Imaging reagents and methods

Abstract

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

Description

TECHNICAL FIELD

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

[0002] Related Applications

[0003] This application claims priority from Australian provisional application AU 2022903922, the contents of which are hereby incorporated in their entirety by reference. BACKGROUND

[0004] Medical imaging methods are commonly used to assist in the diagnosis and staging of the progression of various cancers. Such methods are also advantageous in removing or reducing the need for invasive techniques, such as obtaining a biopsy sample, for confirming a diagnosis, which are not always necessary and can cause complications.

[0005] However, not all cancers can be successfully detected using standard medical imaging. Furthermore, while many imaging techniques are able to detect a lump, they are not able to successfully distinguish between benign and malignant tissue.

[0006] The state of oncological management has evolved in a dichotomous manner over the last few decades. While some types of cancer have significantly benefited from research advances in diagnostic and treatment options, resulting in improved morbidity and mortality rates for their patient populations, other types of cancer have proven more difficult to uncover and continue to portend poor prognoses for their patient populations.

[0007] In the case where this latter class of cancer has exhausted the limitations of existing diagnostic and treatment modalities, innovation based on new approaches to oncological management is required.

[0008] There is a need for improved methods and compositions for in vivo detection and / or imaging of various cancers.

[0009] The reference in this specification to any prior publication (or information derived from it), is not, and should not be taken as an acknowledgment or admission that the prior publication (or information derived from it) forms part of the common general knowledge in the field of endeavour in any jurisdiction, or that the prior publication (or information derived from it) was published in such a way as to make it available to the public. SUMMARY

[0010] The present invention is based, at least in part, on the identification by the present inventors of a subset of solid tumours that express the antigen carbonic anhydrase IX (CAIX), and the discovery that these tumours can be imaged in vivo using an imaging agent that binds to CAIX.

[0011] The present invention therefore provides a method for in vivo imaging or detection of a cancer in a subject in need thereof,

[0012] - 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,

[0013] - detecting the agent in the subject,

[0014] wherein the cancer is selected from:

[0015] • bladder cancer

[0016] • breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER / PR-positive), and HER2-positive breast cancer)

[0017] • cervical cancer

[0018] • colorectal cancer

[0019] • esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / gastroesophageal junction adenocarcinoma)

[0020] • gastric cancer (including gastric adenocarcinoma)

[0021] • glioblastoma multiforme

[0022] • head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer, and nasopharyngeal cancer)

[0023] • liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

[0024] • lung cancer (including non-small cell carcinoma and small cell carcinoma)

[0025] • ovarian cancer (including epithelial ovarian cancer)

[0026] • pancreatic cancer (including pancreatic ductal adenocarcinoma), and

[0027] • soft tissue sarcoma

[0028] wherein detection of the agent above background or standard levels indicates the presence of the cancer, thereby imaging or detecting the cancer in the subject.

[0029] The present application also provides a method for diagnosing a cancer in a subject in need thereof, wherein the method comprises:

[0030] - 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,

[0031] - determining the presence or absence of the agent in the subject,

[0032] wherein the cancer is selected from the group consisting of:

[0033] • bladder cancer

[0034] • breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER / PR-positive), and HER2-positive breast cancer)

[0035] • cervical cancer

[0036] • colorectal cancer

[0037] • esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / gastroesophageal junction adenocarcinoma)

[0038] • gastric cancer (including gastric adenocarcinoma)

[0039] • glioblastoma multiforme

[0040] • head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer, and nasopharyngeal cancer)

[0041] • liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

[0042] • lung cancer (including non-small cell carcinoma and small cell carcinoma)

[0043] • ovarian cancer (including epithelial ovarian cancer)

[0044] • pancreatic cancer (including pancreatic ductal adenocarcinoma), and

[0045] • soft tissue sarcoma

[0046] wherein the determination that the agent is present above a background level or a standard level indicates that the subject has the cancer, thereby diagnosing the cancer in the subject.

[0047] The present application also provides a method for producing an image of a cancer, the method comprising:

[0048] - administering to a subject suspected of having the cancer an effective amount of an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,

[0049] - detecting the agent in the subject,

[0050] wherein the cancer is selected from the group consisting of:

[0051] • bladder cancer

[0052] • breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER / PR-positive), and HER2-positive breast cancer)

[0053] • cervical cancer

[0054] • colorectal cancer

[0055] • esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma)

[0056] • gastric cancer (including gastric adenocarcinoma)

[0057] • glioblastoma multiforme

[0058] • head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer and nasopharyngeal cancer)

[0059] • liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

[0060] • lung cancer (including non-small cell carcinoma and small cell carcinoma)

[0061] • ovarian cancer (including epithelial ovarian cancer)

[0062] • pancreatic cancer (including pancreatic ductal adenocarcinoma) and

[0063] • soft tissue sarcoma

[0064] thereby generating an image of said cancer.

[0065] The present application also provides a method for generating an image of a cancer, said method comprising:

[0066] - infusing an effective amount of an agent for binding to CAIX expressed by said cancer, wherein said agent comprises a detectable moiety for enabling in vivo detection of said agent,

[0067] - detecting said agent,

[0068] wherein said cancer is selected from:

[0069] • bladder cancer

[0070] • breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER / PR-positive) and HER2-positive breast cancer)

[0071] • cervical cancer

[0072] • colorectal cancer

[0073] • esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma)

[0074] • gastric cancer (including gastric adenocarcinoma)

[0075] • glioblastoma multiforme

[0076] • head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal cancer, and nasopharyngeal cancer)

[0077] • liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

[0078] • lung cancer (including non-small cell carcinoma and small cell carcinoma)

[0079] • ovarian cancer (including epithelial ovarian cancer)

[0080] • pancreatic cancer (including pancreatic ductal adenocarcinoma), and

[0081] • soft tissue sarcoma

[0082] An image of the cancer is thereby generated. Optionally, the detectable moiety is a radioisotope, and the detecting comprises detecting radiation, such as positron emission tomography (PET) imaging.

[0083] In any embodiment, the method of the application can further comprise, prior to detecting the agent, allowing the agent to concentrate at sites and / or tissues in the subject where CAIX antigen is found in the subject.

[0084] The administration can be by any suitable means, preferably means that allow for systemic administration (e.g., intravenous infusion) of the agent, such that the agent can accumulate at sites in the subject where CAIX is present on the cell surface. The mode of administration can be dictated by the nature of the agent used to bind to CAIX. For example, in the case of an antibody used to bind to CAIX, preferably the agent is administered by intravenous infusion. A labeled peptide or small molecule can be administered orally or by other means.

[0085] In any embodiment, the diagnostic method of the application does not require additional in vitro diagnosis using a tissue biopsy or other biological sample obtained from the subject.

[0086] In any embodiment, the agent comprises a first moiety for binding to CAIX and a second moiety for enabling in vivo detection of the agent.

[0087] In any embodiment, the moiety for binding to CAIX can be a small molecule, a peptide, or a polypeptide (such as an antibody or antigen-binding fragment thereof).

[0088] 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, sulfonamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, -MIP-1486, MIP-1490, MIP-1504 (especially99m Tc-HEHEHE-Z09781、 99m Tc-MIP-1486、 99m Tc-MIP-1490 or 99m Tc-MIP-1504 / 5) and PHC-102.

[0089] 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.

[0090] In any embodiment, the agent for binding to CAIX is a polypeptide, including an antibody or antigen binding fragment thereof.

[0091] In a particularly preferred embodiment, the agent comprises a first moiety for binding to CAIX, wherein the first moiety is in the form of an antibody or antigen binding fragment thereof.

[0092] In any embodiment, the agent is an antigen binding protein (antibody) that retains the ability to bind to CAIX, such as girentuximab or a functional variant or fragment thereof. 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).

[0093] In alternative embodiments, the antibody for binding to CAIX can comprise BCA-356, BAY-794620 or SLC-0131.

[0094] The agent used according to the methods of the application comprises a moiety for enabling its detection. Any suitable detectable moiety for in vivo detection techniques can be used and will be known to the skilled person.

[0095] The detectable moiety can be directly linked to the moiety for binding to CAIX or can be conjugated through a chelator or other linking moiety. In certain embodiments, the above-mentioned agent for binding to CAIX is detectable without the need for linking an additional detectable moiety thereto.

[0096] 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-111111 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).

[0097] In any embodiment, the agent is an antibody for binding to CAIX, and the detectable portion is a radioisotope, optionally selected from 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).

[0098] In any embodiment, the agent is a gemtuximab antibody (including its chimeric or humanized version), and the detectable portion is a radioisotope, optionally selected from 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).

[0099] In any embodiment, the agent is 89 Zr-geutzximab, 123 I-Gibutuximab 124 I-Gibutuximab 131 I-Gitoximab or 177 Lu-geotuximab.

[0100] In preferred embodiments, the detectable moiety is a radioisotope, and the detecting the agent comprises determining radiation emitted by the radioisotope or detecting the presence or absence of the radiation. In any embodiment, determining radiation or detecting the presence or absence of radiation comprises positron emission tomography (PET) imaging.

[0101] Other suitable detectable moieties include fluorescent labels and dyes. It will be appreciated that in any embodiment of the application, more than one detectable moiety can be utilised in order to maximise imaging or detection of the agent and thereby the CAIX-expressing tumour or cancer.

[0102] The application also provides an agent for CAIX or a composition comprising the agent as described herein for use in a method of detecting, imaging, obtaining an image of, or diagnosing a cancer as described herein.

[0103] Still further, the application provides an agent for CAIX or a composition comprising the agent as described herein for use in a method of detecting, imaging or diagnosing a cancer, or for obtaining an image of a cancer, as described herein.

[0104] Still further, the application provides a kit for use according to any method described herein, wherein the kit comprises: and an agent for binding to CAIX as described herein, and optionally instructions for use thereof in detecting, imaging or diagnosing a cancer.

[0105] As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude additional, unrecited elements or steps.

[0106] Further aspects of the application 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. BRIEF DESCRIPTION OF DRAWINGS

[0107] Figure 1 : In vitro binding of radiolabelled DOTA-GmAb to various cell lines.

[0108] Figure 2 : Representative images of mice bearing As-PC-1 (pancreatic cancer), FaDu (hypopharyngeal cancer) or HT-29 (colorectal cancer) tumour xenografts and subsequent injection of radiolabelled DOTA-GmAb.

[0109] Figure 3 Quantification of the percentage of the injected dose at 24 and 72 hours after injection of radiolabeled DOTA-GmAb. Solid circle = AsPC-1 radiolabeled DOTA-GmAb; solid frame = FaDu radiolabeled DOTA-GmAb; solid triangle = HT-29 radiolabeled DOTA-GmAb.

[0110] Figure 4 Quantification of biodistribution results. This demonstrates the correlation between in vitro biodistribution (i.e., the distribution of radiolabeled DOTA-GmAbs observed in mouse organs after necropsy) and in vivo biodistribution (i.e., the distribution of radiolabeled DOTA-GmAbs observed in mice after whole-body imaging). Solid circles = AsPC-1 radiolabeled DOTA-GmAbs; solid frames = FaDu radiolabeled DOTA-GmAbs; solid triangles = HT-29 radiolabeled DOTA-GmAbs.

