, incorporated herein by reference), G250RIT (when labeled with a suitable detectable moiety), or 90 Y-DOTA-cG250.

[0120] In a particularly preferred embodiment, the agent for binding to CAIX is 89 Zr-girentuximab. 89 Zr-girentuximab is a chimeric monoclonal antibody (INN name: girentuximab (GTX), also known as cG250 and TLX250) with specificity for the CAIX antigen, radiolabeled with the positron-emitting radiometal zirconium-89 via NSuc-DFO-TFP-ester (DFO-TFP) linked to the lysine residue of GTX. 89 Zr results in DFO-TFP-GTX.

[0121] constant region In a preferred embodiment, any antibody and / or antigen-binding fragment thereof described herein for use in the present invention may comprise an antibody constant region, including an antigen-binding fragment of an antibody fused to Fc.

[0122] The sequences of constant regions useful for generating the antibodies or antigen-binding fragments thereof described herein can be obtained from several different sources. In some examples, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one example, the constant region is a human isotype IgG4 or stabilized IgG4 constant region.

[0123] The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG class antibodies in vivo. 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: the 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and the 15 kDa p2-microglobulin (β2ηι). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG class antibodies. The interaction between IgG class antibodies and FcRn is pH-dependent and occurs with 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, AH, et al., J. Mol. Biol. 230 (1993) 1077-1083).

[0124] Therefore, the in vitro FcRn binding properties / characteristics of IgG indicate its in vivo pharmacokinetic properties in the blood circulation. Different amino acid residues in the heavy chain CH2- and CH3-domains are involved in the interaction between FcRn and the Fc region of IgG class antibodies.

[0125] Different mutations that affect FcRn binding and the resulting half-life in the blood circulation have been identified. The Fc region residues important for mouse Fc region-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al., J. Immunol. 169 (2002) 5171-5180). Residues Ile253, His310, His433, Asn434, and His435 (numbered according to the 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, JK, 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 important for the interaction of the human Fc region with mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819-2885).

[0126] More specifically, the antibody or antigen-binding protein can contain one or more amino acid substitutions that reduce the half-life of the protein. For example, the antibody or antigen-binding fragment thereof comprises an Fc region that contains one or more amino acid substitutions that reduce the affinity of the Fc region for the neonatal Fc region (FcRn).

[0127] Preferred modifications In any embodiment, the antibody or antigen-binding fragment thereof (e.g., the G250 antibody or variant thereof 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 class IgG, wherein the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn), thereby decreasing the serum half-life of the modified antibody compared to a wild-type antibody of class IgG.

[0128] 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, thereby reducing the affinity of the IgG molecule for FcRn. Alternatively, the one or more amino acid substitutions can 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 can include at least one substitution in the CH2 region and at least one substitution in the CH3 region of the IgG molecule, thereby reducing the affinity of the IgG molecule for FcRn.

[0129] In certain preferred embodiments, the one or more amino acid substitutions may be at one or more of IgG residues His310, His433, His435, His436, or Ile253. Preferably, the amino acid substitutions comprise a substitution in the heavy chain constant region at position His310 or His435. More preferably, the amino acid substitutions that reduce the affinity of the antibody for FcRn are at both His310 and His435.

[0130] In another preferred embodiment, the antibody and / or antigen-binding fragment thereof has a constant region substantially identical to that of a naturally occurring class IgG antibody, but differs from that present in a naturally occurring class IgG antibody by at least one amino acid residue selected from the group consisting of His310, His435, and Ile253, thereby altering the FcRn binding affinity and / or serum half-life of the antibody compared to the naturally occurring antibody. In a preferred embodiment, the naturally occurring class IgG antibody comprises the heavy chain constant region of a human IgG1, IgG2, IgG2M3, IgG3, or IgG4 molecule.

[0131] Furthermore, in a preferred embodiment, amino acid residue 310 and / or residue 435 from the heavy chain constant region of an antibody having substantially the same constant region as a naturally occurring class IgG antibody is not histidine but any amino acid that reduces the affinity of the constant region for FcRn. For example, the amino acid at residue 310 and / or 435 can be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine.

[0132] The amino acid substitution may include a substitution of a histidine residue with alanine, glutamine, glutamic acid, or aspartic acid. Preferably, the amino acid substitution at His310 is with alanine. Preferably, the amino acid substitution at His435 is with glutamine. Preferably, the amino acid substitution at Ile253 is with alanine.

[0133] In preferred embodiments of the invention, the binding affinity to FcRn and / or serum half-life of the modified antibody is reduced 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 preferred embodiments of the invention, the binding affinity to FcRn and / or serum half-life of the modified antibody is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0134] Additionally, antibodies or fragments thereof for use according to the invention can be modified to contain one or more mutations that alter the affinity of the antibody for any one or more Fc gamma receptors, for example, one or more amino acid modifications that alter the affinity of an antibody constant domain, Fc region, or Fc gamma receptor-binding fragment for any one or more Fc gamma receptors.

[0135] In certain embodiments, the modified antibodies or antigen-binding fragments thereof retain the ability to bind to one or more Fc-gamma receptors, and thus, in certain embodiments, the modified antibodies retain the ability to stimulate an effector response (including ADCC). In one example, the Fc region of the constant region comprises one or more amino acid substitutions that modulate effector function, including increasing effector function compared to wild-type IgG.

[0136] In one example, the Fc region of the constant region has a reduced ability to induce effector function, for example, compared to the Fc region of native or wild-type human IgG1 or IgG3. 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.

[0137] In one example, the amino acid substitution that alters the 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 with any amino acid selected from alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine, and the substitution reduces the ability of the antibody to induce effector function. In a preferred embodiment, the substitution at residue 253 for Ile is with arginine, proline, glutamic acid, or aspartate, more preferably with alanine.

[0138] In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4 constant region), e.g., a human IgG4 Fc region. Sequences of suitable IgG4 Fc regions will be apparent to those skilled in the art and / or are available in publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0139] In one example, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce its tendency to undergo Fab arm exchange or to form half antibodies or to form half antibodies. "Fab arm exchange" refers to a type of protein modification to human IgG4 in which the IgG4 heavy chain and attached light chain (half molecule) are exchanged with a heavy chain-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or a reducing agent such as reduced glutathione. "Half antibodies" are formed when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0140] In one example, a stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to the Kabat system (see 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 IgG4, this residue is generally serine. After substitution of serine for proline, the IgG4 hinge region contains the sequence CPPC. In this regard, those skilled in the art will recognize that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that connects the Fc and Fab regions, conferring mobility to the two Fab arms of the antibody. The hinge region contains the cysteine ​​residues involved in the inter-heavy chain disulfide bond. It is generally defined according to the Kabat numbering system as stretching from Glu226 to Pro243 of human IgG1 (or from Glu216 to Pro230 using the EU index). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same positions (see, for example, WO2010 / 080538).

[0141] In alternative embodiments, the one or more amino acid modifications that reduce affinity for the FcRn receptor also reduce affinity for an Fc gamma receptor. The modified antibody or antigen-binding fragment thereof can further comprise one or more amino acid substitutions compared to a wild-type antibody of class IgG, which amino acid substitutions further reduce the affinity of the antibody for one or more Fc gamma receptors.

