CN121079110A

CN121079110A - Imaging reagents and methods

Info

Publication number: CN121079110A
Application number: CN202380094432.0A
Authority: CN
Inventors: 阿莱西亚·伊瓦什克维奇; 科琳·莱滕德雷; 斯图尔特·肯特·格里布尔; 迈克尔·保罗·惠特克罗夫特
Original assignee: Telix Pharmaceuticals Innovations Pty Ltd
Current assignee: Telix Pharmaceuticals Innovations Pty Ltd
Priority date: 2022-12-20
Filing date: 2023-09-27
Publication date: 2025-12-05
Also published as: JP2026502854A; KR20250124157A; WO2024130293A1; MX2025007180A; AU2023407166A1; EP4637846A1

Abstract

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

Description

Imaging reagents and methods

Technical Field

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

RELATED APPLICATIONS

The present application claims priority from australian provisional application AU 2022903922, the contents of which are hereby incorporated by reference in their entirety.

Background

Medical imaging methods are commonly used to aid in diagnosis and staging of various cancer progression. Such methods are also advantageous in removing or reducing the need for invasive techniques for confirming diagnosis (such as obtaining a biopsy sample), which are not always necessary and may cause complications.

However, not all cancers can be successfully detected using standard medical imaging. Furthermore, while many imaging techniques are capable of detecting a tumor, they have not been successful in distinguishing between benign and malignant tissue.

In recent decades, the state of oncology management has evolved in a dichotomous manner. While some types of cancer have benefited significantly from advances in research of diagnosis and treatment options, resulting in improved morbidity and mortality in their patient populations, other types of cancer have proven more difficult to discover and continue to predict poor prognosis in their patient populations.

With this latter class of cancers already exhausted the limitations of existing diagnostic and therapeutic modalities, innovations based on new methods of oncology management are needed.

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

The reference to any prior art in this specification is not an acknowledgement or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, nor is it an acknowledgement or suggestion that this prior art could be reasonably expected to be understood, considered relevant and/or combined with other prior art by a person skilled in the art.

Disclosure of Invention

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

The present invention thus provides a method for in vivo imaging or detection of cancer in a subject in need thereof,

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

Detecting the agent in the subject,

Wherein the cancer is selected from:

● Bladder cancer

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

● Cervical cancer

● Colorectal cancer

● Esophageal cancer (including esophageal Squamous Cell Carcinoma (SCC) and esophageal/esophageal gastric junction adenocarcinoma)

● Stomach cancer (including gastric adenocarcinoma)

● Glioblastoma multiforme

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

● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

● Lung cancer (including non-small cell cancer and small cell cancer)

● Ovarian cancer (including epithelial ovarian cancer)

● Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

● Sarcoma of soft tissue

Wherein detection of the agent above a background level or standard level is indicative of the presence of the cancer, thereby imaging or detecting the cancer in the subject.

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

Determining the presence or absence of the agent in the subject,

Wherein the cancer is selected from:

● Bladder cancer

● Cervical cancer

● Colorectal cancer

● Stomach cancer (including gastric adenocarcinoma)

● Glioblastoma multiforme

● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

● Lung cancer (including non-small cell cancer and small cell cancer)

● Ovarian cancer (including epithelial ovarian cancer)

● Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

● Sarcoma of soft tissue

Wherein determining that the presence of the agent is above a background level or a standard level indicates that the subject has the cancer, thereby diagnosing the cancer in the subject.

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

Administering to a subject suspected of having said cancer an effective amount of an agent for binding to CAIX expressed by said cancer, wherein said agent comprises a detectable moiety for enabling in vivo detection of said agent in said subject,

Detecting the agent in the subject,

Wherein the cancer is selected from:

● Bladder cancer

● Cervical cancer

● Colorectal cancer

● Stomach cancer (including gastric adenocarcinoma)

● Glioblastoma multiforme

● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

● Lung cancer (including non-small cell cancer and small cell cancer)

● Ovarian cancer (including epithelial ovarian cancer)

● Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

● Sarcoma of soft tissue

Thereby producing an image of the cancer.

infusing an effective amount of an agent for binding to CAIX expressed by the cancer, wherein the agent comprises a detectable moiety for effecting in vivo detection of the agent,

-Detecting the presence of said agent(s),

Wherein the cancer is selected from:

● Bladder cancer

● Cervical cancer

● Colorectal cancer

● Stomach cancer (including gastric adenocarcinoma)

● Glioblastoma multiforme

● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

● Lung cancer (including non-small cell cancer and small cell cancer)

● Ovarian cancer (including epithelial ovarian cancer)

● Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

● Sarcoma of soft tissue

Thereby producing an image of the cancer. Optionally, the detectable moiety is a radioisotope and the detecting comprises detecting radiation, such as Positron Emission Tomography (PET) imaging.