[0111] Figure 5 Imaging of bladder cancer. Day 0: Whole body. 89 Zr-geutzximab scan: A: Coronal PET / CT fusion. B: Maximum intensity projection (MIP) visualization.

[0112] Figure 6 Imaging of bladder cancer. Day 2, A. 89 Zr-geutuzumab pelvic PET / CT fusion image: (arrow) Radiopharmaceutical uptake on different sides of the bladder wall. B. 3D representation of the overlaid image of the bladder. Detailed Implementation

[0113] This invention relates to imaging and diagnosis of cancer, which is challenging due to the lack of robust oncology management options. Diagnostic and therapeutic innovations are needed to improve morbidity and mortality in these oncology indications.

[0114] Detection of CAIX in the context of imaging and diagnosis of renal cell carcinoma is known. However, prior to this invention, it was unknown whether in vivo detection of CAIX could be used for successful imaging and diagnosis of other solid cancers that may express CAIX.

[0115] Although CAIX is typically associated with advanced disease, it is unknown whether CAIX detection and / or imaging is performed during the early stages of certain cancers, or whether cancer can be detected only in advanced stages. Furthermore, some cancers exhibit reduced expression as the disease progresses, and therefore it is unclear whether CAIX imaging is a useful means of detecting these types of cancer.

[0116] Furthermore, given the heterogeneity of many cancers, the mere presence of CAIX expression (e.g. as determined by immunohistochemistry techniques) does not necessarily mean that the cancer can be detected using whole body or partial body imaging methods.

[0117] For example, in a 2006 study by Henrickx et al. (Cancer Biotherapy & Radiopharmaceuticals, 21 :263-268), it was found that radiolabelled antibodies for binding to CAIX were not suitable for imaging of cholangiocarcinoma, despite the fact that cholangiocarcinoma was found to overexpress CAIX. In contrast, the same antibodies are known to be routinely used for imaging and detection of renal cell carcinoma which overexpresses CAIX. Both cholangiocarcinoma and renal cell carcinoma are malignancies of epithelial cells and are characterised by increased expression of CAIX. It is therefore not understood why antibodies that bind to CAIX can be used for imaging and detection of renal cell carcinoma but not cholangiocarcinoma.

[0118] In another example, a recent study published by Huizing et al. (2021, Physics and Imaging in Radiation Oncology, 145-150) found that, 111 The In-labelled CAIX binding antibody, Gemtuzumab, in the form of a F(ab’)2, was not able to distinguish between tumour cells and non-tumour cells and therefore indicated that it could not be used to quantify changes in CAIX expression. In particular, this finding is in stark contrast to the findings reported herein by the present inventors, where it was shown that 89 Zr-GmAb (in the form of a whole IgG antibody) can be used for in vivo imaging and detection of the same cancer cell type.

[0119] The present application is therefore based on the finding that subpopulations of cancers associated with increased CAIX expression can indeed be successfully detected and imaged by using an agent for binding to CAIX and wherein the agent comprises a detectable moiety.

[0120] General definitions

[0121] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, steps or compositions of matter. Thus, as used herein, the singular forms "a", "an" and "the" include plural aspects, unless the context clearly indicates otherwise. For example, reference to "a" encompasses singular and two or more; reference to "an" encompasses singular and two or more; reference to "the" encompasses singular and two or more and so forth.

[0122] Those skilled in the art will appreciate that the application is susceptible to variations and modifications other than those specifically described. It is to be understood that the application includes all such variations and modifications. The application also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0123] Those 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 application. The present application is in no way limited to the methods and materials described.

[0124] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0125] The present application is not limited to the specific examples described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions and methods obviously are within the scope of the present application.

[0126] Unless otherwise expressly stated, any examples or embodiments of the present application herein are to be considered equivalent in terms of the present application when necessary modifications are made.

[0127] Unless specifically stated otherwise, all technical and scientific terms used herein are to be taken in their ordinary, plain meaning as 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).

[0128] The term "and / or", e.g., "X and / or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0129] Carbonic anhydrase IX

[0130] 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, pMWl, RCC-associated antigen G250, RCC-associated protein G250, and renal cell carcinoma-associated antigen G250.

[0131] Cancer cells predominantly express the plasma-membrane associated CA isoforms CAIX and CAXII, as well as intracellular CAs, such as CAI and CAII. Of the cancer-associated CAs, CAIX has received the most attention, as this isoform is only expressed in healthy tissues in the epithelial cells of the stomach and intestine, but is strongly upregulated in renal carcinomas.

[0132] Expression of CAIX is controlled by hypoxia-inducible factor 1 (HIF-1), which is mainly located in chronically hypoxic tumor regions. However, CAIX can also be found in regions of mild hypoxia or even normoxia, as expression of CAIX can be activated by components of the mitogen-activated protein kinase (MAPK) pathway.

[0133] The agent for binding to CAIX and for use in the methods according to the present application can be any compound that specifically recognizes or binds to CAIX, mediates its activity by binding to CAIX or fragments or splice variants thereof; binds irreversibly at the entrance of the active site, and / or inhibits CAIX by coordinating with the zinc ion at the active site of CAIX.

[0134] Preferably, the agent for binding to CAIX specifically interacts with the CAIX polypeptide. Specific interaction (e.g., recognition or binding) means that the agent, e.g., an antibody, has a greater affinity for CAIX than for other polypeptides. In one embodiment, the agent interacts with (i.e., binds to or recognizes) or modulates the activity of the 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. The CAIX inhibitor can act at the protein level or at the nucleic acid level. Examples of CAIX inhibitors acting at the protein level include, but are not limited to, peptides and anti-CAIX antibodies and functional fragments of those antibodies or small organic molecules, preferably having a molecular weight of less than 500 g / mol.

[0135] Examples of anti-CAIX antibodies or antibodies for binding to CAIX are described in EP 637 336, WO93 / 18152, WO 95 / 34650, WO 00 / 24913, WO 02 / 063010, WO 04 / 025302, WO 05 / 037083, WO201 1 / 139375, Murri-Plesko et al., Eur J Pharmacol 201 1, 657: 173-183.

[0136] 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 acting at the nucleic acid level are siRNA molecules, ribozymes and / or antisense molecules.

[0137] As used herein, the term "specifically binds" or "binds specifically" shall be taken to mean that the agent for use according to the application reacts or associates with CAIX or cells expressing it more frequently, more rapidly, for a longer duration and / or with greater affinity than with alternative antigens or cells. For example, the affinity of an antigen-binding protein that binds to CAIX is substantially greater (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 its affinity for other antigens.

[0138] Methods for assessing binding to a protein (e.g., CAIX) are known in the art, for example, as described in Scopes (Protein purification: principles and practice, 3rdedition, Springer Verlag, 1994). Such methods typically involve immobilizing the agent (e.g., an antibody) and contacting it with a labeled target (in the case of an antibody, an antigen). After washing to remove nonspecifically bound proteins, the amount of label is detected, and thus the bound antigen. Of course, the antigen binding site can be labeled and the antigen immobilized. Elutriation-type assays can also be used. Alternatively or additionally, surface plasmon resonance assays can be used.

[0139] Other standard methods for assessing binding to a target such as CAIX are also known in the art.

[0140] Small molecules

[0141] In any embodiment, the moiety for binding to CAIX is the small molecule SLC-0111 (CAS 178606-66-1), SLC-149 (as described in EP 3317255 B1, which is incorporated herein by reference), SLC-0121, or SLC-101.

[0142] In any embodiment, the moiety for binding to CAIX is the small molecule / contrast dye PMI-05 (as described in US 2019 / 0192699 Al, which is incorporated herein by reference).

[0143] In any embodiment, the moiety for binding to CAIX is the small molecule sulfonamide-nitroimidazole (as described in Rami et al., (2013), J. Med. Chem, 56: 8512-8520, which is incorporated herein by reference).

[0144] In any embodiment, the moiety for binding to CAIX is the small molecule JS-403 (as described in WO 2010 / 089752 Al, which is incorporated herein by reference).

[0145] In any embodiment, the moiety for binding to CAIX is the small molecule UB-TT220 (as described in WO 2022 / 015955 Al, which is incorporated herein by reference).

[0146] In any embodiment, the moiety for binding to CAIX is the 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, which are incorporated herein by reference),99m Tc-MIP-1486, 99m Tc-MIP-1490 (4-(2-bis((1 -(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2- oxoethyl)-1 H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide, where X = ethyl) or 99m Tc-MIP-1504 (4-(2-bis((1 -(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2- oxoethyl)-1 H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide, where X = n-butoxy) (Hillier et al. (2012) Journal of Nuclear Medicine, 53 (s1): 217, which is incorporated herein by reference).

[0147] In any embodiment, the moiety for binding to CAIX is the small molecule PHC-102 (as described in WO 2015 / 114171 Al; WO 2018154517 Al; US2014 / 0357650 Al; WO 2015 / 114171 Al, which are incorporated herein by reference).

[0148] Peptide

[0149] In any embodiment, the moiety for binding to CAIX is a peptide. As used herein, a peptide will be understood to comprise a chain of more than 1 amino acid residues. Typically, a peptide can comprise from about 2 to 30 amino acids, for example from about 5 to 30, from about 10 to 30, from about 2 to 25, from about 5 to 25, from about 10 to 25, or from about 10 to 20 amino acids. A peptide can be at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 amino acids in length. Typically, a peptide is no longer than about 40 amino acids, for example no longer than about 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids.

[0150] 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, which are incorporated herein by reference) or 3B-302.

[0151] In any embodiment, the moiety for binding to CAIX is peptide CAIX-P1, having the amino acid sequence YNTNHVPLSPKY (as described in Askoxylakis et al. (2010) PLoS One 5(12):e15962), optionally wherein the peptide is labelled with 125 I or 131 I for enabling detection thereof (although it will be appreciated that any suitable radiolabel or other detectable moiety can be used).

[0152] Polypeptides and antibodies

[0153] According to a particularly preferred embodiment, the agent comprises a first moiety in the form of an anti-CAIX antibody and / or a functional fragment of such an antibody. Fragments of anti-CAIX antibodies can have substantially the same CAIX binding and / or inhibitory activity as full-length anti-CAIX antibodies, and / or be epitope-binding fragments of anti-CAIX antibodies.

[0154] An antibody or antigen-binding fragment thereof referred to herein as "binding to carbonic anhydrase IX (CAIX)" provides textual support for an antibody or fragment thereof that "binds specifically to" or "specifically binds to" CAIX.

[0155] The antibody and / or antibody fragment thereof can 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 antibody can be multivalent, or multivalent and multispecific.

[0156] In particularly preferred embodiments, the antibody is a whole antibody comprising at least one antigen binding domain (Fab) of an antibody and at least one Fc region of an antibody. The antibody can comprise a human constant region of IgGl, IgG2a, IgG3 or IgG4.

[0157] According to further preferred embodiments, the anti-CAIX antibody or epitope-binding fragment thereof for use according to the present application binds to the amino acid sequences LSTAFARV and / or ALGPGREYRAL.

[0158] In any embodiment, the moiety for binding to CAIX is in the form of antibody BAY-794620 or an antigen-binding fragment thereof (as described in WO 2003 / 100029 A2; WO 2003 / 033674 A2; Theiner et al. (2021) Tierarztl Prax Ausg G Grosstiere Nutztiere 49(6):392-402; Kimani et al. (2011) Photochemistry and Photobiology 88(1):175-87; NCT01065623 (v24, September 30, 2014); NCT01028755 (v30, January 19, 2015), which are incorporated herein by reference).