[0142] In a further embodiment, the modified antibody or antigen-binding fragment thereof further comprises one or more amino acid substitutions compared to a wild-type antibody of class IgG, which amino acid substitutions increase the stability of the CH1-CH2 hinge region in the modified antibody compared to a wild-type antibody of class IgG.

[0143] In any embodiment, the heavy chain constant region of the antibody or antigen binding protein thereof comprises amino acid substitutions at both His310 and His435. The antibody may also comprise amino acid substitutions at residues corresponding to Ser228 and Leu235 of the constant heavy chain region.

[0144] In some embodiments, 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.

[0145] An additional example of a stabilized IgG4 antibody is one in which the arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) has been substituted with lysine, threonine, methionine, or leucine (e.g., as described in WO2006 / 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., the CPPC sequence) (as described above).

[0146] 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 IgG1 Fc region containing substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region containing 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 P331S (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. 177:1129-1138 2006, and / or Hezareh J Virol; 75:12161-12168, 2001).

[0147] In another example, the Fc region is a chimeric Fc region comprising, for example, at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, and 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.

[0148] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the sequence shown in any one of SEQ ID NOs: 177 to 180, preferably SEQ ID NO: 178.

[0149] In still further embodiments, the antibody or antigen-binding fragment thereof preferably comprises a heavy chain comprising the sequence set forth in any one of SEQ ID NOs: 182-185, preferably SEQ ID NO: 183.

[0150] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 181. Preferably, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 186.

[0151] 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.

[0152] In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to, or comprises the sequence set forth in SEQ ID NO: 36 or 52, and a VL that comprises a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to, or comprises the sequence set forth in SEQ ID NO: 116, 132, or 148.

[0153] Preferably, the VH comprises a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to, or comprises the 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 the sequence set forth in SEQ ID NO: 132 or 148.

[0154] More preferably, the VH comprises a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to, or comprises the sequence set forth in SEQ ID NO: 36, and the VL comprises a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to, or comprises the sequence set forth in SEQ ID NO: 148.

[0155] Alternatively, the VH comprises a sequence that is at least about 95%, or 96%, or 97%, or 98%, or 99% identical to, or comprises the sequence set forth in SEQ ID NO: 52, and the VL comprises a sequence that is at least about 95%, or 96%, or 97%, or 98%, or 99% identical to, or comprises the sequence set forth in SEQ ID NO: 132 or 148, preferably the sequence set forth in SEQ ID NO: 148.

[0156] Detectable Part Those of skill in the art will be familiar with standard methods for conjugating a detectable moiety to an agent for binding to CAIX.

[0157] In any embodiment of the invention, the small molecules, peptides, proteins, or antibodies described herein for binding to CAIX may be directly or indirectly linked to a detectable moiety, such as a radioisotope, dye, or fluorescent moiety.

[0158] In any embodiment, the detectable moiety is a radioisotope. Examples of suitable isotopes include gallium-67 and gallium-68 ( 67 Ga and 68 Ga), Indium-111( 111 In), iodine-123, iodine-124, or iodine-131 ( 123 I, 124 I, or 131 I), technetium-99( 99m Tc), and zirconium-89 (89 As used herein, the term radionuclide may be used interchangeably with the term radioisotope.

[0159] It will be understood that radioisotopes can be conjugated to polypeptides (e.g., antibodies) directly (via chelators or prosthetic groups or linkers) or indirectly via attachment to single or multiple amino acid residues in the protein (e.g., halogenation of tyrosine residues).

[0160] In alternative embodiments, chelators or linkers may be used to conjugate datable moieties to peptides or proteins for binding to CAIX. In one example, a peptide or protein (e.g., an antibody) can be conjugated to a chelating moiety, such as 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-N-N',N"(N'"-tetraacetic acid, also known as tetraxetane), TCMC (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]tetradecane), NOTA (1,4,7-triazacyclononane-triacetic acid), diacetamide, ... Amsar (3,6,10,13,16,19-hexazabicyclo[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-diaminepentaacetic acid), TETA (1,4,8,11-tetraazabicyclo[6.6.6]eicosane-1,8-diamine) DTPA (pentetic acid or diethylenetriaminepentaacetic acid), The chelating agent is selected from the group consisting of tetraazabicyclotetradecane-1,4,8,11-tetraacetic acid, Te2A (4,11-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 agents described herein.Other known chelating moieties include 3p-C-NETA ({4-[2-(bis-carboxymethylamino)-5-(4-nitrophenyl)pentyl]-7-carboxymethyl-[1,4,7]triazanonan-1-yl}acetic acid), 5p-C-NETA (2-({1-[4,7-bis(carboxymethyl)-1,4,7-triazanonan-1-yl]-7-(4-nitrophenyl)heptan-2-yl}(carboxymethyl)amino)acetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and NODA (1,4,7-triazacyclononane-1,4-diacetic acid).

[0161] In certain non-limiting embodiments discussed below, chelating groups are used to complex metals such as aluminum. 18 F or 19 F may be attached to provide alternative modalities for imaging, detection, and / or diagnosis. Fluorescently labeled molecules may be used more for intraoperative procedures, 18 It is anticipated that F-labeled molecules may be used more extensively for pre- or post-operative imaging, detection, and / or diagnosis of diseased tissue.

[0162] The agent can be modified to include a sulfhydryl group for attaching a maleimide-modified fluorescent probe. Alternatively, fluorescent dyes conjugated to bis-functional crosslinkers or other reactive species can be used to attach the fluorescent probe to a different group on the agent for binding to CAIX. For example, DYLIGHT® 488 and DYLIGHT® 800 are available as amine-reactive dyes derivatized with NHS esters to label primary amines (product numbers 46402 and 46421, Thermo Electric, Rockford, Ill.). Those skilled in the art will recognize that the fluorescent probe used is not limiting and that other DYLIGHT® dyes, or alternative fluorescent probe molecules known in the art, can be used in the claimed methods and compositions.

[0163] In certain embodiments, peptides or proteins (e.g., antibodies) for binding to CAIX can 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 can be utilized. For example, a fluorescent probe can be attached to the hinge region of a reduced antibody component via disulfide bond formation or sulfhydryl-maleimide interactions. Alternatively, such agents can be attached using heterobifunctional crosslinkers 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. 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).

[0164] Alternatively, fluorescent probes can be conjugated via carbohydrate moieties in the Fc region of antibodies. 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. Patent No. 5,057,313, the Examples section of which is incorporated herein by reference. A common method involves reacting an antibody component bearing an oxidized carbohydrate moiety with a fluorescent probe bearing at least one free amine functionality. This reaction results in an initial Schiff base (imine) bond, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0165] When an antibody is used as the antibody component of an immunoconjugate, the Fc region may be absent. 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. Patent Nos. 5,443,953 and 6,254,868, the Examples sections of which are incorporated herein by reference. Engineered carbohydrate moieties are used to attach functional groups to antibody fragments.

[0166] An alternative method for attaching fluorescent probes or other functional groups to target molecules involves the use of click chemistry. The click chemistry approach was initially conceived as a way to rapidly generate complex materials by modularly linking small subunits together (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, such as the copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), are known in the art and are often referred to as "click reactions." Other alternatives include cycloaddition reactions such as Diels-Alder reactions, nucleophilic substitution reactions (especially for small strained rings such as epoxy and aziridine compounds), carbonyl chemistry formation of urea compounds, and reactions involving carbon-carbon double bonds such as alkynes in thiol-yne reactions.

