KR20250124157A - Imaging reagents and methods - Google Patents

Abstract

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

Description

Imaging reagents and methods

The present invention relates to agents for use in in vivo imaging and detection of cancer, and methods of using the same.

Related applications

This application claims priority from Australian Provisional Application AU 2022903922, the contents of which are incorporated herein by reference in their entirety.

Medical imaging methods are often used to aid in the diagnosis and staging of various cancers. These methods can also be beneficial in eliminating or reducing the need for invasive diagnostic techniques (such as biopsy specimen collection), which are not always necessary and can lead to complications.

However, not all cancers can be successfully detected using standard medical imaging. Furthermore, while many imaging techniques can detect tumor masses, they cannot successfully distinguish between benign and malignant tissue.

The state of oncology management has evolved significantly in recent decades. Some types of cancer have benefited significantly from research advances in diagnostic and treatment options, resulting in improved morbidity and mortality rates for their patient populations. However, others have proven more elusive and continue to carry a grim prognosis for their patient populations.

As these latter categories of cancers exceed the limits of existing diagnostic and therapeutic modalities, innovation based on new approaches to oncology management is needed.

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

The reference to any prior art in the specification is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art would be understood and/or considered relevant and/or could reasonably be expected to be combined with other pieces of prior art by a person skilled in the art.

Summary of the invention

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

Accordingly, the present invention provides a method for in vivo imaging or detection of cancer in a subject in need thereof, the method comprising:

- A step of administering to a subject an agent for binding to CAIX expressed by cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,

- Step of detecting the agent in the target

, where cancer is

· Bladder cancer

· Breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER/PR-positive), and HER2-positive breast cancer)

· Cervical cancer

· Colorectal cancer

· Esophageal cancer (including esophageal squamous cell carcinoma (SCC) and 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 and small cell carcinoma)

· Ovarian cancer (including epithelial ovarian carcinoma)

· Pancreatic cancer (including pancreatic ductal adenocarcinoma), and

· Soft tissue sarcoma

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

The present invention also provides a method for diagnosing cancer in a subject in need thereof, the method comprising:

- A step of administering to a subject an agent for binding to CAIX expressed by cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject,

- A step for determining the presence or absence of an agent in a target object.

, where cancer is

· Bladder cancer

· Breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER/PR-positive), and HER2-positive breast cancer)

· Cervical cancer

· Colorectal cancer

· Esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophagogastric junction adenocarcinoma)

· Gastric cancer (including gastric adenocarcinoma)

· Glioblastoma multiforme

· Head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal and nasopharyngeal carcinoma)

· Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

· Lung cancer (including non-small cell and small cell carcinoma)

· Ovarian cancer (including epithelial ovarian carcinoma)

· Pancreatic cancer (including pancreatic ductal adenocarcinoma), and

· Soft tissue sarcoma

, and thereby determining the presence of said agent above background or standard levels indicates that the subject has said cancer, thereby diagnosing cancer in the subject.

The present invention also provides a method for generating an image of cancer, the method comprising:

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

- Step of detecting the agent in the target

, where cancer is

· Bladder cancer

· Breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER/PR-positive), and HER2-positive breast cancer)

· Cervical cancer

· Colorectal cancer

· Esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophagogastric junction adenocarcinoma)

· Gastric cancer (including gastric adenocarcinoma)

· Glioblastoma multiforme

· Head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal and nasopharyngeal carcinoma)

· Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

· Lung cancer (including non-small cell and small cell carcinoma)

· Ovarian cancer (including epithelial ovarian carcinoma)

· Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

· Soft tissue sarcoma

is selected from, thereby generating an image of the cancer.

The present invention also provides a method for generating an image of cancer, the method comprising:

- a step of injecting an agent for binding to CAIX expressed by a cancer in an effective amount, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent;

- Step for detecting the agent

, where cancer is

· Bladder cancer

· Breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER/PR-positive), and HER2-positive breast cancer)

· Cervical cancer

· Colorectal cancer

· Esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophagogastric junction adenocarcinoma)

· Gastric cancer (including gastric adenocarcinoma)

· Glioblastoma multiforme

· Head and neck cancer (including head and neck squamous cell carcinoma, hypopharyngeal and nasopharyngeal carcinoma)

· Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

· Lung cancer (including non-small cell and small cell carcinoma)

· Ovarian cancer (including epithelial ovarian carcinoma)

· Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

· Soft tissue sarcoma

is selected from, thereby generating an image of the cancer. Optionally, the detectable moiety is a radioisotope, and the detection comprises radiation detection, such as positron emission tomography (PET) imaging.

In any embodiment, the method of the present invention may further comprise allowing the agent to concentrate at a site and/or tissue in the subject where the CAIX antigen is found prior to detecting the agent.

Administration may be by any suitable means, preferably one that allows systemic administration of the agent (e.g., intravenous infusion) so that the agent accumulates at sites in the subject where CAIX is present on the cell surface. The mode of administration may depend on the nature of the agent for binding to CAIX. For example, in the case of antibodies for binding to CAIX, the agent is preferably administered by intravenous infusion. Labeled peptides or small molecules may be administered orally or by other means.

In any embodiment, the diagnostic methods of the present invention do not require additional in vitro diagnostics using a tissue biopsy or other biological sample obtained from the subject.

In certain embodiments, the agent comprises a first moiety for binding to CAIX and a second moiety for enabling detection of the agent in vitro.

In any embodiment, the moiety for binding to CAIX can be a small molecule, a peptide or a polypeptide (e.g., an antibody or antigen-binding fragment thereof).

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, sulfamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, -MIP-1486, MIP-1490, MIP-1504 (particularly ^99m Tc-HEHEHE-Z09781, ^99m Tc-MIP-1486, ^99m Tc-MIP-1490 or ^99m Tc-MIP-1504/5) and PHC-102.

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.

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

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 an antigen-binding fragment thereof.

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

In an alternative embodiment, the antibody for binding to CAIX may comprise BCA-356, BAY-794620 or SLC-0131.

Agents for use according to the methods of the present invention include a moiety that facilitates their detection. Any suitable detectable moiety for use in in vivo detection techniques may be used and will be known to those skilled in the art.

The detectable moiety may be directly linked to the moiety for binding to CAIX, or may be conjugated via 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.

In certain embodiments, the detectable moiety is a radioisotope. Examples of suitable isotopes include: gallium-67 and gallium-68 ( ⁶⁷ Ga and ⁶⁸ Ga), indium-111 ( ¹¹¹ In), iodine-123, iodine-124, or iodine-131 ( ¹²³ I, ¹²⁴ I, or ¹³¹ I), lutetium-177 ( ¹⁷⁷ Lu), technetium-99 ( ^99m Tc), yttrium-90 ( ⁹⁰ Y), and zirconium-89 ( ⁸⁹ Zr).

In any embodiment, the agent is an antibody for binding to CAIX, and the detectable moiety is optionally a radioisotope 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).

In any embodiment, the agent is a girentuximab antibody (including a chimeric or humanized version thereof) and the detectable moiety is optionally a radioisotope 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).

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

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

Other suitable detectable moieties include fluorescent labels and dyes. It will be appreciated that in any embodiment of the present invention, more than one detectable moiety may be utilized to maximize imaging or detection of the agent and thereby the CAIX-expressing tumor or cancer.

The present invention also provides an agent for CAIX, or a composition comprising the same, as described herein, for use in a detection, imaging method, for obtaining an image of a cancer, or for diagnosing a cancer.

In addition, the present invention provides an agent for CAIX as described herein, or a composition comprising the same, for use in a method for detecting, imaging or diagnosing cancer, or for obtaining an image of cancer as described herein.

Still further, the present invention provides a kit for use according to any of the methods described herein, comprising: an agent for binding to CAIX as described herein, and optionally, instructions for its use for the detection, imaging or diagnosis of cancer.

As used herein, except where the context requires otherwise, the term "comprises" and variations of the term, such as "comprising," "including," and "included," are not intended to exclude additional additives, components, integers, or steps.

Additional aspects of the present invention and further embodiments of the aspects described in the preceding paragraph will become apparent from the following description, which is provided by way of example only with reference to the accompanying drawings.