[0159] In any embodiment, the moiety for binding to CAIX is in the form of antibody SLC-0131 or an antigen-binding fragment thereof.

[0160] According to a further particularly preferred embodiment, the agent for binding to CAIX is an anti-G250 antibody and / or an antigen binding fragment thereof. Anti-G250 antibodies are described, for example, in EP-B-0 637 336. The antibody or fragment thereof can be a chimeric or humanized G250 antibody. In some embodiments, the antigen binding protein that binds to or specifically binds to CAIX is as described in any of WO 2002 / 062972 A2 (US 2004 / 0219633 Al), WO 2004 / 002526 Al (US 7,632,496 B2), WO 2006 / 002889 A2 (US 7,691,375 B2), WO 2009 / 056342 Al (US 2014 / 0017252 Al), WO 2011 / 032973 Al (US 2012 / 0207672 Al), and WO 2014 / 128258 Al (US 10,620,208 B2), or WO 2021 / 000017 Al, the entire contents of each of these publications are incorporated herein by reference.

[0161] Antibodies for use in the present application can 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.

[0162] A particularly preferred antibody is cG250, preferably gemtuzumab (INN). Another particularly preferred embodiment is the monoclonal antibody G250 produced by the hybridoma cell line DSM ACC 2526. The antibody cG250 is a chimeric version of the IgGl kappa light chain of the original murine monoclonal antibody mG250.

[0163] Variants of the original chimeric G250 (cG250) antibody, including WX-G250 and WX-G250 RIT (131 Iodine) (Janssen Global Services LLC) are known.

[0164] In a particularly preferred embodiment, the antibody is 89 Zr-gemtuzumab (i.e., 89 Zr-cG250), 123 I-gemtuzumab, 124 I-gemtuzumab or 131 I-gemtuzumab or 177 Lu-gemtuzumab.

[0165] In any embodiment, the antibody or antigen binding fragment thereof comprises:

[0166] (a) a heavy chain variable domain (VH) comprising three complementarity determining regions (CDRs) of the amino acid sequence as set forth in SEQ ID NO: 4, 20, 36, 52, or 68; and / or

[0167] (b) a light chain variable domain (VL) comprising three complementarity determining regions (CDRs) of the amino acid sequence as set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0168] In any embodiment, the antibody or antigen-binding fragment thereof comprises an antigen binding domain that specifically binds to carbonic anhydrase IX (CAIX) and comprises:

[0169] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - linker - FRla - CDRla - FR2a - CDR2a - FR3a - CDR3a - FR4a

[0170] wherein:

[0171] each of FR1, FR2, FR3, and FR4 is a framework region;

[0172] each of CDR1, CDR2, and CDR3 is a complementarity determining region;

[0173] each of FRla, FR2a, FR3a, and FR4a is a framework region;

[0174] each of CDRla, CDR2a, and CDR3a is a complementarity determining region;

[0175] wherein the sequence of any of the complementarity determining regions has an amino acid sequence as described in Table 1 below. Preferably, the framework regions have amino acid sequences as also described in Table 1 below, including amino acid variations at specific residues, which can be determined by aligning the various framework regions derived from each antibody. The CDR1, CDR2, and CDR3 can be sequences from the VH, CDRla, CDR2a, and CDR3a can be sequences from the VL, or the CDR1, CDR2, and CDR3 can be sequences from the VL, CDRla, CDR2a, and CDR3a can be sequences from the VH.

[0176] In any embodiment, the antibody or antigen-binding fragment thereof comprises:

[0177] (i) a VH comprising 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, 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: 2, 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: 3;

[0178] (ii) a VH 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 any one of SEQ ID NOs: 4, 20, 36, 52, or 68;

[0179] (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 81, a CDR2 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 83;

[0180] (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;

[0181] (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3;

[0182] (vi) a VH comprising the sequence set forth in any one of SEQ ID NOs: 4, 20, 36, 52, or 68;

[0183] (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;

[0184] (viii) a VL comprising the sequence set forth in any one of SEQ ID NOs:84, 100, 116, 132, 148, or 164;

[0185] (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO:2, and a CDR3 comprising the sequence set forth in SEQ ID NO:3; and 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; or

[0186] (x) a VH comprising the sequence set forth in any one of SEQ ID NOs:4, 20, 36, 52, or 68, and a VL comprising the sequence set forth in any one of SEQ ID NOs:84, 100, 116, 132, 148, or 164.

[0187] In further embodiments, the antibody or antigen-binding fragment thereof comprises:

[0188] (i) a VH comprising 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%, at least 99% identical to a sequence as set forth in any one of SEQ ID Nos: 9, 25, 41, 57, or 73; an FR2 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%, at least 99% identical to a sequence as set forth in any one of SEQ ID Nos: 10, 26, 42, 58, or 74; an FR3 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%, at least 99% identical to a sequence as set forth in any one of SEQ ID Nos: 11, 27, 43, 59, or 75; an FR4 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%, at least 99% identical to a sequence as set forth in any one of SEQ ID Nos: 12, 28, 44, 60, or 76; and

[0189] (ii) a VL comprising 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%, at least 99% identical to a sequence as set forth in any one of SEQ ID Nos: 89, 105, 121, 137, 153, or 169; an FR2 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%, at least 99% identical to a sequence as set forth in any one of SEQ ID Nos: 90, 106, 122, 138, 154, or 170; an FR3 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%, at least 99% identical to a sequence as set forth in any one of SEQ ID Nos: 91, 107, 123, 139, 155, or 171; and an FR4 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%, at least 99% identical to a sequence as set forth in any one of SEQ ID Nos: 92, 108, 124, 140, 156, or 172.

[0190] In further embodiments, the antibody or antigen-binding fragment thereof comprises:

[0191] (i) a VH comprising a framework region (FR) 1 comprising or consisting of the sequence as set forth in SEQ ID No: 9, 25, 41, 57, or 73; a FR2 comprising or consisting of the sequence as set forth in SEQ ID No: 10, 26, 42, 58, or 74; a FR3 comprising or consisting of the sequence as set forth in SEQ ID No: 11, 27, 43, 59, or 75; a FR4 comprising or consisting of the sequence as set forth in SEQ ID No: 12, 28, 44, 60, or 76, and

[0192] (ii) a VL comprising a framework region (FR) 1 comprising or consisting of the sequence as set forth in SEQ ID No: 89, 105, 121, 137, 153, or 169; a FR2 comprising or consisting of the sequence as set forth in any one of SEQ ID No: 90, 106, 122, 138, 154, or 170; a FR3 comprising or consisting of the sequence as set forth in SEQ ID No: 91, 107, 123, 139, 155, or 171; a FR4 comprising or consisting of the sequence as set forth in SEQ ID No: 92, 108, 124, 140, 156, or 172.

[0193] In any embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CAIX comprises an amino acid sequence consisting essentially of, or consisting of, in the order from N- to C-terminus or C- to N-terminus, any one of SEQ ID NO: 4, 20, 36, 52, or 68 and / or any one of SEQ ID NO: 84, 100, 116, 132, 148, 164.

[0194] In any embodiment, the antibody or antigen-binding fragment thereof comprises:

[0195] (a) a heavy chain variable domain (VH) 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%, at least 99% identical to the sequence as set forth in SEQ ID No: 4, 20, 36, 52, or 68; and / or

[0196] (b) a light chain variable domain (VL) 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%, at least 99% identical to a sequence as set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0197] In any embodiment, the antibody or antigen-binding fragment thereof for binding to CAIX can be in the form of:

[0198] (i) a single chain Fv fragment (scFv);

[0199] (ii) a dimeric scFv (di-scFv); or

[0200] (iii) one of (i) or (ii) linked to a constant region of an antibody, Fc, or heavy chain constant domain (CH)2and / or CH3.

[0201] In any embodiment, the antibody or antigen-binding fragment thereof for binding to CAIX can be in the form of:

[0202] (i) a diabody;

[0203] (ii) a triabody;

[0204] (iii) a tetrabody;

[0205] (iv) a Fab;

[0206] (v) a F(ab')2;

[0207] (vi) a Fv; or

[0208] (vii) one of (i) to (vi) linked to a constant region of an antibody, Fc, or heavy chain constant domain (CH)2and / or CH3.

[0209] In any embodiment, the antibody or antigen-binding fragment thereof for use according to the application can be a fusion protein comprising an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab (single domain antibody), a di-scFv, a scFv, a Fab, a Fab', a F(ab')2, a Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody as described herein.

[0210] An antigen binding fragment, immunoglobulin variable domain, antibody, dab, bi-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule or multispecific antibody, fusion protein or conjugate as described herein can be obtained by expression of a nucleic acid encoding the same.

[0211] An antibody or antigen binding fragment thereof as 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 mixtures thereof. In case the antibody or protein comprises 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.

[0212] In one example, an antibody or antigen binding fragment thereof as described herein comprises a heavy chain constant region comprising a stabilised heavy chain constant region comprising a mixture of sequences with or without a C-terminal lysine residue, in whole or in part.

[0213] In one example, an antibody or antigen binding fragment thereof as described herein comprises a VH as disclosed herein linked or fused to an IgG4 constant region or a stabilised IgG4 constant region (e.g. as discussed above) and a VL linked or fused to a kappa light chain constant region.

[0214] Functional properties of antigen binding fragments thereof as described herein are to be considered as applicable to antibodies as described herein mutatis mutandis.

[0215] An antibody or antigen binding fragment thereof for use as described herein can be purified, substantially purified, isolated and / or recombinant.

[0216] Table 1 : Overview of amino acid and nucleotide sequences of preferred CAIX binding antibodies

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] In further embodiments, the moiety for binding to CAIX can comprise BCA-356, a bispecific antibody comprising an affinity matured humanized anti-CAIX antibody linked to an attenuated subunit of IL-12, which is fused at the C-terminus to each of the heavy chains of the anti-CAIX antibody via a linker, forming a “knobs in hole” format (as described in Nair et al., Journal for ImmunoTherapy of Cancer, 10:S2).

[0233] In particularly preferred embodiments of the methods and uses described herein, the agent for binding to CAIX is 89 Zr-Doxilantibody, 124 I-Doxilantibody, 177 Lu-Doxilantibody, or 111 In-Doxilantibody-IRDye800CW (e.g., as described in Stroet et al., (2022), Cancers 14: 861, incorporated herein by reference), G250 RIT (when labeled with an appropriate detectable moiety), or 90 Y-DOTA-cG250.

[0234] In particularly preferred embodiments, the agent for binding to CAIX is 89 Zr-Doxilantibody. 89 Zr-Doxilantibody is a chimeric monoclonal antibody (INN name: Gemtuzumab ozogamicin (GTX), also known as cG250 and TLX250) that is specific for the CAIX antigen, radiolabeled with the positron emitting radioactive metal zirconium-89, which is attached to a lysine residue of GTX via NSuc-DFO-TFP-ester (DFO-TFP) to produce 89Zr DFO-TFP-GTX.