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

Administration may be by any suitable means, preferably means that allow systemic administration (e.g., intravenous infusion) of the agent, such that the agent may accumulate at a site in the subject where CAIX is present on the cell surface. The mode of administration may be determined by the nature of the agent used to bind CAIX. For example, in the case of antibodies for binding to CAIX, the agent is preferably administered by intravenous infusion. The labeled peptide or small molecule 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 tissue biopsies or other biological samples obtained from the subject.

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

In any embodiment, the moiety for binding to CAIX may be a small molecule, peptide or 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, sulfonamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, -MIP-1486, MIP-1490, MIP-1504 (especially ^99mTc-HEHEHE-Z09781、^99mTc-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, including an antibody or 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 antigen-binding fragment thereof.

In any embodiment, the agent is an antigen binding protein (antibody) that retains the ability to bind CAIX, such as gemtuzumab (girentuximab) or a functional variant or fragment thereof. 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 CAIX is a chimeric antibody or antigen binding fragment thereof. In some embodiments, the antigen binding protein that binds or specifically binds CAIX is a humanized antibody or antigen binding fragment thereof. Optionally, the antigen binding protein is humanized G250 (hG 250).

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

The agent used in the method according to the invention comprises a moiety for enabling detection thereof. Any suitable detectable moiety for in vivo detection techniques may be used and will be known to the skilled person.

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-described agents for binding to CAIX are detectable without the need to attach additional detectable moieties thereto.

In any embodiment, the detectable moiety is a radioisotope. Examples of suitable isotopes include gallium-67 and gallium-68 (⁶⁷ Ga and ⁶⁸ Ga), indium-111 (¹¹¹ In), iodine-123, iodine-124 or iodine 131 (¹²³I、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 a radioisotope, optionally selected from gallium-67 and gallium-68 (⁶⁷ Ga and ⁶⁸ Ga), indium-111 (¹¹¹ In), iodine-123, iodine-124 or iodine 131 (¹²³I、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 gemtuximab antibody (including chimeric or humanized versions thereof), and the detectable moiety is a radioisotope, optionally selected from gallium-67 and gallium-68 (⁶⁷ Ga and ⁶⁸ Ga), indium-111 (¹¹¹ In), iodine-123, iodine-124 or iodine 131 (¹²³I、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-gemtuximab, ¹²³ I-gemtuximab, ¹²⁴ I-gemtuximab, ¹³¹ I-gemtuximab, or ¹⁷⁷ Lu-gemtuximab.

In a preferred embodiment, the detectable moiety is a radioisotope and the detecting the agent comprises determining radiation emitted by the radioisotope or detecting the presence or absence of the radiation. In any embodiment, determining the presence or absence of radiation or detecting 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 invention, more than one detectable moiety may be utilized in order to maximize imaging or detection of the agent and thereby maximize CAIX expressing tumors or cancers.

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

Still further, the invention provides an agent for CAIX or a composition comprising the agent as described herein, when used in a method of detecting, imaging or diagnosing cancer, or for obtaining an image of cancer, as described herein.

Still further, the invention provides a kit for use according to any of the methods described herein, wherein the kit comprises an agent for binding to CAIX as described herein, and optionally instructions for its use for detecting, imaging or diagnosing cancer.

As used herein, unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" and "comprises" are not intended to exclude further additions, components, integers or steps.

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

Drawings

FIG. 1 in vitro binding of radiolabeled DOTA-GmAb to various cell lines.

FIG. 2 representative images of mice bearing As-PC-1 (pancreatic carcinoma), faDu (hypopharynx carcinoma) or HT-29 (colorectal carcinoma) tumor xenografts and subsequent injection of radiolabeled DOTA-GmAb.

FIG. 3 quantification of percent of injected doses 24 hours and 72 hours after injection of radiolabeled DOTA-GmAb. Filled circles = AsPC-1 radiolabelled DOTA-GmAb, filled boxes = FaDu radiolabelled DOTA-GmAb, filled triangles = HT-29 radiolabelled DOTA-GmAb.

FIG. 4 quantification of biodistribution results. The in vitro biodistribution (i.e., the distribution of radiolabeled DOTA-GmAb observed in the organs of mice after necropsy) was shown to correlate with the in vivo biodistribution (i.e., the distribution of radiolabeled DOTA-GmAb observed in mice after whole body imaging). Filled circles = AsPC-1 radiolabelled DOTA-GmAb, filled boxes = FaDu radiolabelled DOTA-GmAb, filled triangles = HT-29 radiolabelled DOTA-GmAb.

FIG. 5 imaging of bladder cancer. Day 0, whole body ⁸⁹ Zr-Gituximab scan A coronal PET/CT fusion. Maximum Intensity Projection (MIP) visualization.

FIG. 6 imaging of bladder cancer. Day 2, a. ⁸⁹ Zr-gemtuximab pelvic PET/CT fusion images (arrow) radiopharmaceutical uptake on different sides of the bladder wall. B. A 3D representation of the superimposed image of the bladder.