[0167] Copper-free click reactions have been proposed for the covalent modification of biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc 126:15046-47.) Copper-free reactions use ring strain instead of copper catalyst to promote the [3 + 2] azide-alkyne cycloaddition reaction. For example, cyclooctyne is an eight-carbon ring structure containing an internal alkyne bond. The closed ring structure induces substantial bond angle distortion of the acetylene, which is highly reactive with the azide group to form a triazole. Therefore, cyclooctyne derivatives can be used for copper-free click reactions.

[0168] Another type of copper-free click reaction, reported by Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involves strain-promoted alkyne-nitrone cycloaddition. To address the slow kinetics of the initial cyclooctyne reaction, an electron-withdrawing group is attached adjacent to the triple bond. Examples of such substituted cyclooctynes ​​include difluorinated cyclooctynes, 4-dibenzocyclooctynol, and azacyclooctynes. An alternative copper-free reaction involved strain-promoted alkyne-nitrone cycloaddition to give N-alkylated isoxazolines. The reaction was reported to have very fast kinetics and was used in a one-pot, three-step protocol for site-specific modification of peptides and proteins. Nitrones were prepared by condensation of appropriate aldehydes with N-methylhydroxylamine, and the cycloaddition reaction was carried out in a mixture of acetonitrile and water. These and other known click chemistry reactions can be used to conjugate chelating moieties to antibodies or other CAIX-binding molecules in vitro.

[0169] In certain embodiments, the agent is a radioisotope 124 The isotope may comprise a peptide or protein (e.g., an antibody) covalently bound to the isotope I. The isotope is a positron emitter that can be attached to the antibody, for example, as described by Larsson et al. (J. Nucl. Med. 33 (1992), 2020-2023) or US Pat. No. 5,185,142 (the contents of which are incorporated herein by reference).

[0170] In some embodiments, radiolabeling of proteins or antibodies is achieved by covalent iodination, particularly with the iodogen reagent (1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril). Iodogen labeling is a solid-phase oxidation method similar to the chloramine-T method, but is generally considered milder because the reaction occurs on the surface of the oxidizing agent, minimizing substrate exposure (Salacinzki, PRP, et al., Anal. Biochem. 117:136 (1981)).

[0171] Chelators bearing radiometals and other halogenated radioisotopes may be attached to proteins or antibodies through 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.

[0172] In another example, proteins or antibodies can be conjugated to bifunctional linkers, such as bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, or using any amine or thiol modification chemistry known in the art.

[0173] Those skilled in the art will be familiar with standard methods for conjugating chelators to proteins, including antibodies and derivatives or fragments thereof. In addition, those skilled in the art will be familiar with approaches for selecting relevant chelators for pairing with radiometals, as described, for example, in Chem. Soc. Rev., 2014, 43, 260 (incorporated herein by reference).

[0174] In any embodiment, the dataable moiety can be a fluorescent dye, such as but not limited to those described in US2015 / 0086482 (incorporated herein by reference).

[0175] In any embodiment, the fluorescent dye (which may also be referred to as a fluorescent probe) is 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′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylamino, 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 The dye may be selected from fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptate, europium trisbipyridinediamine, europium cryptate or chelate, diamine, dicyanine, LaJolla blue dye, allopycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas Red.

[0176] Drug administration Those skilled in the art will understand that the dosage of an agent for use in accordance with the methods of the present invention will depend on a variety of factors, including the age, sex, height, and weight of the subject to whom the agent is administered, and will depend on the agent.

[0177] When the agent is an antibody for binding to CAIX, the antibody is preferably administered to the subject, i.e., injected, at a dose of about 1 mg to about 50 mg, preferably at a dose of about 5 mg to about 20 mg, more preferably at a dose of about 10 mg. The specific radioactivity of the radiolabeled antibody is preferably about 15 to about 20 MBq / mg, more preferably about 18 to about 19 MBq / mg.

[0178] In certain embodiments, the agent for binding to CAIX is a radiolabeled girentuximab antibody, and the antibody is administered by slow infusion at a mass dose of about 10 mg of girentuximab.

[0179] The antibody is typically administered as a pharmaceutical composition having a pharmaceutically acceptable carrier, e.g., physiological saline solution, optionally containing a protein stabilizer such as human serum albumin (HSA). The antibody is preferably administered by infusion.

[0180] The CAIX-binding agent, preferably girentuximab or a humanized variant thereof, is preferably administered intravenously, preferably by infusion or intravenous injection. Administration of the antibody by infusion is preferably carried out over a period of up to about 30 minutes, more preferably about 15 minutes. Of course, the CAIX inhibitor can also be administered intraperitoneally or intramuscularly.

[0181] Detection Method It will be appreciated that the method for detecting or imaging an agent for use in accordance with the present invention will depend on the nature of the detectable moiety of the agent.

[0182] The detecting step is preferably carried out using PET, SPECT, fluorescence spectroscopy, or any other suitable method.

[0183] Exemplary in vivo methods for determining the presence or expression of CAIX in tumors include the use of in vivo / regional 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 can involve the use of CAIX-binding molecules conjugated to radioisotopes, which allow for non-invasive imaging of tissues and tumors that express CAIX.

[0184] If the detectable moiety is a radioisotope, then the method includes determining the radiation for the subject to which the agent has been administered.

[0185] The in vivo detection step in the above-described methods can be whole-body imaging or local imaging at a specific site, such as but not limited to the site of expected or potential solid tumor growth.

[0186] For SPECT, agents for binding to CAIX typically include detectable agents in the form of gamma-emitting radioisotopes (radionuclides), usually via injection into the bloodstream. Typically, gamma-emitting radioisotopes for use in SPECT include: 99m Tc (technetium), 123 I or 131 I (iodine), and 68 Ga (gallium) is an example.

[0187] In any embodiment, if the agent comprises a radioisotope, the detection method may include positron emission tomography (PET).

[0188] Optionally, the detection method comprises PET / CT imaging or PET / MRI scanning.

[0189] After administration (preferably injection) of the agent, it may be practical to leave it for a period of time to allow the agent to accumulate at the site of tumor-infecting cancer cells. Typically, the period 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 least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days. Preferably, the period, which is the time between administration of the agent and detection of the agent (e.g., by PET or other methods described herein), is typically about 10 days or less, or about 15 days or less, or about 20 days or less.

[0190] When cancer imaging or detection uses PET, PET imaging may be preferably performed within 7±2 days of injection of the radiolabeled agent, particularly 5±2 days after injection, to obtain optimal imaging results including accumulation of the agent at the site where CAIX is present.

[0191] In situations where the detectable moiety is a fluorescent probe or dye, detection of the moiety can be performed using fluorescence imaging, including during intraoperative, intravascular, or endoscopic procedures, as described in U.S. Patent Nos. 4,932,412, 6,096,289, 6,387,350, and 7,201,890, the Examples section of each of which is incorporated herein by reference. Such imaging methods can be used, for example, to image the distribution of tumor tissue and facilitate its removal. Fluorescence imaging can also be used for diagnostic purposes, for example, to distinguish between malignant, benign, and hyperplastic tissue.