[0235] Constant region

[0236] In preferred embodiments, any antibody and / or antigen-binding fragment thereof as described herein for use in the application can comprise a constant region of an antibody. This includes an antigen-binding fragment of an antibody fused to an Fc.

[0237] Sequences useful for generating a constant region of an antibody or antigen-binding fragment thereof as described herein can be obtained from a number of different sources. In some examples, the constant region of a protein, or portion thereof, is derived from a human antibody. The constant region, or portion thereof, can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgGl, IgG2, IgG3, and IgG4. In one example, the constant region is a human isotype IgG4 or a stabilized IgG4 constant region.

[0238] The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG class antibodies in vivo. The function of FcRn is to salvage IgG from the lysosomal degradation pathway, resulting in decreased clearance rate and prolonged half-life. It is a heterodimeric protein composed of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (a-FcRn) and a 15 kDa p2-microglobulin (β2m). 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 stoichiometry of 1 :2, i.e., one IgG antibody molecule can interact with two FcRn molecules through its two heavy chain Fc region polypeptides (see, e.g., Huber, A.H., et al., J. Mol. Biol. 230 (1993) 1077-1083).

[0239] Accordingly, the in vitro FcRn binding properties / characteristics of IgG are indicative of its in vivo pharmacokinetic properties in the blood circulation. In the interaction between FcRn and the Fc region of IgG class antibodies, different amino acid residues of the heavy chain CH2- and CH3-domains are involved.

[0240] Different mutations that affect FcRn binding and half-life in the circulation in blood are known. Fc region residues critical for mouse Fc region-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, W. F. et al. J. Immunol 169 (2002) 5171-5180). Residues Ile253, 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, J. K. et al., Eur. J. Immunol. 24 (1994) 542-548). (Using the Kabat system, the relevant residues are Ile266, His329, His464, Asn465, and His466). Residues Ile253, His310, and His435 were found to be critical for the interaction of human Fc region with murine FcRn (Kim, J. K. et al., Eur. J. Immunol. 29 (1999) 2819-2885).

[0241] More specifically, the antibody or antigen-binding protein can comprise 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 comprising one or more amino acid substitutions that reduce the affinity of the Fc region for the neonatal Fc region (FcRn).

[0242] Preferred modifications

[0243] In any embodiment, the antibody or antigen-binding fragment thereof (e.g., a G250 antibody or variant thereof as described herein) is a modified IgG antibody or fragment thereof comprising a heavy chain constant region having one or more amino acid substitutions that reduce the affinity of the antibody for the neonatal Fc receptor (FcRn) as compared to a wild-type IgG class antibody, thereby reducing the serum half-life of the modified antibody, as compared to the wild-type IgG class antibody.

[0244] 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 that reduce 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, whereby the substitutions reduce the affinity of the IgG for FcRn.

[0245] In certain preferred embodiments, the one or more amino acid substitutions can be at one or more of residues His310, His433, His435, His436, or Ile253 of the IgG. Preferably, the amino acid substitutions comprise a substitution in the heavy chain constant region at position His310 or at His435. More preferably, the amino acid substitutions that reduce the affinity of the antibody for FcRn are at both His310 and His435.

[0246] In other preferred embodiments, the antibody and / or antigen binding fragment thereof has a constant region that is substantially identical to a constant region of a naturally occurring IgG class antibody, wherein at least one amino acid residue selected from the group consisting of residues His310, His435, and Ile253 is different from the amino acid residue present in the naturally occurring IgG class antibody, thereby altering the FcRn binding affinity and / or serum half-life of the antibody relative to the naturally occurring antibody. In preferred embodiments, the naturally occurring IgG class antibody comprises the heavy chain constant region of a human IgGl, IgG2, IgG2M3, IgG3, or IgG4 molecule.

[0247] Also in preferred embodiments, the amino acid residue 310 and / or residue 435 from the heavy chain constant region of an antibody having a constant region substantially identical to a naturally occurring IgG class antibody is any amino acid other than histidine and 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.

[0248] The amino acid substitution can comprise a substitution from a histidine residue to: alanine, glutamine, glutamic acid, or aspartic acid. Preferably, the amino acid substitution at His310 is a substitution for alanine. Preferably, the amino acid substitution at His435 is a substitution for glutamine. Preferably, the amino acid substitution at Ile253 is alanine.

[0249] In preferred embodiments of the application, the binding affinity of the modified antibody to FcRn and / or serum half-life 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 application, the binding affinity of the modified antibody to FcRn and / or serum half-life is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0250] Additionally, the antibody or fragment thereof for use according to the application can be modified to comprise one or more mutations that alter the affinity of the antibody for any one or more Fc gamma receptors. For example, the one or more amino acid modifications alter the affinity of the antibody constant domain, Fc region, or Fc gamma receptor binding fragment for any one or more Fc gamma receptors.

[0251] In certain embodiments, the modified antibody or antigen binding fragment thereof retains the ability to bind to one or more Fc-gamma receptors, and thus in certain embodiments, the modified antibody retains the ability to stimulate effector responses, including ADCC. In one example, the Fc region of the constant region contains one or more amino acid substitutions that modulate effector function, including increasing effector function compared to wild-type IgG.

[0252] In one example, the Fc region of the constant region has a reduced ability to induce effector function, e.g., compared to a native or wild-type human IgGl or IgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of an Fc region-containing protein are known in the art and / or described herein.

[0253] 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 lie 253 from the heavy chain constant region. In one example, the substitution is a substitution for 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, wherein the substitution reduces the ability of the antibody to induce effector function. In preferred embodiments, the substitution from lie at residue 253 is a substitution for arginine, proline, glutamic acid, or aspartic acid, more preferably alanine.

[0254] 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 the skilled person and / or are available from public databases (e.g., from the National Center for Biotechnology Information).

[0255] In one example, the constant region is a stable IgG4 constant region. The term “stable IgG4 constant region” will be understood to mean an IgG4 constant region that has been modified to reduce the propensity for or propensity to undergo Fab arm exchange or form a half-antibody. “Fab arm exchange” refers to a type of protein modification for human IgG4 in which an IgG4 heavy chain and attached light chain (half-molecule) are exchanged with a heavy-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms recognizing two different antigens (yielding a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. When an IgG4 antibody dissociates to form two molecules, each containing a heavy chain and a light chain, a “half-antibody” is formed.

[0256] In one example, the stabilizing IgG4 constant region comprises a proline at position 241 of the hinge region, according to the system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC United States Department of Health and Human Services, 1987 and / or 1991). According to the EU numbering system, this position corresponds to position 228 of the hinge region. In human IgG4, this residue is typically a serine. Upon substitution of the proline with a serine, the IgG4 hinge region comprises the sequence CPPC. In this regard, the skilled person will be aware that the "hinge region" is the proline-rich part of the constant region of the heavy chain of an antibody that joins the Fc and Fab regions, which confers mobility to the two Fab arms of the antibody. The hinge region includes the cysteine residues involved in inter-heavy chain disulfide bonding. According to the numbering system of Kabat, it is typically defined as extending from Glu226 to Pro243 of human IgGl (or from Glu216 to Pro230 using the EU index). The hinge region of other IgG isotypes can be aligned with the IgGl sequence by placing the first and last cysteine residues forming the inter-heavy chain disulfide bond (S-S) in the same positions (see, e.g., WO2010 / 080538).

[0257] In alternative embodiments, the one or more amino acid modifications that reduce the affinity for the FcRn receptor also reduce the affinity for the Fcy receptor. The modified antibody, or antigen-binding fragment thereof, can further comprise one or more amino acid substitutions compared to a wild-type IgG class antibody, wherein the amino acid substitutions further reduce the affinity of the antibody for one or more Fcy receptors.

[0258] In further embodiments, the modified antibody, or antigen-binding fragment thereof, further comprises one or more amino acid substitutions compared to a wild-type IgG class antibody, wherein the amino acid substitutions increase the stability of the CH1-CH2 hinge region in the modified antibody compared to the wild-type IgG class antibody.

[0259] In any embodiment, the heavy chain constant region of the antibody or antigen-binding protein comprises amino acid substitutions at both His310 and His435. The antibody can also comprise amino acid substitutions at residues equivalent to Ser228 and Leu235 of the constant heavy chain region.

[0260] In any embodiment, the antibody or antigen-binding fragment thereof comprises mutations at Ser228, Leu235, His310, and His435. Preferably, the amino acid modifications are Ser228Pro, Leu235Glu, His310Ala, and His435Gln.

[0261] A further example of a stabilised IgG4 antibody is an antibody in which the arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) is substituted by lysine, threonine, methionine or leucine (e.g. as described in WO2006 / 033386). The Fc region of the constant region can additionally or alternatively comprise a residue selected from the group consisting of alanine, valine, glycine, isoleucine and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e. a CPPC sequence) (as described above).

[0262] 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 IgGl Fc region comprising a substitution at one or more positions selected from the group consisting of 268, 309, 330 and 331. In another example, the Fc region is an IgGl Fc region comprising a deletion of one or more of the following changes E233P, L234V, L235A and 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). Further examples of non-immunostimulatory Fc regions are described in, for example, Dall’Acqua et al., J. Immunol. 177:1129-1138 2006; and / or Hezareh J Virol 75:12161-12168, 2001.

[0263] In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgGl antibody, wherein the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbering) (e.g., as described in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0264] Preferably, the antibody or antigen binding fragment thereof comprises a heavy chain constant region comprising a sequence as set forth in any one of SEQ ID NOs: 177 to 180, preferably as set forth in SEQ ID NO: 178.

[0265] In still further embodiments, the antibody or antigen binding fragment thereof preferably comprises a heavy chain comprising a sequence as set forth in any one of SEQ ID NOs: 182 to 185, preferably as set forth in SEQ ID NO: 183.

[0266] In any embodiment, the antibody or antigen binding fragment thereof comprises a light chain constant region comprising an amino acid sequence as set forth in SEQ ID NO: 181. Preferably, the antibody or antigen binding protein comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 186.

[0267] In any embodiment, the antibody or antigen binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 183 and a sequence as set forth in SEQ ID NO: 186.

[0268] In one embodiment, the antibody or antigen binding fragment thereof comprises: a VH comprising a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to, or comprising a sequence set forth in SEQ ID NO: 36 or 52; and a VL comprising a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to, or comprising a sequence set forth in SEQ ID NO: 116, 132 or 148.

[0269] Preferably, VH contains at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO:36 or 52, or contains the sequence shown therein, and VL contains at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO:132 or 148, or contains the sequence shown therein.

[0270] More preferably, the VH contains at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO:36, or contains the sequence shown therein, and the VL contains at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO:148, or contains the sequence shown therein.

[0271] Alternatively, the VH comprises at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO:52, or includes the sequence shown therein, and the VL comprises the sequence shown in SEQ ID NO:132 or 148, preferably at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO:148, or includes the sequence shown therein.

[0272] Detectable part

[0273] Technicians will become familiar with standard methods for conjugating the detectable portion with a reagent used in conjunction with CAIX.

[0274] In any embodiment of the invention, the small molecule, peptide, protein, or antibody used for binding with CAIX and as described herein may be directly or indirectly linked to a detectable portion (such as a radioisotope, dye, or fluorescent portion).

[0275] In any embodiment, the detectable portion is a radioactive isotope. 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 Zr). As used herein, the term radionuclide may be used interchangeably with the term radioisotope.