Figure 1: In vitro binding of radiolabeled DOTA-GmAb to various cell lines.
Figure 2: Representative images of mice receiving As-PC-1 (pancreatic cancer), FaDu (hypopharyngeal cancer) or HT-29 (colorectal cancer) tumor xenografts and subsequent injections with radiolabeled DOTA-GmAb.
Figure 3: Quantification of the percent injected dose at 24 and 72 hours post-injection of radiolabeled DOTA-GmAb. Closed circles = AsPC-1 radiolabeled DOTA-GmAb; closed boxes = FaDu radiolabeled DOTA-GmAb; closed triangles = HT-29 radiolabeled DOTA-GmAb.
Figure 4: Quantification of biodistribution results. The ex vivo biodistribution (i.e., the distribution of radiolabeled DOTA-GmAb observed in organs of mice after necropsy) was found to correlate with the in vivo biodistribution (i.e., the distribution of radiolabeled DOTA-GmAb observed in mice after whole-body imaging). Closed circles = AsPC-1 radiolabeled DOTA-GmAb; closed boxes = FaDu radiolabeled DOTA-GmAb; closed triangles = HT-29 radiolabeled DOTA-GmAb.
Figure 5: Imaging of bladder cancer. Day 0: Whole-body ⁸⁹ Zr-girentuximab scan: A : Coronal PET/CT fusion. B : Maximum intensity projection (MIP) visualization.
Figure 6: Imaging of bladder cancer. Day 2, A. ⁸⁹ Zr-girentuximab pelvic PET/CT fusion image: (arrows) Radiopharmaceutical uptake on different sides of the bladder wall. B. 3D representation of the bladder as a superimposed image.

Detailed description of the implementation example

The present invention relates to imaging and diagnosis of cancers that are difficult to diagnose due to a lack of robust oncological management options. Diagnostic and therapeutic innovations are needed to improve the morbidity and mortality rates of these oncological indications.

Detection of CAIX in the imaging and diagnosis of renal cell carcinoma is well known. However, prior to the present invention, it was unknown whether in vivo detection of CAIX could be used to successfully image and diagnose other solid tumors that express CAIX.

Although CAIX is typically associated with advanced disease, it is not known whether CAIX detection and/or imaging is feasible during the early stages of certain cancers, or whether it is only detectable in later stages. Furthermore, some cancers exhibit decreased expression as the disease progresses, making it unclear whether CAIX imaging is a useful tool for detecting these types of cancer.

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

For example, a 2006 study by Henrickx et al. (Cancer Biotherapy & Radiopharmaceuticals, 21:263-268) found that a radiolabeled antibody binding to CAIX was not suitable for imaging cholangiocarcinoma, despite the fact that cholangiocarcinoma has been shown to overexpress CAIX. Conversely, the same antibody is generally known to be useful for imaging and detecting CAIX-overexpressing renal cell carcinoma. Both cholangiocarcinoma and renal carcinoma are malignancies of epithelial cells and are characterized by increased expression of CAIX. Therefore, it is not understood why a CAIX-binding antibody is useful for imaging and detecting renal cell carcinoma but not cholangiocarcinoma.

In another example, a recent study published by Huizing et al. (2021, Physics and Imaging in Radiation Oncology , 145-150) found that ^{the 111} In-labeled F(ab')2 form of the CAIX-binding antibody girentuxumab was unable to distinguish between tumor and non-tumor cells and was therefore not useful for quantifying changes in CAIX expression. Notably, this result stands in stark contrast to our results reported herein, where ^{the 89} Zr-GmAb (in the form of a whole IgG antibody) was shown to be useful for imaging and detecting the same cancer cells in vivo.

Accordingly, the present invention is based on the discovery that a subset of cancers associated with increased CAIX expression can indeed be successfully detected and imaged using agents for binding to CAIX, wherein the agents comprise a detectable moiety.

General definition

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter are to be deemed to include one and plural (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the context clearly dictates otherwise. For example, reference to "a" includes one as well as two or more; reference to "an" includes one as well as two or more; reference to "the" includes one as well as two or more, and so on.

Those skilled in the art will appreciate that variations and modifications of the present invention beyond those specifically described herein are possible. It is to be understood that the present invention encompasses all such variations and modifications. The present invention also encompasses all steps, features, and compositions and compounds mentioned or illustrated herein, individually or collectively, and any and all combinations of any two or more of such steps or features.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is not limited in any way to the methods and materials described herein.

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

The present invention is not limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention.

Any example or embodiment of the invention herein is deemed to apply to any other example or embodiment of the invention unless specifically stated otherwise.

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

The term "and/or", e.g., "X and/or Y", is understood to mean "X and Y" or "X or Y" and is considered to provide explicit support for both or either meaning.

carbonic anhydrase IX

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

Cancer cells primarily express the plasma membrane-associated CA isoforms CAIX and CAXII, as well as intracellular CAs such as CAI and CAII. Among cancer-associated CAs, CAIX has received the most attention, as expression of this isoform in healthy tissues is restricted to epithelial cells of the stomach and digestive tract, but is strongly upregulated in renal cancer.

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

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

Preferably, the agent for binding to CAIX specifically interacts with the CAIX polypeptide. Specifically interacting (e.g., recognizing or binding) means that the agent, e.g., an antibody, has a greater affinity for CAIX compared to other polypeptides. In one embodiment, the agent interacts with (i.e., binds to or recognizes) the CAIX polypeptide, 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 may act at the protein level or the nucleic acid level. Examples of CAIX inhibitors that act at the protein level include, but are not limited to, peptides and anti-CAIX antibodies, as well as small organic molecules, preferably having a molecular weight of less than 500 g/mol, or functional fragments of these antibodies.

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

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

As used herein, the terms "specifically bind" or "specifically binds to" are intended to mean that an agent for use according to the invention reacts or associates with CAIX or a cell expressing it more frequently, more rapidly, for a greater duration, and/or with greater affinity than with alternative antigens or cells. For example, an antigen binding protein that binds to CAIX has a substantially greater affinity (e.g., 1.5-fold, or 2-fold, or 5-fold, or 10-fold, or 20-fold, or 40-fold, or 60-fold, or 80-fold, to 100-fold, or 150-fold, or 200-fold) than with other antigens.

Methods for assessing binding to proteins (e.g., CAIX) are well known in the art and are described, for example, in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). These methods typically involve immobilizing an agent (e.g., an antibody) and contacting it with a labeled target (in the case of antibodies, an antigen). After washing to remove non-specific binding proteins, the label, and consequently the amount of bound antigen, is detected. Of course, the antigen binding site can be labeled and the antigen immobilized. Panning-type assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.

Other standard methods for assessing binding to targets such as CAIX are also known in the art.

small molecules

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

In certain embodiments, the moiety for binding to CAIX is the small molecule/contrast dye PMI-05 (described in US2019/0192699A1, which is incorporated herein by reference).

In certain embodiments, the moiety for binding to CAIX is a small molecule sulfamide-nitroimidazole (described in Rami et al., (2013), J. Med. Chem, 56: 8512-8520, incorporated herein by reference).

In certain embodiments, the moiety for binding to CAIX is the small molecule JS-403 (described in WO 2010/089752 A1, which is incorporated herein by reference).

In certain embodiments, the moiety for binding to CAIX is the small molecule UB-TT220 (described in WO 2022/015955 A1, which is incorporated herein by reference).

In any embodiment, the moiety for binding to CAIX is a small molecule ^99m Tc-HEHEHE-Z09781 (Kim et al., (2017) Advanced Science, 4:1600471; Gebauer and Skerra (2009) Current Opin in Chem Biol, 13(3):245-55; Schardt et al., (2017) Mol Pharmaceutics, 14(4):1047-56; Tolmachev et al., (2008) Bioconjugate Chem, 19(8):1579-87; Liu et al., (2022) Analytical and Bioanalytical Chemistry, 414:1095-1104; Grindel et al., (2022) ACS Chem Biol, 17(6): 1543-55, incorporated herein by reference), ^99m Tc-MIP-1486, ^99m Tc-MIP-1490 (4-(2-bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide, 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)-1H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide, where X= n-butyloxy) (Hillier et al., (2012) Journal of Nuclear Medicine, 53(s1):217, incorporated herein by reference).

In certain embodiments, the moiety for binding to CAIX is the small molecule PHC-102 (described in WO 2015/114171 A1; WO 2018154517 A1; US 2014/0357650 A1; WO 2015/114171 A1, which are incorporated herein by reference).

peptides

In certain embodiments, the moiety for binding to CAIX is a peptide. As used herein, a peptide will be understood to comprise a chain of more than one amino acid residue. Typically, the 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. The peptide can have a length of at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 amino acids. Typically, peptides are less than or equal to about 40 amino acids in length, for example less than or equal to about 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids in length.

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., incorporated herein by reference). (2017) Eur J of Medicinal Chem, 136:52-62) or 3B-302.