[0192] Cancer detected or diagnosed The present invention provides methods for identifying or imaging cancer in vivo, which are expected to be useful in diagnosing CAIX-expressing cancers, preferably without the need for additional invasive procedures (such as biopsy collection and testing) to confirm the diagnosis.

[0193] Thus, in a preferred embodiment, the methods of the present invention allow for the diagnosis of any cancer listed herein as the sole, primary, or main mode of diagnosis of cancer, preferably without the need for additional invasive diagnostic methods, including biopsy-related methods.

[0194] The methods of the present invention are also expected to be useful for staging the progression of cancer or the successful treatment of cancer, again offering the advantage of providing a non-invasive means for assessing cancer in a subject.

[0195] As used herein, the term "cancer" refers to a malignant growth or tumor that results from the uncontrolled division of cells. The term "cancer" includes primary tumors and metastatic tumors.

[0196] The methods of the present invention find particular utility in imaging, detecting, and / or diagnosing previously unidentified cancers using in vivo imaging techniques that utilize agents to bind to CAIX.

[0197] The subject for cancer diagnosis, detection, or imaging described herein may be suspected of having cancer or may be at risk of having cancer. A subject suspected of having cancer may exhibit one or more symptoms of cancer, may have a family history of cancer, or may have one or more genetic markers that indicate the risk or likelihood of developing cancer. A subject considered to be at risk of having cancer may exhibit one or more symptoms of cancer, may have a family history of cancer, or may have one or more genetic markers that indicate the risk or likelihood of developing cancer.

[0198] In any embodiment, the cancer to be detected, imaged, or diagnosed is breast cancer. High levels of CAIX have previously been reported in breast cancer, and CAIX expression has also been reported to be associated with resistance to chemotherapy or as an indicator of successful treatment. These observations date back several decades, and to date, diagnosis using CAIX-binding imaging agents has not previously been reported for this patient population.

[0199] The breast cancer may be so-called "triple-negative breast cancer" (TNBC), an aggressive, metastatic, and drug-resistant form of breast cancer with limited treatment options, which is negative for other biomarkers of breast cancer, such as estrogen receptor (ER-positive breast cancer), progesterone receptor (PR-positive breast cancer), and human epidermal growth factor receptor 2 (HER2-positive breast cancer).

[0200] In any embodiment, the breast cancer can be hormone receptor positive breast cancer, such as ER positive, PR positive, ER&PR positive, etc. In any embodiment, the breast cancer can 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.

[0201] In any embodiment, the cancer to be detected, imaged, or diagnosed is cervical cancer. Cervical cancer can be squamous cell carcinoma or adenocarcinoma. In any embodiment, a subject for diagnosis or imaging of cervical cancer can exhibit one or more symptoms of cervical cancer, such as abnormal vaginal bleeding, including contact bleeding, or pelvic pain. A subject considered to be at risk for cervical cancer can have had a previous infection with HPV strains 16 or 18, or have one or more genetic markers indicative of risk for cervical cancer.

[0202] In any embodiment, the cancer to be detected, imaged, or diagnosed is colorectal cancer (including, for example, epithelial colorectal adenocarcinoma). In any embodiment, a subject for diagnosis or imaging of colorectal cancer may exhibit one or more symptoms of colorectal cancer, such as persistent changes in bowel habits, rectal bleeding or blood in the stool, persistent abdominal discomfort, weakness or fatigue, and unexplained weight loss. A subject considered to be at risk for colorectal cancer may have a family history of the disease, or may have one or more genetic markers considered to be associated with an increased risk of colorectal cancer, or may have previously had intestinal polyps.

[0203] In any embodiment, the cancer to be detected, imaged, or diagnosed is esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / gastric junction adenocarcinoma). A subject for diagnosis, imaging, or detection of esophageal cancer may exhibit one or more symptoms selected from difficulty swallowing, unexplained weight loss, chest pain, pressure, or burning, worsening indigestion or heartburn, or cough or hoarseness. A subject considered to be at risk for esophageal cancer may have a family history of the disease, or may have one or more genetic markers considered to be associated with an increased risk of colorectal cancer, or may have previously been diagnosed with Barrett's esophagus.

[0204] In any embodiment, the cancer to be detected, imaged, or diagnosed is gastric cancer (including gastric adenocarcinoma). A subject diagnosed, imaged, or detected with gastric cancer may exhibit one or more symptoms selected from difficulty swallowing, stomach pain, bloating after eating a small amount of food, loss of appetite, indigestion, nausea and vomiting, fatigue, and dark stools. A subject considered to be at risk for gastric cancer may have a family history of the disease or have one or more genetic markers considered to be associated with an increased risk of gastric cancer.

[0205] In any embodiment, the cancer being detected, imaged, or diagnosed is glioblastoma multiforme. Subjects being diagnosed, imaged, or detected for glioblastoma may exhibit one or more symptoms, including vision, hearing, balance, coordination, strength and reflex symptoms, nausea, vomiting, seizures, or other neurological symptoms.

[0206] In some embodiments, the cancer being detected, imaged, or diagnosed is head and neck cancer (including head and neck squamous cell carcinoma and nasopharyngeal and hypopharyngeal cancer). A subject being diagnosed, imaged, or detected for head and neck cancer may exhibit one or more symptoms, such as pain, swelling, hoarseness, sore throat, persistent cough, bad breath, or unexplained weight loss. A subject considered to be at risk for head and neck cancer may have a family history of the disease, possess one or more genetic markers believed to be associated with an increased risk of head and neck cancer, have a previous infection with HPV or Epstein-Barr virus, a weakened immune system, periodontal disease, smoking, or poor oral hygiene, including chewing betel nut, betel nut, gutka, or bread, or have a genetic condition, such as Fanconi anemia or Li-Fraumeni syndrome.

[0207] In any embodiment, the cancer to be detected, imaged, or diagnosed is liver cancer (including cholangiocarcinoma and hepatocellular carcinoma). As used herein, cholangiocarcinoma refers to biliary tract cancer or bile duct cancer. The cholangiocarcinoma can be intrahepatic, distal hilar cholangiocarcinoma. The cancer can be gallbladder cancer or cancer of the ampulla of Vater. In any embodiment, the subject for diagnosis or imaging of liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) can be a subject who exhibits one or more symptoms of liver cancer (including cholangiocarcinoma and hepatocellular carcinoma).

[0208] As used herein, one or more symptoms of cholangiocarcinoma include abdominal pain, yellowish skin (jaundice), weight loss, generalized itching, fever, pale stools, or dark urine.Those skilled in the art will be familiar with various risk factors for cholangiocarcinoma, including primary sclerosing cholangitis (inflammatory disease of the bile duct), ulcerative colitis, liver cirrhosis, hepatitis C, hepatitis B, infection with certain liver flukes, and some congenital liver malformations.However, most people do not have identifiable risk factors.

[0209] In some embodiments, the cancer to be detected, imaged, or diagnosed is lung cancer (including epithelial non-small cell carcinoma and small cell carcinoma). As used herein, the term "lung cancer" includes all types of lung cancer at all stages of progression, such as, but not limited to, non-small cell lung cancer (NSCLC), squamous cell carcinoma, or small cell lung cancer (SCLC), including metastatic lung cancer and lung adenocarcinoma. In some embodiments, the subject is afflicted with non-small cell lung cancer (NSCLC).