[0276] It will be appreciated that the radioisotope can be conjugated to the polypeptide (e.g., antibody) directly (via a chelator or prosthetic group or linker) or indirectly by binding to a single or multiple amino acid residues in the protein (e.g., halogenation of a tyrosine residue).

[0277] In alternative embodiments, a chelator or linker can be used to conjugate a determinable age moiety to a peptide or protein for binding to CAIX. In one example, a peptide or protein (e.g., an antibody) can be conjugated to a chelating moiety selected from the group consisting of TMT (6,6"-bis[N,N",N"'-tetrakis(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 tetraxetan), TCMC (tetra-primary amine 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), Diamsar (3,6,10,13,16,19-hexaazabicyclo[6,6,6]icosane-1,8-diamine), DTPA (pentrane acid or diethylenetriaminepentaacetic acid), CHX-A"-DTPA([(R)-2-amino-3-(4- isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-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-squaraine), and HOPO (3,4,3-(LI-1,2-HOPO)), or other chelators as described herein. Other known chelating moieties include 3p-C-NETA ({4-[2-(bis-carboxy-methylamino)-5-(4-nitrophenyl)pentyl]-7-carboxymethyl-[1,4,7]triazacyclonon-1-yl}acetic acid), 5p-C-NETA (2-({1-[4,7-bis(carboxymethyl)-1,4,7-triazacyclonon-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).

[0278] In certain non-limiting embodiments discussed below, a chelating group can be used to link a metal (such as aluminum) complexed to a18 F or 19 F to provide alternative modalities for imaging, detection, and / or diagnosis. It is contemplated that fluorescently labeled molecules can be used more for intraoperative procedures, while 18 F labeled molecules can be used more for preoperative or postoperative imaging, detection, and / or diagnosis of diseased tissue.

[0279] The agent can be modified to contain a thiol group for attachment of a maleimide-modified fluorescent probe. Alternatively, a bifunctional crosslinking agent or a fluorescent dye conjugated to other reactive species can be used to attach a fluorescent probe to a different group on the agent for binding to CAIX. For example, 488 and 800 can be obtained as amine-reactive dyes for labeling primary amines derivatized with NHS esters (product numbers 46402 and 46421, Thermo Electric, Rockford, Ill.). The skilled artisan will recognize that the fluorescent probes used are not limiting and other dyes or alternative fluorescent probe molecules known in the art can be used in the claimed methods and compositions.

[0280] In certain embodiments, a peptide or protein (e.g., an antibody) for binding to CAIX can be conjugated to a fluorescent probe (to form an immunoconjugate). Methods for covalently conjugating fluorescent probes and other functional groups are known in the art, and any such known methods can be utilized. For example, a fluorescent probe can be attached at the hinge region of a reduced antibody component by disulfide bond formation or mercapto-maleimide interaction. Alternatively, such agents can be linked using heterobifunctional cross-linking agents such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such conjugation are well known in the art. See, e.g., 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.), pp. 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.), pp. 60-84 (Cambridge University Press 1995).

[0281] Alternatively, the fluorescent probe can be conjugated through a carbohydrate moiety in the Fc region of the antibody. See, e.g., 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 example section of which is incorporated herein by reference. The general method involves reacting an antibody component having an oxidized carbohydrate moiety with a fluorescent probe having at least one free amine function. The reaction produces an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0282] If the antibody used as the antibody component of the immunoconjugate is an antibody fragment, the Fc region can not be present. However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full-length antibody or antibody fragment. See, e.g., Leung et al., J. Immunol. 154:5919 (1995); U.S. Patent Nos. 5,443,953 and 6,254,868, the example sections of which are incorporated herein by reference. Engineered carbohydrate moieties are used to link functional groups to antibody fragments.

[0283] Alternative methods for linking fluorescent probes or other functional groups to targeting molecules involve the use of click chemistry reactions. Click chemistry methods were originally conceived as a method for rapidly producing complex substances by linking small subunits together in a modular fashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistry reactions are known in the art, such as the Huisgen 1,3-dipolar cycloaddition copper-catalyzed reaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), which is commonly referred to as the "click reaction." Other alternatives include cycloaddition reactions, such as Diels-Alder, nucleophilic substitution reactions (especially small strained rings, such as epoxide and aziridine compounds), carbonyl chemistry formation of urea compounds, and reactions involving carbon-carbon double bonds, such as alkyne in thiol-alkyne reactions.

[0284] 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 a copper catalyst to facilitate [3+2] azide-alkyne cycloaddition reactions. For example, cyclooctyne is an 8-carbon ring structure that contains an internal alkyne bond. The closed ring structure causes a significant bond angle distortion of the acetylene, which is highly reactive with an azido group to form a triazole. Thus, cyclooctyne derivatives can be used in copper-free click reactions.

[0285] Ning et al. (2010, Angew. Chem. Int. Ed. 49:3065-68) reported another type of copper-free click reaction involving a strain-promoted alkyne-nitrone cycloaddition reaction. To address the slow rate of the initial cyclooctyne reaction, an electron-withdrawing group was attached adjacent to the triple bond. Examples of such substituted cyclooctynes include difluorocyclooctyne, 4-dibenzo cyclooctyne alcohol, and azacyclooctyne. The alternative copper-free reaction involves a strain-promoted alkyne-nitrone cycloaddition reaction to produce N-alkylated isoxazolidines. The reaction was reported to have exceptionally fast reaction kinetics and was used in a one-pot three-step protocol for site-specific modification of peptides and proteins. Nitrone was prepared by condensation of the appropriate aldehyde with N-methylhydroxylamine, and the cycloaddition reaction was performed in a mixture of acetonitrile and water. These and other known click chemistry reactions can be used to link chelating moieties to antibodies or other CAIX binding molecules in vitro.

[0286] In certain embodiments, the pharmaceutical agent can comprise a peptide or protein (e.g., an antibody) covalently coupled to a radioisotope 124 l. The isotope is a positron emitter that can be attached to the antibody, e.g., as described in Larsson et al. (J. Nucl. Med. 33 (1992), 2020-2023) or US 5,185,142, the contents of which are incorporated herein by reference.

[0287] In any embodiment, the radiolabeling of the protein or antibody is accomplished by covalent iodination, particularly with chloroguanidine reagent (1,3,4,6-tetrachloro-3a,6a-diphenylguanidine). While chloroguanidine labeling is a solid-phase oxidation method similar to the chloramine-T method, it is generally considered to be more mild because the reaction is carried out on the surface of the oxidizing agent, thereby minimizing the exposure of the substrate (Salacinzki, P. R. P. et al., Anal. Biochem. 117: 136 (1981)).

[0288] Chelators with radiometals and other halogenated radioisotopes can be conjugated 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.

[0289] In another example, the protein or antibody can be conjugated to a bifunctional linker, such as a bromoacetyl, thiol, succinimidyl ester, TFP ester, maleimide, or using any amine or thiol modification chemistry known in the art.

[0290] The skilled person will be familiar with standard methods for conjugating chelators to proteins, including antibodies and derivatives or fragments thereof. In addition, the skilled person will be familiar with methods for selecting relevant chelators for pairing with radiometals, for example as described in Chem. Soc. Rev., 2014, 43, 260, which is incorporated herein by reference.

[0291] In any embodiment, the dating moiety can be a fluorescent dye, such as but not limited to the fluorescent dyes described in US20150086482, which is incorporated herein by reference.

[0292] In any embodiment, the fluorescent dye (which can also be referred to as a fluorescent probe) can be selected from the group consisting of: Alexa 350, Alexa 430, AMCA, Aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-Carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein, 5-Carboxy-2',4',5',7'-tetrachlorofluorescein, 5-Carboxyfluorescein, 5-Carboxyrhodamine, 6-Carboxyrhodamine, 6-Carboxytetramethylamino, Cascade Blue, Cy2, Cy3, Cy5, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresyl fast violet, Cresyl blue violet, Brilliant cresyl blue, p-Aminobenzoic acid, Erythrosin, Phthalocyanine, Azomethine, Fluorin, Xanthine, Succinylfluorescein, Rare earth cryptate, Europium trisbipyridine diamine, Europium cryptate or chelate, Diamine, Dicyanin, La Jolla blue dye, Allopycocyanin, Allococyanin B, C-Phycocyanin, R-Phycocyanin, Thiamine, Phycoerythrobilin, Phycoerythrin R, REG, Rhodamine Green, Rhodamine isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), Tetramethyl rhodamine, and Texas Red.

[0293] Medicament administration

[0294] The skilled person will understand that the dosage of medicament for use in accordance with the methods of the application will depend on various factors, including the age, sex, height and weight of the subject to whom the medicament is administered, and on the medicament.

[0295] In the case where the medicament is an antibody for binding to CAIX, the antibody is preferably administered or infused to the subject at a dose of about 1 mg to about 50 mg, preferably at a dose of about 5 mg to about 20 mg, and more preferably at a dose of about 10 mg. The specific activity of the radiolabelled antibody is preferably about 15 MBq / mg to about 20 MBq / mg, more preferably about 18 Mbq / mg to about 19 Mbq / mg.

[0296] 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 girentuximab.

[0297] The antibody is typically administered as a pharmaceutical composition with a pharmaceutically acceptable carrier (e.g., a physiological saline solution), optionally including a protein stabilizer, such as human serum albumin (HSA). The antibody is preferably administered by infusion.

[0298] The CAIX-binding agent (preferably girentuximab) or humanized variant thereof is preferably administered intravenously, preferably by infusion or intravenous injection. Administration of the antibody by infusion is preferably performed over a period of up to about 30 minutes, more preferably over about 15 minutes. Of course, the CAIX inhibitor can also be administered intraperitoneally or intramuscularly.

[0299] Detection methods

[0300] It will be appreciated that the method for detecting or imaging the agent for use according to the application will depend on the nature of the detectable moiety of the agent.

[0301] The detection step is preferably performed using PET, SPECT, fluorescence spectroscopy or any other suitable method.

[0302] Examples of in vivo methods for determining the presence or expression of CAIX in a tumor include the use of in vivo / partial or whole body imaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging. Immuno-PET and immuno-SPECT imaging can involve the use of CAIX-binding molecules conjugated to a radioisotope to enable non-invasive imaging of CAIX-expressing tissues and tumors.

[0303] Where the detectable moiety is a radioisotope, the method will thus involve determining the radiation for the subject to which the agent is administered.

[0304] The in vivo detection step in the methods described above can be whole body imaging or local imaging of a specific site, such as but not limited to a site of expected or possible solid tumor growth.

[0305] In the case of SPECT, the agent for binding to CAIX typically comprises a detectable agent in the form of a gamma-emitting radioisotope (radionuclide), usually by injection into the bloodstream. Typically, the gamma-emitting radioisotope for SPECT includes 99m Tc (technetium), 123 I or 131 I (iodine) and 68Ga (gallium).

[0306] In any embodiment where the agent comprises a radioisotope, the detection method can comprise positron emission tomography (PET).

[0307] Optionally, the detection method comprises PET / CT imaging or PET / MRI scanning.