In certain embodiments, the moiety for binding to CAIX is the peptide CAIX-P1 having the amino acid sequence YNTNHVPLSPKY (described in Askoxylakis et al., (2010), PLoS One, 5(12): e15962), optionally wherein the peptide is labeled with ¹²⁵ I or ¹³¹ I to enable its detection (although it will be appreciated that any suitable radiolabel or other detectable moiety may be used).

Polypeptides and antibodies

In a particularly preferred embodiment, the agent comprises a first moiety of an anti-CAIX antibody and/or a functional fragment of such an antibody. The fragment of the anti-CAIX antibody may have essentially the same CAIX-binding and/or inhibitory activity as the full-length anti-CAIX antibody and/or is an epitope-binding fragment of the anti-CAIX antibody.

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

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

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

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

In any embodiment, the moiety for binding to CAIX is in the form of the antibody BAY-794620, or an antigen-binding fragment thereof (described in 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).

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

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 may be a chimeric or humanized G250 antibody. In some embodiments, the antigen binding protein that binds or specifically binds to CAIX is as described in any of the following, the entire contents of each of which are incorporated herein by reference: WO 2002/062972 A2 (US 2004/0219633 A1), WO 2004/002526 A1 (US 7,632,496 B2), WO 2006/002889 A2 (US 7,691,375 B2), WO 2009/056342 A1 (US 2014/0017252 A1), WO 2011/032973 A1 (US 2012/0207672 A1), and WO 2014/128258 A1 (US 10,620,208 B2), or WO 2021/000017 A1.

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

A particularly preferred antibody is cG250, preferably girentuximab (INN). Another particularly preferred embodiment is the monoclonal antibody G250 produced by the hybridoma cell line DSM ACC 2526. Antibody cG250 is an lgG1 kappa light chain chimeric version of the original murine monoclonal antibody mG250.

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

In particularly preferred embodiments, the antibody is ⁸⁹ Zr-girentuximab (i.e., ⁸⁹ Zr-cG250), ¹²³ I-, ¹²⁴ I-, or ¹³¹ I-girentuximab, or ¹⁷⁷ Lu-girentuximab.

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

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

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

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

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - Linker - FR1a - CDR1a - FR2a - CDR2a - FR3a - CDR3a - FR4a

Here:

FR1, FR2, FR3 and FR4 are framework regions, respectively;

CDR1, CDR2, and CDR3 are complementarity determining regions, respectively;

FR1a, FR2a, FR3a and FR4a are framework regions, respectively;

CDR1a, CDR2a, and CDR3a are complementarity determining regions, respectively;

wherein the sequence of any of the complementarity determining regions has an amino acid sequence as set forth in Table 1 below. Preferably, the framework region has an amino acid sequence as set forth in Table 1 below, including amino acid mutations at specific residues that can be determined by aligning various framework regions derived from each antibody. CDR1, CDR2 and CDR3 may be sequences from the VH, and CDR1a, CDR2a and CDR3a may be sequences from the VL, or CDR1, CDR2 and CDR3 may be sequences from the VL, and CDR1a, CDR2a and CDR3a may be sequences from the VH.

In any embodiment, the antigen or antigen-binding fragment thereof comprises:

(i) a complementarity determining region (CDR) 1 comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 1, and 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 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least A VH comprising a CDR2 comprising a sequence that is 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, 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;

(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 a sequence set forth in any of SEQ ID NOs: 4, 20, 36, 52, or 68;

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

(iv) a VL comprising a sequence 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 a sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164;

(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;

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

(vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 81, a CDR2 comprising the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising the sequence set forth in SEQ ID NO: 83;

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

(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

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

In a further embodiment, the antibody or antigen-binding fragment thereof comprises:

(i) 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 set forth in any 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 set forth in any 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 set forth in any of SEQ ID NOs: 11, 27, 43, 59, or 75; A VH comprising 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 set forth in any of SEQ ID NOs: 12, 28, 44, 60, or 76; and

(ii) 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 set forth in any 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 set forth in any 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 set forth in any of SEQ ID NOs: 91, 107, 123, 139, 155, or 171; A VL comprising 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 set forth in any of SEQ ID NOs: 92, 108, 124, 140, 156, or 172.

In a further embodiment, the antibody or antigen-binding fragment thereof comprises:

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

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

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 N-terminal to C-terminal or C-terminal to N-terminal order) any one of SEQ ID NOs: 4, 20, 36, 52, or 68 and/or any one of SEQ ID NOs: 84, 100, 116, 132, 148, 164.

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

(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 set forth in SEQ ID NO: 4, 20, 36, 52 or 68; and/or

(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 the sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

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

(i) single chain Fv fragment (scFv);

(ii) dimeric scFv (di-scFv); or

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

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

(i) Diabody;

(ii) Triabadi;

(iii) tetrabodies;

(iv) Fab;

(v) F(ab')2;

(vi) Fv; or

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

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

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

An antibody or antigen-binding fragment thereof as described herein may comprise a human constant region, for example, an IgG constant region, such as an IgG1, IgG2, IgG3, or IgG4 constant region, or a mixture thereof. For antibodies or proteins comprising a VH and a VL, the VH may be linked to a heavy chain constant region and the VL may be linked to a light chain constant region.

In one example, an antibody or antigen-binding fragment thereof as described herein comprises a heavy chain constant region comprising a stabilized heavy chain constant region comprising a mixture of sequences that have or do not have a C-terminal lysine residue, either completely or partially.

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

The functional characteristics of its antigen-binding fragments as described herein will be considered to apply to antibodies as described herein.

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

Table 1: Summary of amino acid and nucleotide sequences of preferred CAIX-binding antibodies.

In a further embodiment, the moiety for binding to CAIX is BCA-356, a bispecific antibody comprising an affinity matured humanized anti-CAIX antibody linked to an attenuated subunit of IL-12, each of which is fused at its C-terminus to a heavy chain of an anti-CAIX antibody via a linker in a "knobs in hole" configuration (described in Nair et al., Journal for ImmunoTherapy of Cancer, 10:S2).

In a particularly preferred embodiment of the methods and uses described herein, the agent for binding to CAIX is ⁸⁹ Zr -Girentuximab, ¹²⁴ I-girentuximab, ¹⁷⁷ Lu-girentuximab, or 1 ¹¹ In-girentuximab-IRDye800CW (e.g., as described in Stroet et al., (2022), Cancers 14: 861, incorporated herein by reference), G250RIT (when labeled with an appropriate detectable moiety), or ⁹⁰ Y-DOTA-cG250.

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

Invariant region

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

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

The neonatal Fc receptor (FcRn) is crucial for the metabolic fate of IgG class antibodies in vivo. FcRn functions to retrieve IgG from the lysosomal degradation pathway, thereby reducing clearance and increasing its half-life. It is a heterodimeric protein composed of two polypeptides: the 50 kDa class I major histocompatibility complex-like protein (a-FcRn) and the 15 kDa p2-microglobulin (β2ηι). FcRn binds with high affinity to the CH2-CH3 region of the Fc region of IgG class antibodies. The interaction between antibodies of class IgG and FcRn is pH dependent and occurs in a 1:2 stoichiometry, i.e. one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc-region polypeptides (see, e.g., Huber, A.H. et al., J. Mol. Biol. 230 (1993) 1077-1083).

Therefore, the in vitro FcRn binding properties/characteristics of IgG are indicative of its in vivo pharmacodynamic properties in the bloodstream. The interaction between the Fc region of IgG class antibodies and FcRn involves different amino acid residues in the heavy chain CH2- and CH3-domains.

Several mutations are known that affect FcRn binding and therefore its half-life in the bloodstream. Fc-domain residues important for mouse Fc-domain-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 shown to be important for the interaction of human Fc-domain with murine FcRn (Kim, J.K. et al., Eur. J. Immunol. 29 (1999) 2819-2885).

More specifically, the antibody or antigen-binding protein may comprise one or more amino acid substitutions that reduce the half-life of the protein. For example, the antibody or antigen-binding fragment thereof may comprise an Fc region comprising one or more amino acid substitutions that reduce the affinity of the Fc region for the neonatal Fc region (FcRn).

Desirable transformation

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 compared to a wild-type antibody of class IgG, wherein the one or more amino acid substitutions decrease the affinity of the antibody for neonatal Fc receptor (FcRn), thereby decreasing the serum half-life of the modified antibody compared to a wild-type antibody of class IgG.