[0210] In any embodiment, the cancer to be detected, imaged, or diagnosed is ovarian cancer (including ovarian epithelial cancer).

[0211] In any embodiment, the cancer to be detected, imaged, or diagnosed is pancreatic cancer (including pancreatic ductal adenocarcinoma).

[0212] In any embodiment, the cancer to be detected, imaged, or diagnosed is a soft tissue sarcoma.

[0213] In any embodiment, the cancer to be detected, imaged, or diagnosed is bladder cancer. The bladder cancer can be non-muscle-invasive bladder cancer (NMIBC).

[0214] In particularly preferred embodiments, the cancer being detected, imaged, or diagnosed is not kidney cancer (including clear cell renal cell carcinoma).

[0215] Cancer imaging or diagnosis is typically assessed after administration of an agent and its detection by qualitatively assessing the detection of the agent compared to conventional imaging. Quantitative assessments can be lesion-specific and include standardized uptake values ​​(SUV) (SUVmax and SUVmean), SUV corrected for lean mass (SUL), metabolic tumor volume (MTV), and tumor-to-background ratio (TBR).

[0216] The tumor-to-background ratio (TBR) is typically defined as the ratio of the lesion-standardized uptake value (SUVmax) to the reference region SUV (liver, blood pool, etc.). Comparisons will be made of the number, size, and other characteristics of lesions detected by PET scans with standard imaging modalities, including high-resolution CT / MRI and other possible imaging, for each patient (depending on tumor type), lesion type, and indication.

[0217] Qualitative visual analysis of imaging (presence or absence of tumor-associated localized drug uptake as shown by contrast-enhanced CT, MRI, or FDG PET / CT) can be used to assess the agreement in tumor lesion detection between drug-specific PET / CT and conventional imaging. RECIST 1.1 criteria for conventional imaging can be used as the primary tool for agreement comparison to PET.

[0218] In addition to the above, any visible tumor lesions on conventional imaging can also be compared with the PET imaging results. [Example]

[0219] Example 1: Clinical Trial Protocol The exam is 89 Assessment of CAIX expression in a subset of solid tumors using Zr-labeled girentuximab deferoxamine PET / CT imaging.

[0220] Main Objective CAIX tumor expression in different solid tumors 89 To evaluate Zr-girentuximab PET / CT imaging noninvasively. Formal imaging studies are available for these tumor types. 89 No studies have been performed on Zr-girentuximab uptake.

[0221] Primary endpoint: Qualitative (presence / absence) and quantitative assessment of 89Zr-girentuximab uptake compared with conventional imaging. Descriptive statistics will be reported for each tumor type. Lesion-specific analyses include SUVmax, SUVmean, SUL (SUV corrected for lean mass), tumor-to-background ratio (TBR), and metabolic tumor volume (MTV).

[0222] Secondary Objectives in patients with different tumor types 89 To assess the tolerability and safety of Zr-girentuximab administration.

[0223] Secondary Endpoints: Patient safety will be assessed based on the incidence and nature of adverse events (AEs) and serious adverse events (SAEs), as well as clinically significant changes in laboratory values, vital signs, or physical examination findings. Laboratory abnormalities will be assessed according to the NCI CTCAE v. 5.0. Patients will be informed that any abnormal physical signs occurring within 24 hours of this test must be reported to the investigator for enrollment. The NCI Common Toxicity Criteria, version 5.0 reference will be used.

[0224] Tertiary goal 89 Standardized uptake value (SUV) of Zr girentuximab and 89 To evaluate the correlation between Zr-girentuximab imaging and CAIX histologic expression in patients undergoing biopsy or surgery within 90 days of imaging.

[0225] Tertiary endpoint: biopsy or surgical specimens 89 Zr-girentuximab imaging, if available within 90 days before or after administration (and tissue samples available); 89 The correlation between the standardized uptake value (SUV) of Zr-girentuximab and CAIX histological expression will be evaluated by comparing the semiquantitative data of 89Zr-girentuximab with the immunohistochemical results (IHC) of biopsied / resected tumors at the site.

[0226] All patients will be followed for safety until the EOS visit (days 15-25).

[0227] Overall Study Design An open-label, non-randomized study was conducted to compare the efficacy and safety of EGFR-1 in different tumor types. 89 To assess CAIX expression via Zr-girentuximab PET / CT imaging and evaluate the feasibility of targeting CAIX for potential diagnostic and therapeutic applications.

[0228] A minimum of five subjects will be enrolled for each tumor type, including, but not limited to, cervical cancer, colorectal cancer, esophageal cancer (esophageal SCC and esophageal / gastric 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 (ovarian epithelial carcinoma), pancreatic cancer (pancreatic adenocarcinoma), and soft tissue sarcoma.

[0229] This test is 89 It involves a single administration of Zr-girentuximab (37 MBq [1 mCi] ± 10%, containing a mass dose of 10 mg of girentuximab).

[0230] PET / CT imaging will be performed 5±2 days after administration. Image data analysis of PET / CT imaging will be performed by a nuclear medicine reader, and up to 10 most active lesions will be analyzed. 89 Tumor uptake of Zr-girentuximab will be assessed and also according to conventional imaging per RECIST 1.1.

[0231] Qualitative visual analysis (localized tumor-related lesions as shown by contrast-enhanced CT, MRI, or FDG PET / CT) 89 The presence or absence of Zr-girentuximab uptake was used to evaluate the agreement of tumor lesion detection between 89Zr-girentuximab PET / CT and conventional imaging. Lesions revealed by 89Zr-girentuximab alone are described.

[0232] Tissue samples (from patient biopsies or surgery) will be collected whenever possible and sent to a central laboratory for CAIX expression analysis.

[0233] The test evaluation is performed as shown in the table below.

[0234] [Table 2]

[0235] dosage Used here 89 The Zr-girentuximab dose (37 MBq [1 mCi] ± 10%, including a 10 mg girentuximab mass dose) is consistent with the Zr-girentuximab dosing regimen in an ongoing phase 3 clinical trial, which Merkx et al. (2021) demonstrated allows PET imaging 4–7 days after administration.

[0236] 89 Zr-girentuximab is a radiolabeled chimeric monoclonal antibody (INN name: Zirconium Zr 89 Girentuximab deferoxamine. Girentuximab is a chimeric monoclonal antibody (INN: girentuximab, synonyms: cG250, TLX250) with specificity for the CAIX (carbonic anhydrase 9) antigen, radiolabeled with the positron-emitting radiometal zirconium-89 via NSuc-DFO-TFP-ester (DFO-TFP) linked to a lysine residue in girentuximab. 89 This results in Zr-DFO-girentuximab.

[0237] 89 Zr-girentuximab is formulated as a solution for intravenous administration in a nominal dosage strength of 37 MBq (±10%) (1 mCi ±10%) containing a total of 10 mg of girentuximab for a single intravenous use. 89The Zr-girentuximab solution will be supplied in either glass vials or syringes (depending on the geographic region) in appropriate packaging (lead-shielded containers with radioactive warning symbols in accordance with radiopharmaceutical requirements).