[0308] It can be useful to wait a period of time after administration (preferably infusion) of the agent to allow the agent to accumulate at sites of cancer cells expressing the tumor. Typically, the period of time is 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, at least about 10 days. Preferably, the period of time between administration of the agent and detection of the agent (e.g., by PET or other method described herein) is typically no more than about 10 days, or no more than about 15 days, or no more than about 20 days.

[0309] Where PET is used to image or detect cancer, PET imaging can preferably be performed within 7 ± 2 days of infusion of the radiolabeled agent, particularly within 5 ± 2 days after infusion, in order to obtain optimal imaging results, including accumulation of the agent at sites where CAIX is present.

[0310] In the context where the detectable moiety is a fluorescent probe or dye, fluorescence imaging can be used to detect the moiety, including during intraoperative, intravascular, or endoscopic procedures, as described in U.S. Patents Nos. 4,932,412; 6,096,289; 6,387,350; 7,201,890; the Example sections of each of which patents are incorporated herein by reference. Such imaging methods can be used, for example, to image the distribution of tumor tissue, to facilitate removal of the tumor tissue. Fluorescence imaging can also be used for diagnostic purposes, for example, to distinguish between malignant, benign, and proliferative tissue.

[0311] Cancer to be detected or diagnosed

[0312] The present invention provides methods for in vivo identification or imaging of cancer. Such methods are expected to be useful in the diagnosis of CAIX-expressing cancers, preferably without the need for additional invasive techniques (such as biopsy collection and testing) to confirm the diagnosis.

[0313] Thus, in preferred embodiments, the methods of the present invention are capable of diagnosing any of the cancers recited herein as the sole, primary, or principal mode of cancer diagnosis, and preferably without the need for additional invasive diagnostic methods, including biopsy-related methods.

[0314] It is also contemplated that the methods of the present application are useful for staging of cancer progression or success of cancer treatment. In addition, such methods provide the benefit of providing a non-invasive means for assessing cancer in a subject.

[0315] As used herein, the term "cancer" refers to a malignant growth or tumor resulting from uncontrolled cell division. The term "cancer" includes primary tumors and metastatic tumors.

[0316] The methods of the present application have particular use in imaging, detecting and / or diagnosing cancers that have not previously been identified using in vivo imaging techniques that utilize an agent for binding to CAIX.

[0317] A subject for whom a cancer diagnosis or detection or imaging as described herein is performed can be suspected of having cancer or at risk for having cancer. A subject suspected of having cancer can exhibit one or more symptoms of cancer, can have a family history of cancer or can have one or more genetic markers indicative of a risk or likelihood of developing cancer. A subject deemed to be at risk for cancer can exhibit one or more symptoms of cancer, can have a family history of cancer or can have one or more genetic markers indicative of a risk or likelihood of developing cancer.

[0318] In any embodiment, the cancer being detected, imaged or diagnosed is breast cancer. High levels of CAIX in breast cancer have been previously reported, and CAIX expression has also been reported to be associated with resistance to chemotherapy or an indicator of treatment success. These observations date back several decades, and prior to that, this use of a CAIX binding imaging agent for diagnosis of this patient group has not been previously reported.

[0319] The breast cancer can be a so-called "triple negative breast cancer" (TNBC), an aggressive, metastatic and drug resistant form of breast cancer, with limited treatment options and 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).

[0320] In any embodiment, the breast cancer can be a hormone receptor positive breast cancer, such as ER positive, PR positive, ER & PR positive. In any embodiment, the breast cancer can be positive for HER2, including HER2 and hormone receptor positive breast cancer.

[0321] In certain embodiments, the cancer being detected, imaged or diagnosed is not breast cancer.

[0322] In any embodiment, the cancer detected, imaged, or diagnosed is cervical cancer. The cervical cancer can be squamous cell carcinoma or adenocarcinoma. In any embodiment, the subject diagnosed or imaged for cervical cancer can exhibit one or more symptoms of cervical cancer, such as abnormal vaginal bleeding, including contact bleeding, or pelvic pain. The subject deemed at risk for developing cervical cancer can have previously been infected with HPV 16 or 18 strains or have one or more genetic markers indicative of risk for cervical cancer.

[0323] In any embodiment, the cancer detected, imaged, or diagnosed is colorectal cancer (including, for example, epithelial colorectal cancer). In any embodiment, the subject diagnosed or imaged for colorectal cancer can exhibit one or more symptoms of colorectal cancer, such as a persistent change in bowel habits, rectal bleeding or blood in the stool, persistent abdominal discomfort, weakness or fatigue, and unexplained weight loss. The subject deemed at risk for developing colorectal cancer can have a family history of the disease or have one or more genetic markers deemed to be associated with increased risk for colorectal cancer or can have previously had intestinal polyps.

[0324] In any embodiment, the cancer detected, imaged, or diagnosed is esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophageal / esophagogastric junction adenocarcinoma). The subject diagnosed or imaged or detected for esophageal cancer can exhibit one or more symptoms selected from the group consisting of difficulty swallowing, unexplained weight loss, chest pain, pressure or burning, worsening indigestion or heartburn, or cough or hoarseness. The subject deemed at risk for developing esophageal cancer can have a family history of the disease or have one or more genetic markers deemed to be associated with increased risk for colorectal cancer or can have previously been diagnosed with Barrett's esophagus.

[0325] In any embodiment, the cancer detected, imaged, or diagnosed is gastric cancer (including gastric adenocarcinoma). The subject diagnosed or imaged or detected for gastric cancer can exhibit one or more symptoms selected from the group consisting of difficulty swallowing, stomach pain, feeling bloated after eating small amounts, loss of appetite, indigestion, nausea and vomiting, fatigue, black stools. The subject deemed at risk for developing gastric cancer can have a family history of the disease or have one or more genetic markers deemed to be associated with increased risk for gastric cancer.

[0326] In any embodiment, the cancer detected, imaged, or diagnosed is glioblastoma multiforme. The subject diagnosed or imaged or detected for glioblastoma can exhibit one or more symptoms, including visual, auditory, balance, coordination, strength, and reflex symptoms, nausea, vomiting, seizures, or other neurological symptoms.

[0327] In any embodiment, the cancer being detected, imaged, or diagnosed is head and neck cancer (including head and neck squamous cell carcinoma and nasopharyngeal and hypopharyngeal cancer). The subject being diagnosed or imaged or detected for head and neck cancer can exhibit one or more symptoms such as pain, swelling, hoarseness, sore throat, persistent cough, bad breath, unexplained weight loss. The subject deemed at risk for developing head and neck cancer can have a family history of the disease, have one or more genetic markers deemed to be associated with increased risk of head and neck cancer, can have been previously infected with HPV or Epstein-Barr virus, have a weakened immune system, poor oral hygiene (including gum disease), smoke or chew betel nut, arecanut, gutka, or pan, or have a genetic condition such as Fanconi anemia or Li-Fraumeni syndrome.

[0328] In any embodiment, the cancer being detected, imaged, or diagnosed is liver cancer (including cholangiocarcinoma and hepatocellular carcinoma). As used herein, cholangiocarcinoma refers to cancer of the bile duct or biliary tract. The cholangiocarcinoma can be intrahepatic, perihilar, or distal. The cancer can be gallbladder cancer or cancer of the ampulla of Vater. In any embodiment, the subject being diagnosed or imaged for liver cancer (including cholangiocarcinoma and hepatocellular carcinoma) can be a subject exhibiting one or more symptoms of liver cancer (including cholangiocarcinoma and hepatocellular carcinoma).

[0329] As used herein, one or more symptoms of cholangiocarcinoma include abdominal pain, yellowing of the skin (jaundice), weight loss, generalized itching, fever, light-colored stools, or dark-colored urine. The skilled artisan will be familiar with various risk factors for cholangiocarcinoma, including primary sclerosing cholangitis (an inflammatory disease of the bile duct), ulcerative colitis, cirrhosis, hepatitis C, hepatitis B, certain liver fluke infections, and some congenital liver malformations. However, most people do not have identifiable risk factors.

[0330] In any embodiment, the cancer being 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, but is not limited to, all types of lung cancer at all stages of progression, such as lung carcinoma, metastatic lung carcinoma, non-small cell lung cancer (NSCLC) such as lung adenocarcinoma, squamous cell carcinoma, or small cell lung cancer (SCLC). In some embodiments, the subject has non-small cell lung cancer (NSCLC).

[0331] In any embodiment, the cancer being detected, imaged, or diagnosed is ovarian cancer (including epithelial ovarian cancer).

[0332] In any embodiment, the cancer being detected, imaged, or diagnosed is pancreatic cancer (including pancreatic ductal adenocarcinoma).

[0333] In any embodiment, the cancer being detected, imaged or diagnosed is a soft tissue sarcoma.

[0334] In any embodiment, the cancer being detected, imaged or diagnosed is bladder cancer. The bladder cancer can be non-muscle invasive bladder cancer (NMIBC).

[0335] In a particularly preferred embodiment, the cancer being detected or imaged or diagnosed is not a kidney cancer (including clear cell renal carcinoma).

[0336] Imaging or diagnosis of cancer will typically be assessed by qualitative assessment of detection of the agent compared to conventional imaging after administration of the agent and its detection. Quantitative assessment can be based on each lesion, including standardized uptake value (SUV) (SUVmaxand SUVmean), SUV corrected for lean body mass (SUL), metabolic tumor volume (MTV), and tumor-to-background ratio (TBR).

[0337] Tumor-to-background ratio (TBR) will typically be defined as the ratio of the lesion standardized uptake value (SUVmax) to the reference region SUV (liver, blood pool, etc.). Comparison will be made by PET scan and standard imaging modalities (including high resolution CT / MRI and other potential imaging per patient (depending on tumor type)), type of lesion, and number, size, and other characteristics of the detected lesions will be indicated.

[0338] Qualitative visual analysis of imaging (presence or absence of localized agent uptake associated with tumors, as seen on contrast-enhanced CT, MRI, or FDG PET / CT) can be used to assess the concordance of tumor lesion detection between specific agent PET / CT and conventional imaging. RECIST 1.1 criteria for conventional imaging can be used as the primary tool for concordance comparison with PET.

[0339] In addition to the above, all visible tumor lesions in conventional imaging can also be compared to PET imaging results.

[0340] Examples

[0341] Example 1 : Clinical trial protocol

[0342] The trial involves the use of 89 Zr-labeled gemtuzumab ozogamicin PET / CT imaging to assess CAIX expression in a subset of solid tumors.

[0343] Primary objective

[0344] To non-invasively assess CAIX tumor expression in different solid tumors 89 Zr-gemtuzumab ozogamicin PET / CT imaging. These tumor types have not yet been shown to take up 89Zr-doxilamab formal imaging study.

[0345] Primary endpoint: Qualitative (yes / no) and quantitative assessment of89Zr-doxilamab uptake compared to conventional imaging. Descriptive statistics are reported for each tumor type. Each lesion analysis includes SUVmax, SUVmean, SUL (SUV corrected for lean body mass), tumor-to-background ratio (TBR), and metabolic tumor volume (MTV).

[0346] Secondary objectives

[0347] To evaluate 89 The tolerability and safety of Zr-doxilamab administration in patients with different tumor types.