In one embodiment, the one or more amino acid substitutions are selected from substitutions in the heavy chain constant region 2 (CH2) of the IgG molecule, thereby decreasing the affinity of the IgG molecule for FcRn. Alternatively, the one or more amino acid substitutions can be substitutions in the heavy chain constant region 3 (CH3) of the IgG molecule, thereby decreasing the affinity of the IgG molecule for FcRn. Still further, the amino acid substitutions can comprise at least one substitution within the CH2 region and at least one substitution within the CH3 region of the IgG molecule, thereby decreasing the affinity of the IgG molecule for FcRn.

In certain preferred embodiments, the one or more amino acid substitutions may be at one or more of residues His310, His433, His435, His436, or Ile253 of an IgG. Preferably, the amino acid substitution comprises a substitution within the heavy chain constant region at position His310 or His435. More preferably, the amino acid substitution that reduces the affinity of the antibody for FcRn is a substitution at both His310 and His435.

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

Also in a preferred embodiment, amino acid residues 310 and/or 435 from the heavy chain constant region of the antibody having a constant region substantially identical to a naturally occurring class IgG antibody are any amino acid that is not histidine and that decreases the affinity of the constant region for FcRn. For example, the amino acids at residues 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.

Amino acid substitutions may include substitutions of histidine residues with alanine, glutamine, glutamic acid, or aspartic acid. Preferably, the amino acid substitution at His310 is alanine. Preferably, the amino acid substitution at His435 is glutamine. Preferably, the amino acid substitution at Ile253 is alanine.

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

Additionally, antibodies or fragments thereof for use according to the present invention may be modified to include one or more mutations that alter the affinity of the antibody for any one or more Fc gamma receptors. For example, one or more amino acid modifications alter the affinity of an antibody constant domain, Fc region, or Fc gamma receptor binding fragment for any one or more Fc gamma receptors.

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 an effector response (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.

In one example, the Fc region of the constant region has a reduced ability to induce effector function, for example, compared to a native or wild-type human IgG1 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 are described herein.

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

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. The sequence of a suitable IgG4 Fc region will be apparent to the skilled person and/or is available in publicly available databases (e.g., those available from the National Center for Biotechnology Information).

In one example, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce the tendency for Fab arm exchange or Fab arm exchange or the formation of half-antibodies or anti-antibodies. "Fab arm exchange" refers to a type of protein modification to human IgG4 in which an IgG4 heavy chain and attached light chain (half-molecule) are exchanged for a heavy chain-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two distinct Fab arms that recognize two distinct antigens (making it 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. "Anti-antibodies" are formed when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

In one example, a stabilized IgG4 constant region includes a proline at position 241 of the hinge region according to Kabat's system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and/or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system. In human IgG4, this residue is typically serine. If the serine is replaced with proline, the IgG4 hinge region includes the sequence CPPC. In this regard, the skilled person will recognize that the "hinge region" is the proline-rich portion of an antibody heavy chain constant region that connects the Fc and Fab regions, which imparts mobility to the two Fab arms of the antibody. The hinge region includes cysteine residues that participate in inter-heavy chain disulfide bonds. It is generally defined as the stretch from Glu226 to Pro243 of human IgG1 (or from Glu216 to Pro230 using the EU index) according to Kabat's numbering system. The hinge region of other IgG isotypes can be aligned with the IgG1 sequence by positioning the first and last cysteine residues that form the inter-heavy chain disulfide (S-S) bond at the same position (see, e.g., WO2010/080538).

In an alternative embodiment, one or more amino acid modifications that decrease affinity for the FcRn receptor also decrease affinity for the Fc gamma receptor. The modified antibody or antigen-binding fragment thereof may further comprise one or more amino acid substitutions compared to a wild-type antibody of class IgG, wherein the amino acid substitutions further decrease the affinity of the antibody for one or more Fc gamma receptors.

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

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

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

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

In another example, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region." For example, the Fc region is an IgG1 Fc region 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 IgG1 Fc region comprising a deletion 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). Additional examples of non-immunostimulatory Fc regions are described, for example, in the following references: Dall'Acqua et al., J Immunol. 177: 1129-1138 2006; and/or Hezareh J Virol ;75: 12161-12168, 2001).

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

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

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

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

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

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 comprises 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 comprises a sequence set forth in SEQ ID NO: 116, 132 or 148.

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

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

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

detectable moiety

Skilled practitioners will be familiar with standard methods for conjugating a detectable moiety to an agent for binding to CAIX.

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

In certain embodiments, the detectable moiety is a radioisotope. Examples of suitable isotopes include: gallium-67 and gallium-68 ( ⁶⁷ Ga and ⁶⁸ Ga), indium-111 ( ¹¹¹ In), iodine-123, iodine-124, or iodine-131 ( ¹²³ I, ¹²⁴ I, or ¹³¹ I), technetium-99 ( ^99m Tc), and zirconium-89 ( ⁸⁹ Zr). As used herein, the term radionuclide may be used interchangeably with the term radioisotope.

It will be appreciated that radioisotopes may be conjugated to polypeptides (e.g., antibodies) either directly (via a chelating agent or a prosthetic group or linker) or indirectly via binding to single or multiple amino acid residues within the protein (e.g., halogenation of a tyrosine residue).

In alternative embodiments, a chelating agent or linker may be used to conjugate the detectable 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'"-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1, 4,7,10-tetraazacyclododecane-NN',N"(N'"-tetraacetic acid, also known as tetraxetane), TCMC (tetra-primary amide of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), NOTA (1,4,7-triazacyclononane-triacetic acid), Diamsar (3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine), DTPA (pentetic acid or diethylenetriaminepentaacetic acid), CHX-A"-DTPA ([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid), TETA (1,4,8,11-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-squaramide) and HOPO (3,4,3-(LI-1,2-HOPO) or other chelating agents 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]triazanonan-1-yl} acetic acid), 5p- C -NETA (2-({1-[4,7-bis(carboxymethyl)-1,4,7-triazanonan-1-yl]-7-(4-nitrophenyl)heptan-2-yl}(carboxymethyl) (amino)acetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and NODA (1,4,7-triazacyclononane-1,4-diacetic acid).

In certain non-limiting embodiments discussed below, a chelating group may be used to attach ¹⁸ F or ¹⁹ F complexed with a metal, such as aluminum, to provide an alternative modality for imaging, detection, and/or diagnosis. It is anticipated that fluorescently-labeled molecules may be more useful in intraoperative procedures, and ¹⁸ F-labeled molecules may be more useful for preoperative or postoperative imaging, detection, and/or diagnosis of diseased tissue.

The agent may be modified to contain a sulfhydryl group for attachment of a maleimide-modified fluorescent probe. Alternatively, a bifunctional cross-linking agent, or a fluorescent dye conjugated to another reactive species, may be used to attach the fluorescent probe to a different group on the agent for CAIX binding. For example, DYLIGHT® 488 and DYLIGHT® 800 are available as amine-reactive dyes derivatized with NHS esters for primary amine labeling (product numbers 46402 and 46421, Thermo Electric, Rockford, Ill.). Those skilled in the art will recognize that the fluorescent probe used is not limited and that other DYLIGHT® dyes or alternative fluorescent probe molecules known in the art may be used in the claimed methods and compositions.

In certain embodiments, a peptide or protein (e.g., an antibody) for binding to CAIX can be conjugated to a fluorescent probe (forming an immunoconjugate). Methods for covalently attaching fluorescent probes and other functional groups are well known in the art, and any such well-known methods can be utilized. For example, the fluorescent probe can be attached to the hinge region of the reduced antibody component via disulfide bond formation or sulfhydryl-maleimide interactions. Alternatively, such agents can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2-paradithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for such conjugations 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,” MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pp. 60-84 (Cambridge University Press 1995).

Alternatively, the fluorescent probe can be conjugated via a carbohydrate moiety within 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. Pat. No. 5,057,313 (the Examples sections of which are incorporated herein by reference). A general method involves reacting an antibody component having an oxidized carbohydrate moiety with a fluorescent probe having at least one free amine functional group. This reaction creates an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

If the antibody used as the antibody component of the immunoconjugate is an antibody fragment, the Fc region may be absent. However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full-length antibody or antibody fragment. See, e.g., Leung et al., J. Immunol. 154: 5919 (1995); U.S. Patent Nos. 5,443,953 and 6,254,868 (the Examples sections of which are incorporated herein by reference). Engineered carbohydrate moieties are used to attach functional groups to antibody fragments.

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

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 utilize ring strain instead of copper catalysis to catalyze [3+2] azide-alkyne cycloaddition reactions. For example, cyclooctyne is an eight-carbon ring structure containing an internal alkyne bond. The closed ring structure induces significant bond angle strain in acetylene, which is highly reactive with the azide group to form triazoles. Therefore, cyclooctyne derivatives can be used in copper-free click reactions.