[0238] Compounds were administered as a single dose by slow intravenous infusion over 3 minutes via a single peripherally placed intravenous cannula. 89 It is administered as Zr-girentuximab (37 megabecquerels ± 10%, [1 mCi ± 0.1 mCi] containing a mass dose of 10 mg of girentuximab). The injection volume is approximately 10 ml, depending on the radioactivity administered.

[0239] Inclusion criteria All participants meet the following criteria: 1. Written and freely obtained informed consent. 2. Male or female, aged 18 years or older at the time of consent. 3. Ability to understand the study and willingness to comply with all protocol requirements. 4. Participant must have histologically or cytologically proven solid tumors of the following types (including but not limited to): Cervical cancer Colorectal cancer Esophageal cancer (esophageal SCC and esophageal / esophagogastric junction adenocarcinoma) ● Stomach cancer (gastric adenocarcinoma) Glioblastoma multiforme Head and neck cancer (head and neck SCC and nasopharyngeal cancer) ● Liver cancer (cholangiocarcinoma and hepatocellular carcinoma) Lung cancer (non-small cell and small cell) ●Ovarian cancer (ovarian epithelial cancer) Pancreatic cancer (pancreatic ductal adenocarcinoma) Soft tissue sarcoma 5. At least one non-CNS, measurable target lesion per RECIST 1.1, documented by conventional imaging, performed within 30 days prior to Day 0. 6. Participants agree not to participate in any other interventional studies while participating in this study, as defined when signing the Informed Consent Form (ICF), until the final study visit is completed. 7. A negative serum pregnancy test in female patients of childbearing potential at screening and confirmed negative urine pregnancy test result within 24 hours prior to receiving study drug. Female patients of non-childbearing potential must provide evidence by meeting one of the following criteria at screening: Postmenopausal is defined as age greater than 50 years and amenorrhea for at least 12 months after cessation of all exogenous hormone therapy. • Women under 50 years of age are considered postmenopausal if they have been amenorrhea for 12 months or more since discontinuation of exogenous hormone therapy and their luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels are within the institutional postmenopausal range. • Documentation of irreversible sterilization by hysterectomy, bilateral oophorectomy, or bilateral salpingectomy without tubal ligation. 8. For all participants: 89 Agree to use double barrier contraception for at least 42 days after Zr-girentuximab administration.

[0240] Exclusion criteria Patients will be excluded from participation in the study if they meet one or more of the following criteria: 1. Exposure to murine or chimeric antibodies within the past 5 years. 2. 89 Prior administration of any radionuclide within 10 half-lives (of the radionuclide) prior to the intended administration of Zr-girentuximab (i.e., within 10 half-lives from day 0). 3. Exposure to any CAIX targeting compound (diagnostic / therapeutic) in the past 3 months 4. Serious non-malignant disease (e.g., psychiatric, infectious, autoimmune, or metabolic disease) that, in the investigator's judgment, may interfere with the objectives of the study or the safety or compliance of the subject. 5. Any clinically significant abnormality detected during the screening laboratory or physical examination that, in the opinion of the investigator, may adversely affect the participant's ability to participate in the study. The investigator will assess the patient's suitability for inclusion based on pathology and tumor type. 6. Psychiatric disorders that may impair the ability to grant informed consent and comply with the requirements of the study. 7. 89 Exposure to any antitumor treatment within 14 days of the planned date of administration of Zr-girentuximab (i.e., within 14 days of day 0). 8. Pregnant or breastfeeding women. 9. Known allergy, hypersensitivity, or irritability to girentuximab, DFO (desferrioxamine), or intolerance to any of the components of the investigational drug. Renal failure with a glomerular filtration rate (GFR) of 10.45 ml / min / 1.73 m2 or less 11. Vulnerable patients (e.g., in custody).

[0241] Efficacy evaluation Imaging noninvasively assesses CAIX tumor expression in patients 89 Based on the performance of Zr-girentuximab in PET / CT imaging. 89 A single dose of Zr-girentuximab is followed by a whole-body PET / CT scan on day 5±2 after administration, according to the table above and the imaging manual. Patients with metastatic (suspected or confirmed) disease may undergo an optional additional whole-body PET / CT scan if clinically indicated (e.g., if the tumor-to-background ratio makes tumor lesions difficult to identify and improvement is expected).

[0242] 89 Zr-girentuximab tumor uptake will be assessed qualitatively (present / absent) compared with conventional imaging for up to the 10 most active lesions in each patient. Quantitative assessments may be per lesion and include SUVmax, SUVmean, SUL, MTV, and TBR.

[0243] For patients who cannot undergo a CT scan for any reason and / or for whom a CT scan is contraindicated, PET / MRI can be performed instead of PET / CT if available at the investigational site. PET / MRI can also be performed in patients whose disease state can be better visualized by MRI (e.g., GBMR).

[0244] The tumor-to-background ratio (TBR) is defined as the ratio of the lesion SUVmax to the reference region SUV (liver, blood pool, etc.). 89 Comparisons will be made of the number, size, and other characteristics of lesions detected by Zr-girentuximab PET scans with standard imaging modalities, including high-resolution CT / MRI and other possible imaging, by patient (according to tumor type), lesion type, and indication.

[0245] Qualitative visual analysis (localized tumor-related lesions as shown by contrast-enhanced CT, MRI, or FDG PET / CT) 89 Zr-girentuximab uptake) was used to 89 To evaluate the agreement of tumor lesion detection between Zr-girentuximab PET / CT and conventional imaging. RECIST 1.1 criteria for conventional imaging should be used as the primary tool for agreement comparison to PET whenever possible. For radiological evaluation of tumors using different recommendations from the Clinical Guidelines of Scientific Oncology Societies, these guidelines should also be followed. In addition, all visible tumor lesions on conventional imaging can be compared with PET imaging results.

[0246] For each patient, SUVmax, SUVmean, SUL, MTV, TBR, and agreement with conventional imaging will be calculated locally by a nuclear medicine specialist at each site and included in the eCRF. Details are included in the imaging manual.

[0247] Example 2: Preparation of radiolabeled girentuximab Radiolabeled girentuximab was prepared as previously described (see, e.g., WO2021 / 000017). Briefly, radioisotopes useful for imaging (e.g., 89 Prior to labeling with Zr), bioconjugated girentuximab was prepared using standard techniques to yield DOTA-girentuximab or DFO-girentuximab).

[0248] Example 3: In vitro and in vivo binding of radiolabeled girentuximab to various cancers Imaging studies using radiolabeled DOTA-girentuximab were performed to evaluate the ability of the imaging reagent to detect non-RCC cancer types.

[0249] First, the ability of radiolabeled DOTA-girentuximab to bind to various cell lines. These data, presented in Figure 1, demonstrate the ability of the antibody to bind to various cell types expressing CAIX, although the extent of in vitro binding varies.