[0348] Secondary endpoint: Patient safety is assessed based on the incidence and nature of adverse events (AEs) and serious adverse events (SAEs) and clinically significant changes in laboratory test values, vital signs, or physical examination findings. Laboratory abnormalities are assessed according to NCI CTCAE v.5.0. Patients are informed that they must report and register with the primary investigator in case of abnormal physical signs within 24 hours of examination. The NCI Common Toxicity Criteria, version 5.0, are used.

[0349] Tertiary objectives

[0350] To evaluate 89 The correlation between standardized uptake value (SUV) of89Zr-doxilamab and CAIX histology in patients who underwent biopsy or surgery within 90 days of Zr-doxilamab imaging 89 The correlation between standardized uptake value (SUV) of89Zr-doxilamab and CAIX histology.

[0351] Tertiary endpoint: If biopsy or surgery samples are available (and their tissue samples are available) within 90 days of dosing or 89 The correlation between standardized uptake value (SUV) of89Zr-doxilamab and CAIX histology in patients who underwent biopsy or surgery within 90 days of Zr-doxilamab imaging 89 The correlation between standardized uptake value (SUV) of89Zr-doxilamab and CAIX histology.

[0352] Safety follow-up is performed for all patients until the EOS visit (day 15-25).

[0353] Overall study design

[0354] An open-label, non-randomized study is conducted to evaluate the expression of CAIX by 89 Zr-doxilamab PET / CT imaging in different tumor types and to assess the feasibility of targeting CAIX for potential diagnostic and therapeutic applications.

[0355] For each of the tumor types, including but not limited to: cervical cancer, colorectal cancer, esophageal cancer (esophageal SCC and esophagus / gastroesophageal junction adenocarcinoma), gastric 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 carcinoma and small cell carcinoma), ovarian cancer (epithelial ovarian cancer), pancreatic cancer (pancreatic adenocarcinoma), and soft tissue sarcoma, a minimum of 5 subjects were enrolled.

[0356] The study involved a single administration 89 Zr-cetuximab (37 MBq [1 mCi] ± 10% containing cetuximab with a mass dose of 10 mg).

[0357] PET / CT imaging was performed 5 ± 2 days after administration. Image data analysis of PET / CT imaging was performed by a nuclear medicine reader to assess 89 Tumor uptake of Zr-cetuximab, up to 10 most active lesions, and also conventional imaging according to RECIST 1.1.

[0358] Qualitative visual analysis (localization of tumor related 89 The concordance of 89Zr-cetuximab PET / CT with conventional imaging in the detection of tumor lesions was assessed using the presence or absence of Zr-cetuximab uptake as seen on contrast-enhanced CT, MRI or FDG PET / CT. Lesions shown by 89Zr-cetuximab alone were described.

[0359] Tissue samples (biopsies or surgical procedures from patients) were collected whenever possible and sent to a central laboratory for CAIX expression analysis.

[0360] The study was evaluated as shown in the following table:

[0361]

[0362]

[0363] 1 The following procedures were performed outside the study

[0364] 2 Only in case of clinical indication

[0365] 3 Remote visits (telephone follow-up)

[0366] 4 To be performed within 30 days after Day 0

[0367] 5 If a patient cannot undergo PET / CT, then CT is contraindicated, or PET / MRI may be the preferred option for the patient, and PET / MRI can be performed instead of PET / CT.

[0368] Dosage

[0369] The use of here 89 The Zr-geutuximab dose (37 MBq [1 mCi] ± 10%, containing a mass dose of 10 mg of gemutuximab) is consistent with the dosing regimen of 89Zr-geutuximab in the ongoing phase 3 clinical trial, and Merkx et al. (2021) showed that PET imaging was permitted 4–7 days after administration.

[0370] 89 Zr-geutzoma antibody is a radiolabeled chimeric monoclonal antibody (INN name: Zr). 89 Gelatinumab (deferroamine). Gelatinumab is a chimeric monoclonal antibody (INN: gemutuximab, synonyms: cG250, TLX250) that is specific for the CAIX (carbonic anhydrase 9) antigen. It is radiolabeled with positron-emitting radioactive zirconium-89, which is linked to the lysine residues of gemutuximab via an NSuc-DFO-TFP ester (DFO-TFP) to produce… 89 Zr-DFO - Gelatinumab.

[0371] Will 89 Zr-geutuximab is formulated as a solution containing a total of 10 mg of gemutuximab for a single intravenous administration at a nominal dose strength of 37 MBq (±10%) (1 mCi ±10%). 89 Zr-geutuzumab solution is supplied in glass vials or syringes (depending on the geographic region) in appropriate packaging (lead-shielded containers with radiation warning symbols, depending on radiopharmaceutical requirements).

[0372] The compound was administered as a single dose via slow intravenous injection over 3 minutes through a single peripherally placed intravenous cannula. 89 Zr-geutuximab (37 MbCi ± 10%, [1 mCi ± 0.1 mCi], containing a mass dose of 10 mg of gemutuximab) is administered. The injection volume is approximately 10 ml, depending on the activity administered.

[0373] Inclusion criteria

[0374] All participants will meet the following criteria:

[0375] 1. Written and voluntary informed consent.

[0376] 2. Male or female age > 18 years at the time of consent.

[0377] 3. Able to understand and willing to sign a written informed consent form.

[0378] 4. Participants must have histologically or cytologically documented solid tumors of the following types, but not limited to:

[0379] • Cervical cancer

[0380] • Colorectal cancer

[0381] • Esophageal cancer (esophageal SCC and esophagus / gastroesophageal junction adenocarcinoma)

[0382] • Gastric cancer (gastric adenocarcinoma)

[0383] • Glioblastoma multiforme

[0384] • Head and neck cancer (head and neck SCC and nasopharyngeal cancer)

[0385] • Liver cancer (cholangiocarcinoma and hepatocellular carcinoma)

[0386] • Lung cancer (non-small cell carcinoma and small cell carcinoma)

[0387] • Ovarian cancer (epithelial ovarian cancer)

[0388] • Pancreatic cancer (pancreatic ductal adenocarcinoma)

[0389] • Soft tissue sarcoma

[0390] 5. At least one non-CNS, measurable target lesion according to RECIST 1.1 documented on conventional imaging performed within 30 days prior to Day 0.

[0391] 6. Participants agree not to participate in another interventional study while participating in this study, defined as signing an informed consent form (ICF), until completion of the last study visit.

[0392] 7. Negative serum pregnancy test in female patients of childbearing potential within 24 hours of receiving study product at screening and confirmed negative pregnancy test result from urine. Female patients of non-childbearing potential must provide evidence by meeting one of the following criteria at screening:

[0393] • Postmenopausal defined as greater than 50 years of age and amenorrhea for at least 12 months after cessation of all exogenous hormonal treatments.

[0394] • If women < 50 years of age have been amenorrheic for 12 months or longer after stopping exogenous hormone therapy and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels are within the institutionally defined postmenopausal range, they will be considered postmenopausal.

[0395] • Documentation of irreversible sterilization surgery by hysterectomy, bilateral oophorectomy, or bilateral salpingectomy, but not tubal ligation.

[0396] 8. For all participants, agreement to implement dual barrier contraception until at least 28 days after 89 Minimum 42 days after Zr-gemtuzumab administration.

[0397] Exclusion Criteria

[0398] Patients will be excluded from participating in the trial if one or more of the following criteria are met:

[0399] 1. Exposure to murine or chimeric antibodies within the last 5 years.

[0400] 2. Prior administration of any radionuclide within 10 half-lives of the (radionuclide) (i.e., within 10 half-lives after Day 0) prior to anticipated administration of 89 Zr-gemtuzumab.

[0401] 3. Exposure to any CAIX-targeting compound (diagnostic / therapeutic) within the last 3 months.

[0402] 4. Serious non-malignant illness that in the investigator’s judgment could interfere with the ability of the subject to participate in or complete the study or the safety of the subject (e.g., psychiatric illness, infectious disease, autoimmune disease, or metabolic disease).

[0403] 5. Any clinically significant abnormality detected during screening laboratory tests or physical examination that in the opinion of the investigator would adversely affect the participant’s ability to participate in the study. The principal investigator assesses patients for inclusion based on pathology and tumor type.

[0404] 6. Psychiatric impairment likely to impair the ability of the subject to give informed consent or to comply with study requirements.

[0405] 7. Exposure to any antineoplastic therapy within 14 days of the planned administration of 89 Zr-gemtuzumab (i.e., within 14 days after Day 0).

[0406] 8. Pregnant or lactating women.

[0407] 9. Known hypersensitivity, intolerance, or allergy to gemtuzumab, DFO (deferoxamine), or any component of the study agent.

[0408] 10. Renal insufficiency, glomerular filtration rate (GFR) < 45 mL / min / 1.73 m2.

[0409] 11. Vulnerable patients (e.g. detained).

[0410] Efficacy assessment

[0411] Imaging is based on the use of 89 The ability of Zr-Doxil to non-invasively assess CAIX tumor expression in patients was evaluated by PET / CT imaging. A single administration of Zr-Doxil was performed at Day 0 89 Following Zr-Doxil, a whole body PET / CT scan was performed at 5 ± 2 days post administration according to the above table and the imaging manual. Patients with metastatic (suspected or confirmed) disease can receive an optional additional whole body PET / CT scan if clinically indicated (e.g. if the tumor to background ratio makes the tumor lesions difficult to identify and is expected to improve).

[0412] Qualitative assessment will be performed compared to conventional imaging 89 Zr-Doxil tumor uptake (yes / no) in up to 10 most active lesions in individual patients. Quantitative assessment will be performed on a per lesion basis (including SUVmax, SUVmean, SUL, MTV and TBR).

[0413] For patients who cannot perform a CT scan for any reason and / or patients who have a contraindication for CT scan, PET / MRI can be performed instead of PET / CT if PET / MRI is available at the study site. PET / MRI can also be performed in patients in which the disease condition can be better visualized by MRI (e.g. GBMR).

[0414] Tumor to background ratio (TBR) will be defined as the ratio of the lesion SUVmax to the reference region SUV (liver, blood pool, etc.). The comparison will be made by 89 Zr-Doxil PET scan and standard imaging modalities (including high resolution CT / MRI and other potential imaging per patient (depending on tumor type)), the type of lesions and the number, size and other characteristics of the detected lesions will be compared.

[0415] Qualitative visual analysis (localization of the tumor related to the contrast enhanced CT, MRI or FDG PET / CT) 89 The presence or absence of Zr-Doxil uptake, as seen on contrast enhanced CT, MRI or FDG PET / CT, will be assessed 89Consistency of tumor lesion detection between Zr- gemtuzumab PET / CT and conventional imaging. The RECIST 1.1 criteria used for conventional imaging must be used as the primary tool for consistency comparison with PET as much as possible. For tumor radiological assessment with different recommendations according to the Clinical Guidelines of Scientific Oncology Societies, these guidelines should also be followed. In addition to the above, all visible tumor lesions in conventional imaging can also be compared with the results of PET imaging.

[0416] For each patient, the SUVmax, SUVmean, SUL, MTV, TBR, and conventional imaging concordance rate will be calculated locally by the nuclear medicine specialist at each site and entered into the eCRF. Detailed information will be included in the imaging manual.