Another type of copper-free click reaction was reported by Ning et al. (2010, Angew Chem Int Ed 49:3065-68), which involves a strain-catalyzed alkyne-nitrone cycloaddition. To overcome the slow kinetics of the original cyclooctyne reaction, an electron-withdrawing group is attached adjacent to the triple bond. Examples of such substituted cyclooctynes include difluorinated cyclooctynes, 4-dibenzocyclooctynes, and azacyclooctynes. An alternative copper-free reaction involved a strain-catalyzed alkyne-nitrone cycloaddition that produced N-alkylated isoxazolines. The reaction was reported to have very fast kinetics and was used in a one-pot, three-step protocol for the site-specific modification of peptides and proteins. Nitrones were prepared by condensing 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 attach chelating moieties to antibodies or other CAIX-binding molecules in vitro.

In certain embodiments, the agent may comprise a peptide or protein (e.g., an antibody) covalently bonded to a radioisotope ¹²⁴ l. The isotope may be a positron emitter, which may be attached to the antibody, as described, for example, 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.

In certain embodiments, radiolabeling of proteins or antibodies is achieved by covalent iodination, particularly with an iodogene reagent (1,3,4,6-tetrachloro-3a,6a-diphenyl glycoluril). Iodogene labeling is a solid-phase oxidation method similar to the chloramine-T method, but is generally considered milder because the reaction occurs on the surface of the oxidizing agent, minimizing exposure of the substrate (Salacinzki, P.R.P. et al., Anal. Biochem. 117:136 (1981)).

Chelaters having radioactive metals and other halogenated radioisotopes can be bound to proteins or antibodies via one or more amino acid residues or reactive moieties in the protein/antibody, including but not limited to one or more lysine residues, tyrosine residues or thiol moieties.

In another example, the protein or antibody may be conjugated to a bifunctional linker, e.g., bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, or any amine or thiol-modifying chemistry known in the art.

Skilled artisans will be familiar with standard methods for conjugating chelating agents to proteins, including antibodies and their derivatives and fragments. Furthermore, skilled artisans will be familiar with approaches for selecting relevant chelating agents for pairing with radiometals, such as those described, for example, in Chem. Soc. Rev., 2014, 43, 260, which is incorporated herein by reference.

In any embodiment, the detectable moiety can be a fluorescent dye, such as (but not limited to) those described in US 20150086482, which is incorporated herein by reference.

In any embodiment, the fluorescent dye (which may also be referred to as a fluorescent probe) may be selected from: Alexa 350, Alexa 430, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein, 5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptate, europium trisbipyridine diamine, europium cryptate or chelate, diamine, dicyanine, La Jolla blue dye, allophycocyanin, allophycocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin, phycoerythrin R, REG, rhodamine green, rhodamine Isothiocyanates, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), tetramethylrhodamine, and Texas Red.

Administration of the agent

The skilled person will appreciate that the dosage of an agent for use according to the methods of the present invention will vary depending on various factors, including the age, sex, height and weight of the subject to whom the agent is administered, and also depending on the agent.

When the agent is an antibody for binding to CAIX, the antibody is preferably administered or injected into the subject in a dose of about 1 mg to about 50 mg, preferably in a dose of about 5 mg to about 20 mg, and more preferably in a dose of about 10 mg. The specific activity of the radiolabeled antibody is preferably about 15 to about 20 MBq/mg, more preferably about 18 to about 19 MBq/mg.

In certain embodiments, the agent for binding to CAIX is a radiolabeled girentuximab antibody, and the antibody is administered as a large dose of about 10 mg of girentuximab by slow infusion.

Antibodies are typically administered as pharmaceutical compositions with a pharmaceutically acceptable carrier, e.g., saline, optionally including a protein stabilizer such as human serum albumin (HSA). Antibodies are preferably administered by infusion.

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

Detection method

It will be appreciated that the method for detecting or imaging an agent for use according to the present invention will vary depending on the nature of the detectable moiety of the agent.

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

Examples of in vivo methods for determining the presence or expression of CAIX within 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 may involve the use of CAIX-binding molecules conjugated to radioisotopes to enable non-invasive imaging of CAIX-expressing tissues and tumors.

Thus, if the detectable moiety is a radioisotope, the method will include determining the radiation for the subject to which the agent has been administered.

In the methods described above, the in vivo detection step may be local imaging at a specific site, such as (but not limited to) a site of expected or probable solid tumor growth, or whole body imaging.

For SPECT, the agent for binding to CAIX typically includes a detectable agent in the form of a gamma-emitting radioisotope (radionuclide), usually administered by injection into the bloodstream. Typical gamma-emitting radioisotopes for use in SPECT include ^99m Tc (technetium), ¹²³ I or ¹³¹ I (iodine), and ⁶⁸ Ga (gallium).

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

Optionally, the detection method includes PET/CT imaging or PET/MRI scanning.

After administration of the agent (preferably by injection), it may be practical to wait a period of time to allow the agent to accumulate at the site of the cancer cells expressing the tumor. Typically, the period of time will be at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or 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 methods described herein) is typically no more than about 10 days, or no more than about 15 days, or no more than about 20 days.

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

When the detectable moiety is a fluorescent probe or dye, detection of the moiety can be accomplished using fluorescence imaging, including during intraoperative, intravascular, or endoscopic procedures, as described in U.S. Patent Nos. 4,932,412; 6,096,289; 6,387,350; and 7,201,890; the Example sections of each of the cited patents being incorporated herein by reference. Such imaging methods can be useful, for example, for imaging the distribution of tumor tissue to facilitate its removal. Fluorescence imaging can also be useful for diagnostic purposes, for example, for distinguishing between malignant, benign, and hyperplastic tissue.

Cancer detected or diagnosed

The present invention provides a method for identifying or imaging cancer in vivo. This method is expected to be useful for diagnosing cancers expressing CAIX, preferably without the need for additional invasive techniques (e.g., biopsy collection and testing) to confirm the diagnosis.

Thus, in a preferred embodiment, the method of the present invention enables the diagnosis of any cancer mentioned herein as the sole, primary or main method of cancer diagnosis, and preferably without the need for additional invasive methods, including biopsy-related methods.

The method of the present invention is also expected to be useful in determining the success of cancer treatment or the stage of cancer progression. Furthermore, this method offers the advantage of providing a non-invasive means for assessing cancer in a subject.

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

The methods of the present invention are particularly useful in the imaging, detection and/or diagnosis of previously unidentified cancers using in vivo imaging techniques that utilize agents for binding to CAIX.

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

In certain embodiments, the cancer being detected, imaged, or diagnosed is breast cancer. High levels of CAIX have been previously reported in breast cancer, and CAIX expression has also been reported to be associated with chemotherapy resistance or treatment success. These observations have been ongoing for decades, and until now, no diagnosis using a CAIX-binding imaging agent has been reported for this patient group.

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

In any embodiment, the breast cancer can be hormone receptor positive breast cancer, such as ER positive, PR positive, or ER&PR positive. In any embodiment, the breast cancer can be HER2 positive (including HER2 and hormone receptor positive breast cancer).

In certain embodiments, the cancer detected, imaged, or diagnosed is not breast cancer.

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

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

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

In any embodiment, the cancer being detected, imaged, or diagnosed is gastric cancer (including gastric adenocarcinoma). A subject being diagnosed, imaged, or detected for gastric cancer may exhibit one or more symptoms selected from the following: dysphagia, abdominal pain, abdominal distension after eating a small amount of food, loss of appetite, indigestion, nausea and vomiting, fatigue, and black stool. A subject considered at risk for gastric cancer may have a family history of the disease or one or more genetic markers considered to be associated with an increased risk of gastric cancer.

In any embodiment, the cancer being detected, imaged, or diagnosed is glioblastoma multiforme. A subject being diagnosed, imaged, or detected with glioblastoma may exhibit one or more symptoms, including impaired vision, hearing, balance, coordination, muscle strength, and reflexes, nausea, vomiting, seizures, or other neurological symptoms.

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

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

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

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

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

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

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

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

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

Imaging or diagnosis of cancer will typically be assessed by qualitatively assessing the detection of the agent after administration and its detection compared to conventional imaging. Quantitative assessments can be performed on a lesion-by-lesion basis, including standardized uptake values (SUVs) (SUVmax and SUVmean), lean mass-adjusted SUV (SUL), metabolic tumor volume (MTV), and tumor-to-background ratio (TBR).