[0250] Three groups of mice, each bearing a different tumor xenograft, were then tested. The groups were as follows: Group 1: Mice bearing AsPc-1 cell xenografts (pancreatic cancer line), n=4 Group 2: Mice bearing FaDu cell xenografts (pharyngeal squamous cell carcinoma / hypopharyngeal carcinoma cell line), n=4 Group 3: Mice bearing HT-29 xenografts (colorectal cancer cell line), n=4

[0251] Radiolabeled girentuximab was administered intravenously and imaging was performed at 24 and 72 hours. Biodistribution was assessed 72 hours after administration. The radioactivity and dose of antibody administered are summarized in the table below:

[0252] [Table 3]

[0253] Representative images for mice from each of the three groups are shown in FIG.

[0254] Figure 3 shows quantification of the percentage of injected dose in the tumor (24 and 72 hours after injection). The results confirm the observations made using flow cytometry and demonstrate the ability of the radiolabeled antibodies to bind to each respective cancer cell line. In other words, the results show that (with the exception of FaDu cells - see further comments below) the antibodies are able to bind to target tumor cells in an in vivo situation with similar affinity as in vitro.

[0255] The results also show that 72 hours after administration, radiolabeled antibody is still detectable in the circulation and spleen.

[0256] The ex vivo biodistribution of radiolabeled DOTA-GmAb was compared with the in vivo biodistribution. Briefly, the ex vivo biodistribution corresponds to the distribution of radiolabeled DOTA-GmAb in mouse organs as assessed after autopsy. The in vivo biodistribution corresponds to the biodistribution observed in whole-mouse imaging experiments (e.g., as shown in Figure 1).

[0257] FIG. 4 shows that there was a high degree of correlation between in vivo and ex vivo quantification of the signal in the tumor.

[0258] The results show that the positive in vitro binding results (eg, with good binding to HT-29 and AsPc-1 cells) were reproduced with the positive binding observed in vivo.

[0259] Interestingly, we observed that although the radiolabeled antibody bound very poorly to FaDu cells in vitro, the antibody was able to bind to tumor cells in vivo. These results demonstrate that negative findings from in vitro binding may not predict in vivo binding, and thus demonstrate the potential utility of radiolabeled DOTA-G mAb for imaging specific cancer types.

[0260] Example 4: Imaging of alternative cancer types An experiment similar to that carried out in Example 2 was carried out 89 This was performed using Zr-DFO-GmAb to detect the presence of tumor xenografts in mice as follows. Group 1: Mice bearing A-549 cell line xenografts (lung cancer) Group 2: Mice bearing MDA-MB-468 cell line xenografts (triple-negative breast cancer) Group 3: Mice bearing HeLa cell line xenografts (cervical cancer) Group 4: Mice bearing AGS cell line xenografts (gastric cancer) Group 5: Mice bearing HepG2 cell line xenografts (liver cancer) Group 6: Mice bearing A2780 cell line xenografts (ovarian cancer) Group 7: Mice bearing SK-LMS1 cell line xenografts (soft tissue sarcoma - vulvar leiomyosarcoma)

[0261] into xenograft-bearing mice 89 24 and 72 hours after intravenous injection of Zr-DFO-GmAb, mice are subjected to PET / CT scans to determine the ability of the radiolabel to detect cancer cells in vivo.

[0262] The results show that the radiolabeled antibody is capable of binding to tumor xenografts, or in other words, that the antibody is capable of binding to target tumor cells in an in vivo setting with similar affinity as in vitro.

[0263] These results indicate that radiolabeled GmAb is suitable for use in imaging various types of cancer in vivo and is therefore useful as a non-invasive diagnostic reagent for the diagnosis and detection of cancers other than renal cell carcinoma.

[0264] Example 4: Imaging of triple-negative breast cancer Triple-negative breast cancer (TNBC) is an aggressive, metastatic, and drug-resistant cancer with limited treatment options.

[0265] We believe that CAIX, a hypoxia-mediated breast tumor growth regulator, may be important for maintaining breast cancer stem cells within hypoxic regions. Therefore, we performed a randomized controlled trial of CAIX in 12 metastatic TNBC patients. 89 We evaluated the imaging of TNBC using PET / CT imaging with Zr-labeled girentuximab.

[0266] Patients were randomly assigned to receive fludeoxyglucose F18 (FDG) and 89 The patient underwent Zr-girentuximab PET-CT and CT imaging. 89 Patients received a single slow intravenous infusion of Zr-girentuximab (10 mg). On the third day after administration, PET / CT scans were acquired from the skull to mid-thigh with a 10-minute acquisition time per bed position. The gold standard was determined by FDG PET / CT, CT, and follow-up, and lesions detected by at least two modalities were considered true positives. Tumor SUV [max、mean

[0001] , total lesion glycolysis (TLG), and metabolic tumor volume (MTV) were measured. Immunohistochemistry (IHC) was performed using a Bond RX automated research stain with an anti-CAIX antibody (Leica, clone TH22). Staining was evaluated by semiquantitative analysis (percentage and intensity of tumor cell expression) and SUV values ​​compared with the degree of CAIX expression assessed by IHC.

[0267] We examined preliminary results from four patients and included data drawn from a total of 49 lesions (lymph node, bone, lung, breast) detected in these patients ( 89 (41 cases with Zr-girentuximab, 42 with CT, and 49 with FDG PET / CT). Forty-four lesions were identified by the gold standard in lymph nodes, lungs, bones, skin, and breasts in 24, 5, 4, 2, and 9 cases, respectively.

[0268] 89The overall sensitivity of Zr-girentuximab PET / CT was 93.2%, with sensitivities of 100% for bone, lung, breast, and skin, and 87.5% for lymph nodes. The overall sensitivity of both CT and FDG-PET / CT was 82.7%. Tumor SUV max The median of 89 Zr-girentuximab and 89 For Zr-FDG, the IQ was 3.45 [IQ: 2.03-4.69] and 4.68 [IQ: 3.27-10.71], respectively. IHC showed two CAIX-high-expressing lesions [100%, 20%] in two patients, while two patients showed the respective low profiles [3%, 0%]. IHC CAIX cell status and 89 Zr-Girentuximab SUV mean There was a weak correlation with the values ​​(rho=0.80, p=0.20). 89 No safety issues were reported with Zr-girentuximab.

[0269] The results are: 89 We demonstrate that Zr-girentuximab is useful for PET / CT imaging and diagnosis of TNBC in patients, providing superior results to biopsy IHC.

[0270] Example 6: Imaging of bladder cancer Patients with non-muscle-invasive bladder cancer (NMIBC) are typically treated with cystectomy, therefore, there is a need for new treatment options that allow for bladder preservation.

[0271] CAIX is expressed on the luminal surface of papillary structures that are in direct contact with the bladder cavity. 89 A pilot prospective study was conducted to ensure intravesical radioactivity containment and tumor targeting after intravesical instillation of Zr-girentuximab.

[0272] Patients had a 37±10%MBq 89Patients received a single intravesical instillation of Zr-girentuximab (10 mg) and a 2-hour urine retention period. Four PET / CT scans were then performed: three single-step scans of the pelvis (H+2, days 1 and 2) and one scan from skull to mid-thigh (H+4) to observe the evolution of intravesical radioactivity over time.