[0417] Example 2: Preparation of radiolabeled gemtuzumab ozogamicin

[0418] Radiolabeled gemtuzumab is prepared as previously described (see, e.g., WO 2021 / 000017). Briefly, bioconjugated gemtuzumab is prepared using standard techniques to obtain DOTA-gemtuzumab or DFO-gemtuzumab, which is then labeled with a radioisotope useful for imaging (e.g., Zr). 89

[0419] Example 3: In vitro and in vivo binding of radiolabeled gemtuzumab ozogamicin to various cancers

[0420] Imaging studies are performed using radiolabeled DOTA-gemtuzumab to assess the ability of the imaging agent to detect non-RCC cancer types.

[0421] First, the ability of radiolabeled DOTA-gemtuzumab to bind to various cell lines. These data, shown in Table 1, demonstrate the ability of the antibody to bind to various cell types expressing CAIX, but the extent of binding in vitro is variable. Figure 1

[0422] Subsequently, three groups of mice are tested, each bearing a different tumor xenograft. These groups are as follows:

[0423] Group 1: Mice bearing AsPc-1 cell xenografts (pancreatic cancer cell line); n = 4

[0424] Group 2: Mice bearing FaDu cell xenografts (squamous cancer pharyngeal / hypopharyngeal cancer cell line); n = 4

[0425] Group 3: Mice bearing HT-29 xenografts (colorectal cancer cell line); n = 4

[0426] ​​Radiolabeled gemtuximab was administered intravenously, and imaging was performed at 24 and 72 hours. Biodistribution was assessed 72 hours post-administration. The table below summarizes the radioactivity and dose of the administered antibody:

[0427]

[0428]

[0429] Figure 2 The image shows representative images of mice from each of the three groups.

[0430] Figure 3 The percentage of the injected dose in the tumor (24 and 72 hours post-injection) is quantified. The results confirm the observations obtained using flow cytometry, demonstrating the ability of the radiolabeled antibody to bind to the respective cancer cell lines. In other words, the results demonstrate (except for FaDu cells – see further comment below) that the antibody can bind to target tumor cells in vivo with similar affinity to that in vitro.

[0431] The results also showed that the radiolabeled antibodies remained detectable in circulation and spleen 72 hours after administration.

[0432] The in vitro biodistribution of radiolabeled DOTA-GmAb was compared with its in vivo biodistribution. In simple terms, the in vitro biodistribution corresponds to the distribution of radiolabeled DOTA-GmAb in mouse organs, as assessed after necropsy. The in vivo biodistribution corresponds to the biodistribution observed in whole-mouse imaging experiments (e.g., as shown in...). Figure 1 (as shown in the image).

[0433] Figure 4 The results showed a high correlation between in vivo and in vitro quantification of signals in tumors.

[0434] The results showed that positive in vitro binding results (e.g., good binding with HT-29 and AsPc-1 cells) could be generalized to positive binding observed in vivo.

[0435] Interestingly, the inventors observed that although the radiolabeled antibody bound very poorly to FaDu cells in vitro, it was able to bind to tumor cells in vivo. These results suggest that a negative in vitro binding finding may not predict in vivo binding, and therefore, radiolabeled DOTA-GmAb has potential utility for imaging specific cancer types.

[0436] Example 4: Imaging of alternative cancer types

[0437] use 89Zr-DFO-GmAb was used in experiments similar to those in Example 2 to detect the presence of tumor xenografts in the following mice:

[0438] Group 1: Mice carrying A-549 cell line xenografts (lung cancer)

[0439] Group 2: Mice carrying MDA-MB-468 cell line xenografts (triple-negative breast cancer)

[0440] Group 3: Mice carrying HeLa cell line xenografts (cervical cancer)

[0441] Group 4: Mice carrying AGS cell line xenografts (gastric cancer)

[0442] Group 5: Mice carrying HepG2 cell line xenografts (liver cancer)

[0443] Group 6: Mice carrying A2780 cell line xenografts (ovarian cancer)

[0444] Group 7: Mice carrying SK-LMS1 cell line xenografts (soft tissue sarcoma-vulvar leiomyosarcoma)

[0445] In 89 PET / CT scans were performed on mice 24 and 72 hours after intravenous injection of Zr-DFO-GmAb into mice carrying xenografts to determine the ability of the radiolabeled marker to detect cancer cells in vivo.

[0446] The results will show that the radiolabeled antibodies can bind to tumor xenografts. In other words, the results will demonstrate that the antibodies can bind to target tumor cells in vivo with similar affinity to that in vitro.

[0447] These results indicate that radiolabeled GmAbs are suitable for generating images of different cancer types in vivo and can therefore be used as non-invasive diagnostic reagents for diagnosing and detecting cancers other than renal cell carcinoma.

[0448] Example 4: Imaging of triple negative breast cancer

[0449] Triple-negative breast cancer (TNBC) is an aggressive, metastatic, and drug-resistant cancer with limited treatment options.

[0450] The inventors believe that CAIX (a hypoxia-mediated breast tumor growth regulator) may be important for maintaining breast cancer stem cells in hypoxic environments. Therefore, the inventors used it in 12 patients with metastatic TNBC. 89 Zr-labeled gemutuximab was used to evaluate TNBC imaging using PET / CT imaging.

[0451] patients received fluorodeoxyglucose F18 (FDG) and 89 Zr-rituximab PET-CT and CT imaging. Patients received a single slow intravenous administration of 37 ± 10% MBq 89 Zr-rituximab (10 mg). On day 3 post administration, PET / CT from skull to mid-thigh were acquired, with 10 minutes acquisition time per bed position. The gold standard was determined by FDG PET / CT, CT and follow-up; lesions detected by at least 2 modalities were considered true positives. Tumor SUV [最大值，平均值 ], total lesion glycolysis (TLG) and metabolic tumor volume (MTV). Immunohistochemistry (IHC) was performed with Bond RX fully automated research staining with anti-CAIX antibody (Leica, clone TH22). Staining was evaluated with semi-quantitative analysis (percentage and intensity of tumor cell expression) and SUV values were compared to the degree of CAIX expression assessed by IHC.

[0452] Preliminary results from 4 patients were examined and included data from a total of 49 lesions (lymph nodes, bone, lung and breast) detected in these patients, 41 by 89 Zr-rituximab, 42 by CT and 49 by FDG PET / CT. Forty-four lesions were confirmed by the gold standard: 24, 5, 4, 2, 9 in lymph nodes, lung, bone, skin and breast, respectively.

[0453] 89 The overall sensitivity of Zr-rituximab PET / CT was 93.2% with a sensitivity of 100% for bone, lung, breast, skin and 87.5% for lymph nodes. The overall sensitivity of both CT and FDG-PET / CT was 82.7%. For 89 Zr-rituximab and 89 Zr-rituximab FDG, median SUV in tumors 最大值 were 3.45 [IQ: 2.03-4.69] and 4.68 [IQ: 3.27-10.71], respectively. IHC showed two patients with two CAIX-high expressing lesions [100%, 20%] while two patients presented a corresponding low profile [3%, 0%]. IHC CAIX cell status and 89 Zr-rituximab SUV 平均值 presented a weak correlation (rho = 0.80; p = 0.20). No 89 Zr-rituximab safety issues were reported.

[0454] The results demonstrate that, 89Zr-Doxilant is useful for PET / CT imaging of patients and diagnosis of TNBC and provides results superior to biopsy IHC.

[0455] Example 6: Imaging of bladder cancer

[0456] Patients with non-muscle invasive bladder cancer (NMIBC) are usually treated with cystectomy. Therefore, there is a need for new treatment options that can preserve the bladder.

[0457] CAIX is expressed on the luminal surface of the endocervical lining of the papillary structures in direct contact with the bladder lumen. The inventors performed a study aimed at ensuring the intravesical instillation of Zr-Doxilant in patients with NMIBC. 89 Pre-trial prospective study of intravesical radioactivity limitation and tumor targeting after Zr-Doxilant.

[0458] Patients received one 37 ± 10% MBq of Zr-Doxilant 89 Intravesical instillation of Zr-Doxilant (10 mg) and retention of urine for 2 hours. Then 4 PET / CT scans were performed, 3 one-step scans at the pelvis (H+2, day 1 and day 2) and one one-step scan from the skull to the middle of the thigh at H+4 to observe the evolution of intravesical radioactivity over time.

[0459] Blood samples were taken on day 1 to determine the number of radioactivity that can pass through the blood vessels. For all collections, a collection time of 10 minutes was used per bed. In 89 The gold standard was determined by a second cystoscopy and transurethral resection of the bladder (TURB) on the Zr-Doxilant PET / CT positive sites. Immunohistochemistry (IHC) was performed with an anti-CAIX antibody (Leica, clone TH22). Staining was evaluated with a semi-quantitative analysis (percentage and intensity of tumor cell expression) and the 89 The Zr-Doxilant PET / CT bladder pattern was compared to the degree of CAIX expression evaluated by IHC.

[0460] Results were obtained from 4 / 6 patients. Each patient was identified with recurrent pTaG3, despite previous multiple intravesical instillations of Bacillus Calmette-Guerin (BCG), a common intravesical immunotherapy used to treat bladder cancer.

[0461] 89 Zr-Doxilant PET / CT showed no extravasation outside the bladder. In 2 / 4 patients with positive IHC, one patient had uptake spots on the bladder wall confirmed by TURB with a corresponding recurrent lesion and the other patient had an inflammatory scar reaction. For the other two patients, no uptake was observed consistent with negative IHC. No adverse radiation contamination was observed during the procedure and no specific worker exposure was observed.

[0462] Results (as shown in Figure 5 and 6 indicated) show that intravesical instillation of 89 Zr-gemtuzumab showed localization of radioactivity in the bladder and indicated 89 Zr-gemtuzumab was useful for detection and imaging of the tumor in this patient set.

[0463] It is to be understood that the application disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the application.

Claims

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

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

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

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

5. The method of any one of claims 1 to 4, wherein the method does not require an additional in vitro method for detection, diagnosis, or imaging of the cancer.

6. The method of any one of claims 1 to 4, wherein the method is the only method required to achieve detection, diagnosis, or imaging of the cancer.

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

8. The method of any one of claims 1 to 7, wherein the agent for binding to CAIX is a small molecule, optionally selected from the group consisting of SLC-0111, SLC-149, SLC-0121, SLC-101, PMI-05, sulfonamide-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 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 of any one of claims 1 to 7, wherein the agent for binding to CAIX is a polypeptide.

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

12. The method of claim 11, wherein the antibody or antigen-binding fragment thereof is girentuximab, including 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. The method of any one of the preceding claims, wherein the detectable moiety of the agent is conjugated to the agent directly or through a chelator or linker.

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

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

17. The method of claim 16, wherein the radioisotope is selected from: gallium-67 and gallium-68 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).

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

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

20. The method of any one of claims 1 to 14, wherein the agent is selected from: 89 Zr-gemtuzumab ozogamicin, 123 I-gemtuzumab ozogamicin, 124 I-gemtuzumab ozogamicin or 131 I-gemtuzumab ozogamicin.

21. The method of any one of claims 1 to 14, wherein the agent is 89 Zr-gemtuzumab.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

48. The method of any one of claims 1 to 47, wherein the agent is a radiolabeled gemtuzumab, the radiolabeled gemtuzumab 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 method of any one of claims 35 to 48, wherein the radiolabeled gemtuzumab comprises gemtuzumab conjugated to a radioisotope selected from the group consisting of 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).

Images