Tumor-to-background ratio (TBR) will typically be defined as the ratio of the lesion standardized uptake value (SUVmax) to a reference region SUV (e.g., liver, blood pool). Comparisons will be made between the number, size, and other characteristics of lesions detected by standard imaging modalities, including PET scans and high-resolution CT/MRI, as well as other available patient-specific imaging modalities (depending on tumor type, lesion type, and indication).

Qualitative visual analysis (the presence or absence of localized agent uptake, as seen on contrast-enhanced CT, MRI, or FDG PET/CT) can be used to assess the agreement in tumor lesion detection between agent-specific PET/CT and conventional imaging. The RECIST 1.1 criteria for conventional imaging can be used as a primary tool for comparing agreement with PET.

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

Example

Example 1: Clinical Trial Protocol

The trial involved the assessment of CAIX expression in a subset of solid tumors using ⁸⁹ Zr-labeled girentuximab deferoxamine PET/CT imaging.

Primary objective

To noninvasively assess CAIX tumor expression in different solid tumors using ⁸⁹ Zr-girentuximab PET/CT imaging. No formal imaging studies have been performed on the uptake of ⁸⁹ Zr-girentuximab by these tumor types.

Primary endpoint: Qualitative (yes/no) and quantitative assessment of 89Zr-girentuximab uptake compared with conventional imaging. Descriptive statistics for each tumor type are reported. Lesion-specific analyses include SUVmax, SUVmean, SUL (lean-mass adjusted SUV), tumor-to-background ratio (TBR), and metabolic tumor volume (MTV).

Secondary objective

To evaluate the tolerability and safety of ⁸⁹ Zr-girentuximab administration in patients with different solid tumors.

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

3rd objective

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

Tertiary endpoint: To assess the correlation between the standardized uptake value (SUV) of ⁸⁹ Zr-girentuximab and CAIX histological expression by comparing the semi-quantitative data of 89 Zr-girentuximab with immunohistochemistry (IHC) results of biopsy or surgical samples (and corresponding tissue samples) from the biopsy/resection tumor at that site within 90 days before dosing or 90 days after ⁸⁹ Zr-girentuximab imaging.

All patients are followed for safety until their EOS visit (days 15-25).

Overall study design

To conduct an open-label, non-randomized study to evaluate the expression of CAIX using ⁸⁹ Zr-girentuximab PET/CT imaging in different tumor types and to assess the feasibility of targeting CAIX for potential diagnostic and therapeutic applications.

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

The study involved a single administration of ⁸⁹ Zr-girentuximab (37 MBq [1 mCi] ± 10%, containing a bulk dose of 10 mg of girentuximab).

PET/CT imaging is performed 5 ± 2 days after administration. Image data analysis of PET/CT imaging is performed by a nuclear medicine reader to assess tumor uptake of ⁸⁹ Zr-girentuximab in a lesion-by-lesion analysis of up to the 10 most active lesions, and according to RECIST 1.1 conventional imaging.

Qualitative visual analysis (presence or absence of localized ⁸⁹ Zr-girentuximab uptake as seen on contrast-enhanced CT, MRI, or FDG PET/CT) was used to assess the agreement in tumor lesion detection between 89 Zr-girentuximab PET/CT and conventional imaging. Lesions revealed by 89 Zr-girentuximab alone are described.

Tissue samples (from patient biopsy or surgery) are collected when possible and sent to a central laboratory for CAIX expression analysis.

The study evaluation is conducted as shown in the table below:

¹ Obtained from outside the study within 28 days prior to day 0

² Only when clinically indicated

³ Remote visit (phone follow-up)

⁴ Pre-study procedures performed within 30 days of Day 0

⁵ If a patient cannot undergo PET/CT, if CT is contraindicated, or if PET/MRI may be a preferable option for the patient, PET/MRI may be performed instead of PET/CT.

administration

The ⁸⁹ Zr-girentuximab dose used here (37 MBq [1 mCi] ± 10%, containing a bulk dose of 10 mg of girentuximab) is consistent with the dosing regimen of 89 Zr-girentuximab in an ongoing phase 3 clinical trial and has been shown to enable PET imaging 4-7 days post-dose by Merkx et al. (2021).

⁸⁹ Zr-girentuximab is a radiolabeled chimeric monoclonal antibody (INN name: Zirconium Zr ⁸⁹ girentuximab deferoxamine. Girentuximab is a chimeric monoclonal antibody (INN: girentuximab, synonyms: cG250, TLX250) with specificity for CAIX (carbonic anhydrase 9) antigen, radiolabeled with positron-emitting radio-metal zirconium- ⁸⁹ via NSuc-DFO-TFP-ester (DFO-TFP), which is linked to a lysine residue of girentuximab to yield 89 Zr-DFO-girentuximab.

⁸⁹ Zr-girentuximab is formulated as a solution for intravenous administration at a nominal dosage strength of 37 MBq (±10%) (1 mCi±10%) containing a total of 10 mg of girentuximab for a single intravenous use. ^{The 89} Zr-girentuximab solution for IV administration is supplied in glass vials or syringes (depending on the geographic region) in appropriate packaging (lead-shielded containers with radioactive warning symbol in accordance with radioactive constraint requirements).

The compound is administered as a single dose of ⁸⁹ Zr-girentuximab (37 megabecquerel ± 10%, [1 mCi ± 0.1 mCi] containing a bulk dose of 10 mg of girentuximab) by slow intravenous infusion over 3 minutes through a single peripherally placed intravenous cannula. The injection volume is approximately 10 ml, depending on the activity of the infusion.

Inclusion criteria

All participants will meet the following criteria:

1. Informed consent voluntarily provided in writing.

2. Male or female aged ≥18 years at the time of consent.

3. Have the capacity to understand the study and be willing and able to comply with all protocol requirements.

4. Participants must have a histologically or cytologically proven solid tumor of the following types (but not limited to):

· Cervical cancer

· Colorectal cancer

· Esophageal cancer (esophageal SCC and esophagogastric junction adenocarcinoma)

· Gastric cancer (gastric adenocarcinoma)

· Glioblastoma multiforme

· Head and neck cancer (head and neck SCC and nasopharyngeal carcinoma)

· Liver cancer (cholangiocarcinoma and hepatocellular carcinoma)

· Lung cancer (non-small cell and small cell)

· Ovarian cancer (epithelial ovarian carcinoma)

· Pancreatic cancer (pancreatic ductal adenocarcinoma)

· Soft tissue sarcoma

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

6. Participants agree not to participate in any other intervention study while participating in this study, as defined by signing the Informed Consent Form (ICF) by the completion of the final study visit.

7. A negative serum pregnancy test result and a negative urine pregnancy test result within 24 hours prior to receiving the investigational product are required for screening in female patients of childbearing potential. Female patients of non-childbearing potential must provide evidence by meeting one of the following criteria at screening:

· Postmenopausal, defined as being over 50 years of age and having had amenorrhea for at least 12 months after discontinuation of all exogenous hormone therapy.

· Women younger than 50 years are considered postmenopausal if they have been amenorrheic for more than 12 months after discontinuation of exogenous hormone therapy and their luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels are in the institution's postmenopausal range.

· Documentation of irreversible surgical sterilization by hysterectomy, bilateral oophorectomy, or bilateral salpingectomy (but not fallopian tube ligation).

8. For all participants, agree to use double-barrier contraception for at least 42 days after administration of ⁸⁹ Zr-girentuximab.

Exclusion criteria

Patients will be excluded from the trial if they meet one or more of the following criteria:

1. Exposure to murine or chimeric antibodies within the past 5 years.

2. Previous administration of any radionuclide within 10 half-lives (i.e., within 10 half-lives of day 0) prior to the intended administration of ⁸⁹ Zr-girentuximab.

3. Exposure to any CAIX targeting compound (diagnostic/therapeutic) within the past 3 months

4. Serious non-malignant disease (e.g., psychiatric, infectious, autoimmune, or metabolic) that, in the judgment of the investigator, may interfere with the study purpose or the safety or compliance of the subject.

5. Any clinically significant abnormality detected during screening laboratory tests or physical examination that, in the investigator's opinion, would negatively impact the participant's ability to participate in the study. The principal investigator will assess patient eligibility for inclusion based on pathology and tumor type.

6. Mental impairment that may impair the ability to provide informed consent and comply with the requirements of the study.

7. Exposure to any antineoplastic treatment within 14 days of the planned dosing date of ⁸⁹ Zr-girentuximab (i.e., within 14 days of Day 0).