[0273] Blood samples were taken on day 1 to quantify potential vascular passage of radioactivity. A 10-minute acquisition time per bed position was used for all acquisitions. The gold standard was 89 Zr-girentuximab PET / CT positive sites were determined by second-look cystoscopy and transurethral resection of the bladder (TURB). Immunochemistry (IHC) was performed using an anti-CAIX antibody (Leica, clone TH22). Staining was analyzed semiquantitatively (percentage and intensity of tumor cell expression) and compared with the degree of CAIX expression assessed by IHC. 89 The Zr-girentuximab PET / CT bladder pattern was evaluated.

[0274] Results were available for 4 / 6 patients. Recurrent pTaG3 was identified in each patient despite multiple previous intravesical instillations with Bacillus Calmette-Guerin (BCG—an intravesical immunotherapy commonly used to treat bladder cancer).

[0275] 89 Zr-girentuximab PET / CT showed no extravesical leakage. In 2 / 4 patients with positive IHC, uptake spots in the bladder wall were confirmed by TURB in one patient with a corresponding recurrent lesion, and an inflammatory scarring reaction was confirmed in the second patient. In the other two patients, uptake was not observed, consistent with negative IHC. No harmful radiation contamination was observed during the process, and no specific worker exposure was observed.

[0276] The results (shown in Figures 5 and 6) show that 89 Intravesical instillation of Zr-girentuximab demonstrated radioactivity containment in the bladder in patients with positive IHC. 89We demonstrate that Zr-girentuximab is useful for detecting and imaging tumors in this patient population.

[0277] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

Claims

1. 1. A method for in vivo imaging or detection of cancer in a subject in need thereof, said method comprising: - administering to the subject an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety to allow in vivo detection of the agent in the subject; - detecting said agent in said subject; The cancer is 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 cancer) Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) Lung cancer (including non-small cell and small cell lung cancer) Ovarian cancer (including ovarian epithelial cancer) Pancreatic cancer (including pancreatic ductal adenocarcinoma) and Selected from soft tissue sarcomas, wherein detection of said agent above background or standard levels indicates the presence of said cancer, thereby imaging or detecting said cancer in said subject.

2. 1. A method for diagnosing cancer in a subject in need thereof, said method comprising: - administering to the subject an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety to allow in vivo detection of the agent in the subject; - determining the presence or absence of the agent in the subject; The cancer is 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 cancer) Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) Lung cancer (including non-small cell and small cell lung cancer) Ovarian cancer (including ovarian epithelial cancer) Pancreatic cancer (including pancreatic ductal adenocarcinoma) and Selected from soft tissue sarcomas, wherein detection of said agent above background or standard levels indicates that said subject has said cancer, thereby diagnosing said cancer in said subject.

3. 1. A method for producing an image of cancer, said method comprising: administering to a subject suspected of having said cancer an effective amount of an agent for binding to CAIX expressed by said cancer, said agent comprising a detectable moiety to allow in vivo detection of said agent in said subject; - detecting said agent in said subject; The cancer is 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 cancer) Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) Lung cancer (including non-small cell and small cell lung cancer) Ovarian cancer (including ovarian epithelial cancer) Pancreatic cancer (including pancreatic ductal adenocarcinoma) and Selected from soft tissue sarcomas, thereby generating an image of said cancer.

4. The method of any one of claims 1 to 3, wherein the method further comprises concentrating the agent at sites and / or tissues in the subject where CAIX antigen is found in the subject before detecting the agent in the subject or determining the presence or absence of the agent.

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 said method is the only method required to enable detection, diagnosis or imaging of said cancer.

7. The method of any one of claims 1 to 6, wherein the agent for binding to CAIX is a small molecule, a peptide, or a polypeptide (such as an antibody or an antigen-binding fragment thereof).

8. The agent for binding to CAIX is a small molecule, optionally selected from the group consisting of SLC-0111, SLC-149, SLC-0121, SLC-101, PMI-05, sulfamido-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, MIP-1486, MIP-1490, MIP-1504 (especially ９９ｍ Tc-HEHEHE-Z09781, ９９ｍ Tc-MIP-1486, ９９ｍ Tc-MIP-1490, or ９９ｍ 8. The method according to claim 1, wherein the anti-inflammatory agent is selected from the group consisting of Tc-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-P1.

10. The method according to any one of claims 1 to 7, wherein the agent for binding to CAIX is a polypeptide.

11. The method according to any one of claims 1 to 7, wherein the agent for binding to CAIX is an antibody or an antigen-binding fragment thereof.

12. 12. The method of claim 11, wherein the antibody or antigen-binding fragment thereof is girentuximab, including chimeric or humanized variants thereof.

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

14. 10. 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 label or a dye.

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

17. The radioisotopes are gallium-67 and gallium-68 ( ６７ Ga and ６８ Ga), Indium-111 ( １１１ In), iodine-123, iodine-124, or iodine-131 ( １２３ I, １２４ I, or １３１ I), lutetium-177 ( １７７ Lu), technetium-99 ( ９９ｍ Tc), yttrium-90 ( ９０ Y), and zirconium-89 ( ８９ 17. The method of claim 16, wherein the metal is selected from the group consisting of Zr.

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

19. 20. The method of claim 18, wherein said determining or detecting the presence or absence of radiation comprises positron emission tomography (PET) imaging.

20. The drug is ８９ Zr-girentuximab, １２３ I-, １２４ I-, or １３１ 15. The method according to any one of claims 1 to 14, wherein the IL-1 receptor agonist is selected from I-girentuximab.

21. The drug is ８９ The method according to any one of claims 1 to 14, wherein the compound is Zr-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 according to any one of claims 1 to 6, wherein the cancer is esophageal cancer.

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

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

30. The method according to 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 cancer.

32. 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 a soft tissue sarcoma.

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

35. 23. The method of claim 22, wherein the agent is a radiolabeled girentuximab antibody.

36. 24. The method of claim 23, wherein the agent is a radiolabeled girentuximab antibody.

37. 25. The method of claim 24, wherein the agent is a radiolabeled girentuximab antibody.

38. 26. The method of claim 25, wherein the agent is a radiolabeled girentuximab antibody.

39. 27. The method of claim 26, wherein the agent is a radiolabeled girentuximab antibody.

40. 28. The method of claim 27, wherein the agent is a radiolabeled girentuximab antibody.

41. 29. The method of claim 28, wherein the agent is a radiolabeled girentuximab antibody.

42. 30. The method of claim 29, wherein the agent is a radiolabeled girentuximab antibody.

43. 31. The method of claim 30, wherein the agent is a radiolabeled girentuximab antibody.

44. 32. The method of claim 31, wherein the agent is a radiolabeled girentuximab antibody.

45. 33. The method of claim 32, wherein the agent is a radiolabeled girentuximab antibody.

46. 34. The method of claim 33, wherein the agent is a radiolabeled girentuximab antibody.

47. 35. The method of claim 34, wherein the agent is a radiolabeled girentuximab antibody.

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

49. The radiolabeled girentuximab is radiolabeled with gallium-67 and gallium-68 ( ６７ Ga and ６８ Ga), Indium-111 ( １１１ In), iodine-123, iodine-124, or iodine-131 ( １２３ I, １２４ I, or １３１ I), lutetium-177 ( １７７ Lu), technetium-99 ( ９９ｍ Tc), yttrium-90 ( ９０ Y), and zirconium-89 ( ８９ 49. The method of any one of claims 35 to 48, comprising girentuximab conjugated to a radioisotope selected from:

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