8. Pregnant or breastfeeding women.

9. Known allergy, hypersensitivity, or intolerance to girentuximab, DFO (desferrioxamine), or any of the components of the investigational agent.

10. Renal failure with a glomerular filtration rate (GFR) ≤ 45 milliliters/min/1.73 m2

11. Vulnerable patients (e.g., in custody).

Validity assessment

Imaging is based on the ability of PET/CT imaging with ⁸⁹ Zr-girentuximab to noninvasively assess CAIX tumor expression in patients. After a single dose of ⁸⁹ Zr-girentuximab on day 0, a whole-body PET/CT scan is performed on days 5 ± 2 post-dose according to the table above and the imaging manual. Patients with metastatic disease (suspected or confirmed) may undergo an optional additional whole-body PET/CT scan if clinically indicated (e.g., if the tumor-to-background ratio makes identification of tumor lesions difficult and is expected to improve).

Tumor uptake of ^Zr -girentuximab will be qualitatively assessed (yes/no) in up to the 10 most active lesions in each patient compared to conventional imaging. Quantitative assessments will be performed on a lesion-by-lesion basis, including SUVmax, SUVmean, SUL, MTV, and TBR.

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

Tumor-to-background ratio (TBR) will be defined as the ratio of lesion SUVmax to reference region SUV (liver, blood pool, etc.). Comparisons will be made between the number, size, and other characteristics of lesions detected by standard imaging modalities, including ⁸⁹ Zr-girentuximab PET scans and high-resolution CT/MRI, as well as other available patient-specific imaging modalities (depending on tumor type, lesion type, and indication).

Qualitative visual analysis (presence or absence of localized ⁸⁹ Zr-girentuximab uptake associated with the tumor, as seen on contrast-enhanced CT, MRI, or FDG PET/CT) will be used to assess the agreement between ⁸⁹ Zr-girentuximab PET/CT and conventional imaging in tumor lesion detection. RECIST 1.1 criteria for conventional imaging should be used as the primary tool for comparing agreement with PET, when possible. For radiological evaluation of tumors, which have different recommendations according to the Society of Scientific Oncology clinical guidelines, these guidelines should also be followed. In addition, all visible tumor lesions on conventional imaging can also be compared with PET imaging results.

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

Example 2: Preparation of radiolabeled girentuximab

Radiolabeled girentuximab was prepared as previously described (see, e.g., WO 2021/000017). In brief, bioconjugated girentuximab was prepared using standard techniques prior to labeling with a radioisotope useful for imaging (e.g., ⁸⁹ Zr) to obtain DOTA-girentuximab or DFO-girentuximab.

Example 3: In vitro and in vivo binding of radiolabeled girentuximab to various cancers.

Imaging studies using radiolabeled DOTA-girentuximab were performed to evaluate the ability of the imaging agent to detect non-RCC cancer types.

First, the ability of radiolabeled DOTA-girentuximab to bind to various cell lines. These data, shown in Figure 1, demonstrate the ability of the antibody to bind to various cell types expressing CAIX, although the degree of binding in vitro is variable.

Subsequently, three groups of mice, each bearing a different tumor xenograft, were tested. The groups were as follows:

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

Group 2: Mice bearing FaDu cell xenografts (squamous cell carcinoma pharyngeal/hypopharyngeal cell line); n = 4

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

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

Representative images of mice from each of the three groups are shown in Figure 2.

Figure 3 shows the quantification of the percent injected dose in tumors (at 24 and 72 hours post-injection). The results confirm observations made using flow cytometry to demonstrate the ability of the radiolabeled antibody to bind to each cancer cell line. In other words, the results demonstrate that the antibody can bind to target tumor cells in vivo with similar affinity as in vitro (except for FaDu cells—see additional comments below).

Results also show that 72 hours after administration, radiolabeled antibodies were still detectable in the circulation and spleen.

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

Figure 4 shows that there was a high degree of correlation between in vivo and ex vivo quantification of signals in tumors.

The results show that the positive in vitro binding results (e.g., good binding to HT-29 and AsPc-1 cells) are consistent with the positive binding observed in vivo.

Interestingly, the present inventors, in vitro Despite very weak binding of the radiolabeled antibody to FaDu cells, we observed that the antibody was able to bind to tumor cells in vivo. These results demonstrate that negative in vitro binding may not be predictive of in vivo binding, thus highlighting the potential utility of radiolabeled DOTA-GmAb for imaging certain cancer types.

Example 4: Imaging of Alternative Cancer Types

Experiments similar to those performed in Example 2 were performed using ⁸⁹ Zr-DFO-GmAb to detect the presence of tumor xenografts in mice as follows:

Group 1: Mice bearing A-549 cell line xenografts (lung cancer)

Group 2: Mice bearing MDA-MB-468 cell line xenografts (triple-negative breast cancer)

Group 3: Mice bearing HeLa cell line xenografts (cervical cancer)

Group 4: Mice with AGS cell line xenografts (gastric cancer)

Group 5: Mice with HepG2 cell line xenografts (liver cancer)

Group 6: Mice bearing A2780 cell line xenografts (ovarian cancer)

Group 7: Mice bearing SK-LMS1 cell line xenografts (soft tissue sarcoma - vulvar leiomyosarcoma)

At 24 and 72 hours after intravenous injection of ⁸⁹ Zr-DFO-GmAb into xenograft-bearing mice, the mice were subjected to PET/CT scanning to determine the ability of the radiolabel to detect cancer in vivo.

The results will demonstrate that the radiolabeled antibody can bind to tumor xenografts. In other words, the results will demonstrate that the antibody can bind to target tumor cells in vivo with similar affinity as in vitro.

These results suggest that radiolabeled GmAb is suitable for use in generating images of various cancer types in vivo and thus may be useful as a non-invasive diagnostic reagent for the diagnosis and detection of cancers other than renal cell carcinoma.

Example 4: Imaging of triple-negative breast cancer

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

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

The patient underwent imaging with fludeoxyglucose F18 (FDG) and ⁸⁹ Zr-girentuximab PET-CT and CT. The patient received a single, slow intravenous dose of 37±10% MBq ⁸⁹ Zr-girentuximab (10 mg). On day 3 after administration, PET/CT was acquired from the skull to the mid-thigh with 10-minute acquisitions per bed position. The gold standard was determined by FDG PET/CT, CT, and follow-up; lesions detected by at least two modalities were considered true positive. Tumor SUV _{[max, mean} ], total lesion glycolysis (TLG), and metabolic tumor volume (MTV) were measured. Immunohistochemistry (IHC) was performed using an anti-CAIX antibody (Leica, clone TH22) using a Bond RX fully automated laboratory stain. Staining was evaluated semiquantitatively (percentage and intensity of tumor cell expression), and SUV values were compared with the intensity of CAIX expression assessed by IHC.

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

The overall sensitivity of ⁸⁹ Zr-girentuximab PET/CT was 93.2%, 100% for bone, lung, breast, and skin, and 87.5% for lymph nodes. The overall sensitivity for both CT and FDG-PET/CT was 82.7%. The median tumor SUV _max was 3.45 [IQ: 2.03-4.69] and 4.68 [IQ: 3.27-10.71] for ⁸⁹ Zr-girentuximab and ⁸⁹ Zr-girentuximab FDG, respectively. IHC showed two CAIX-high-expressing lesions in two patients [100%, 20%], whereas two patients each had a low profile [3%, 0%]. IHC CAIX cell status and ⁸⁹ Zr-girentuximab SUV _mean showed a weak correlation (rho=0.80; p=0.20). No safety concerns were reported for ⁸⁹ Zr-girentuximab.

Results demonstrate that ⁸⁹ Zr-girentuximab is useful for PET/CT imaging and diagnosis of TNBC in patients and provides superior results to biopsy IHC.

Example 6: Imaging of bladder cancer

Patients with non-muscle-invasive bladder carcinoma (NMIBC) are typically treated with cystectomy. Therefore, new treatment options that allow bladder preservation are needed.

CAIX is expressed on the luminal surface of papillary structures that directly contact the bladder cavity. We conducted a pilot prospective study aimed at ensuring intravesical radioactivity limitation and tumor targeting after intravesical injection of ⁸⁹ Zr-girentuximab.

The patient received a single intravesical instillation of ⁸⁹ Zr-girentuximab (10 mg) at 37±10% MBq and retained urine for 2 hours. Four subsequent PET/CT scans were performed, three in the pelvis at one stage (H+2, Day 1 and D2) and one at H+4 from the skull to the mid-thigh to monitor the evolution of intravesical radioactivity over time.

Blood samples were collected on D1 to quantify possible vascular penetration of radioactivity. For all acquisitions, a 10-minute acquisition time per bed position was used. The gold standard was determined by second-look cystoscopy and transurethral resection of the bladder (TURB) on ^{the 89} Zr-girentuximab PET/CT-positive areas. Immunohistochemistry (IHC) was performed with an anti-CAIX antibody (Leica, clone TH22). Staining was evaluated semi-quantitatively (percentage and intensity of tumor cell expression) and the ⁸⁹ Zr-girentuximab PET/CT bladder pattern was compared with the degree of CAIX expression assessed by IHC.

Results were obtained from 4/6 patients. Despite multiple prior intravesical instillations with bacille Calmette-Guérin (BCG - a commonly used intravesical immunotherapy for bladder cancer), recurrent pTaG3 was identified in each patient.

PET/CT of ⁸⁹ Zr-girentuximab did not reveal any extravesical leakage. In 2/4 patients with positive IHC, uptake sites on the bladder wall were identified by TURB in 1 patient, and a corresponding recurrent lesion and inflammatory scarring were identified in the second patient. In the other 2 patients, no uptake was observed, consistent with negative IHC. No hazardous radiation contamination was observed during the procedure, and no specific worker exposure was observed.

The results (shown in Figures 5 and 6) indicate that intravesical instillation of ⁸⁹ Zr-girentuximab showed radioactivity confined to the bladder, and in patients with positive IHC, ⁸⁹ Zr-girentuximab is useful for detection and imaging of tumors in this patient group.

It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text and drawings. All of these various combinations constitute various alternative aspects of the invention.

Attach an electronic file of the sequence list

Claims (49)

A method for in vivo imaging or detection of cancer in a subject in need thereof, said method comprising:
- A step of administering to a subject an agent for binding to CAIX expressed by cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject;
- Step of detecting the agent in the target
, where cancer is
· Bladder cancer
· Breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER/PR-positive), and HER2-positive breast cancer)
· Cervical cancer
· Colorectal cancer
· Esophageal cancer (including esophageal squamous cell carcinoma (SCC) and esophagogastric junction adenocarcinoma)
· Gastric cancer (including gastric adenocarcinoma)
· Glioblastoma multiforme
· Head and neck cancer (including head and neck squamous cell carcinoma and nasopharyngeal carcinoma)
· Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)
· Lung cancer (including non-small cell and small cell carcinoma)
· Ovarian cancer (including epithelial ovarian carcinoma)
· Pancreatic cancer (including pancreatic ductal adenocarcinoma) and
· Soft tissue sarcoma
A method of imaging or detecting cancer in a subject, wherein detection of said agent above background or standard levels indicates the presence of cancer. As a method for diagnosing cancer in a subject in need thereof, the method comprises:
- A step of administering to a subject an agent for binding to CAIX expressed by cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject;
- A step for determining the presence or absence of an agent in a target object.
, where cancer is
· Bladder cancer
· Breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER/PR-positive), and HER2-positive breast cancer)
· Cervical cancer
· Colorectal cancer
· Esophageal cancer (including esophageal squamous cell carcinoma (SCC) and 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 and small cell carcinoma)
· Ovarian cancer (including epithelial ovarian carcinoma)
· Pancreatic cancer (including pancreatic ductal adenocarcinoma) and
· Soft tissue sarcoma
A method wherein detection of said agent above background or standard levels indicates that the subject has said cancer, thereby diagnosing cancer in the subject. As a method for generating an image of cancer, the method comprises:
- a step of administering to a subject suspected of having cancer an effective amount of an agent for binding to CAIX expressed by a cancer, wherein the agent comprises a detectable moiety for enabling in vivo detection of the agent in the subject;
- Step of detecting the agent in the target
, where cancer is
· Bladder cancer
· Breast cancer (including triple-negative breast cancer, hormone receptor-positive breast cancer (ER-positive, PR-positive, ER/PR-positive), and HER2-positive breast cancer)
· Cervical cancer
· Colorectal cancer
· Esophageal cancer (including esophageal squamous cell carcinoma (SCC) and 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 and small cell carcinoma)
· Ovarian cancer (including epithelial ovarian carcinoma)
· Pancreatic cancer (including pancreatic ductal adenocarcinoma) and
· Soft tissue sarcoma
A method of selecting from and thereby generating an image of cancer. A method according to any one of claims 1 to 3, wherein the method further comprises a step of allowing the agent to be concentrated at a site and/or tissue in the subject where the CAIX antigen is found before detecting the agent in the subject or determining the presence or absence of the agent in the subject. A method according to any one of claims 1 to 4, wherein the method does not require an additional in vitro method for detecting, diagnosing, or imaging cancer. A method according to any one of claims 1 to 4, wherein the method is the only method necessary to enable detection, diagnosis or imaging of cancer. A method according to any one of claims 1 to 6, wherein the agent for binding to CAIX is a small molecule, peptide or polypeptide (e.g., an antibody or an antigen-binding fragment thereof). A method according to 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, sulfamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, -MIP-1486, MIP-1490, MIP-1504 (in particular ^99m Tc-HEHEHE-Z09781, ^99m Tc-MIP-1486, ^99m Tc-MIP-1490 or ^99m Tc-MIP-1504/5) and PHC-102. A method according to any one of claims 1 to 7, wherein the agent for binding to CAIX is a peptide selected from the group consisting of: 3B-301, 3B-302 or CAIX-P1. A method according to any one of claims 1 to 7, wherein the agent for binding to CAIX is a polypeptide. A method according to any one of claims 1 to 7, wherein the agent for binding to CAIX is an antibody or an antigen-binding fragment thereof. A method according to claim 11, wherein the antibody or antigen-binding fragment thereof is girentuximab, including a chimeric or humanized variant thereof. A method according to claim 11, wherein the antibody or antigen-binding fragment thereof is BCA-356, BAY-794620 or SLC-0131. A method according to any one of claims 1 to 13, wherein the detectable moiety of the agent is conjugated directly to the agent or via a chelator or linker. A method according to any one of claims 1 to 14, wherein the detectable moiety is a fluorescent label or dye. A method according to any one of claims 1 to 14, wherein the detectable moiety is a radioisotope. A method according to claim 16, wherein the radioisotope is selected from the following: 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). A method according to claim 1 to claim 14, claim 16 or claim 17, wherein the detectable moiety is a radioisotope, and the detection of the agent or the detection of the presence or absence of the agent comprises determining or detecting the presence or absence of radiation emitted by the radioisotope. A method according to claim 18, wherein the determination or detection of the presence or absence of radiation comprises positron emission tomography (PET) imaging. A method according to any one of claims 1 to 14, wherein the agent is selected from: ⁸⁹ Zr-girentuximab, ¹²³ I-, ¹²⁴ I-, or ¹³¹ I-girentuximab. A method according to any one of claims 1 to 14, wherein the agent is ⁸⁹ Zr-girentuximab. A method according to 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). A method according to any one of claims 1 to 6, wherein the cancer is cervical cancer. A method according to any one of claims 1 to 6, wherein the cancer is colorectal cancer. A method according to any one of claims 1 to 6, wherein the cancer is esophageal cancer. A method according to any one of claims 1 to 6, wherein the cancer is gastric cancer. A method according to any one of claims 1 to 6, wherein the cancer is glioblastoma multiforme. A method according to any one of claims 1 to 6, wherein the cancer is head and neck cancer (e.g., hypopharyngeal cancer, pharyngeal cancer). A method according to any one of claims 1 to 6, wherein the cancer is liver cancer. A method according to any one of claims 1 to 6, wherein the cancer is lung cancer. A method according to any one of claims 1 to 6, wherein the cancer is ovarian cancer. A method according to any one of claims 1 to 6, wherein the cancer is pancreatic cancer. A method according to any one of claims 1 to 6, wherein the cancer is soft tissue sarcoma. A method according to any one of claims 1 to 6, wherein the cancer is bladder cancer. A method according to claim 22, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 23, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 24, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 25, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 26, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 27, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 28, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 29, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 30, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 31, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 32, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 33, wherein the agent is a radiolabeled girentuximab antibody. A method according to claim 34, wherein the agent is a radiolabeled girentuximab antibody. A method according to any one of claims 1 to 47, wherein the agent is radiolabeled girentimax comprising one or more amino acid substitutions in the Fc region of the antibody that reduce the serum half-life of the antibody. The method of any one of claims 35 to 48, wherein the radiolabeled girentuximab comprises girentuximab 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).