Detailed Description

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

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

Although CAIX is typically associated with advanced disease, it is not known whether detection and/or imaging of CAIX is performed during the early stages of certain cancers, or whether cancer may be detected only in advanced disease. In addition, some cancers exhibit reduced expression as the disease progresses, and thus it is not clear whether CAIX imaging is a useful means for detecting these types of cancers.

Furthermore, given the heterogeneity of many cancers, the simple presence of CAIX expression (e.g., as determined by immunohistochemical techniques) does not necessarily suggest that whole-body or partial-body imaging methods can be used to detect cancer.

For example, in the 2006 study of Henrickx et al (cancer biotherapy and radiopharmaceuticals (Cancer Biotherapy & Radiopharmaceuticals), 21:263-268), radiolabeled antibodies for binding to CAIX were found to be unsuitable for imaging of biliary cancer, despite the fact that biliary cancer is found to overexpress CAIX. In contrast, it is known that the same antibodies are commonly used for imaging and detection of kidney cell carcinomas that overexpress CAIX. Both biliary and renal cancers are malignant tumors of epithelial cells and are characterized by increased expression of CAIX. Thus, it is not understood why antibodies that bind CAIX can be used for imaging and detection of renal cell carcinoma rather than biliary carcinoma.

In another example, a recent study published by Huizing et al (2021, physical and imaging In radiooncology (PHYSICS AND IMAGING IN Radiation Oncology), 145-150) found that the ¹¹¹ In-labeled F (ab') 2 form of CAIX-binding antibody, gemtuximab, was unable to distinguish between tumor cells and non-tumor cells and thus indicated that it was not possible to quantify changes In CAIX expression. In particular, this finding is in sharp contrast to the inventors' findings reported herein, where ⁸⁹ Zr-GmAb (in the form of whole IgG antibodies) was shown to be useful for in vivo imaging and detection of the same cancer cell type.

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

General definition

Throughout this specification, unless the context clearly indicates otherwise, reference to a single step, composition of matter, group of steps, or group of compositions of matter should be taken to encompass one or more (i.e., one or more) of those steps, compositions of matter, group of steps, or group of compositions of matter. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. For example, references to "a" and "an" include a single and two or more, references to "the" include a single and two or more, and the like.

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

Those skilled in the art will recognize many methods and materials that may be similar or equivalent to those described herein, which may be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

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

The present invention is not to be 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 invention.

Any examples or embodiments of the invention herein, mutatis mutandis, are to be considered as applicable to any other examples or embodiments of the invention unless explicitly stated otherwise.

Unless specifically stated otherwise, all technical and scientific terms used herein shall be regarded as having 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") should be understood to mean "X and Y" or "X or Y" and should be considered as providing explicit support for both or either meaning.

Carbonic anhydrase IX

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

Cancer cells predominantly express plasma-membrane associated CA isoforms CAIX and CAXII, as well as intracellular CAs, such as CAI and CAII. Of the cancer-associated CAs, CAIX has gained the most attention, as expression of this isoform in healthy tissues is limited to gastric and intestinal epithelial cells, but is strongly upregulated in renal cancer.

CAIX expression is controlled by hypoxia-inducible factor 1 (HIF-1), which is located primarily in chronically hypoxic tumor areas. CAIX, however, can also be found in regions of mild hypoxia or even normoxic conditions, as CAIX expression can be activated by components of the Mitogen Activated Protein Kinase (MAPK) pathway.

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

Preferably, the agent for binding to CAIX specifically interacts with CAIX polypeptides. Specific interaction (e.g., recognition or binding) means that an agent, such as an antibody, has a greater affinity for CAIX than other polypeptides. In one embodiment, the agent interacts with (i.e., binds to or recognizes) or modulates the activity of a CAIX polypeptide and/or mediates antibody-dependent cellular cytotoxicity (ADCC) and/or complement-mediated cytotoxicity (CDC). Thus, according to one embodiment, the agent is a CAIX inhibitor. The CAIX inhibitor may act at the protein level or the nucleic acid level. Examples of CAIX inhibitors that act, for example, at the protein level include, but are not limited to, peptides and anti-CAIX antibodies, as well as those antibodies or functional fragments of small organic molecules, preferably having a molecular weight of less than 500g/mol.

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

Examples of small organic molecules for binding to CAIX include, but are not limited to, sulfonamides, heteroaromatic sulfonamides, sulfamates, coumarin 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 term "specific binding (SPECIFICALLY BINDS)" or "specific binding (binds specifically)" shall be taken to mean that the agent for use according to the invention reacts or associates more frequently, more rapidly, for a longer duration, and/or with greater affinity with CAIX or cells expressing it than with alternative antigens or cells. For example, the affinity of an antigen binding protein that binds CAIX is substantially much greater (e.g., 1.5-fold, or 2-fold, or 5-fold, or 10-fold, or 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 150-fold or 200-fold) than its affinity for other antigens.

Methods for assessing binding to proteins (e.g., CAIX) are known in the art, for example, as described in scens (protein purification: principle and practice (Protein purification: PRINCIPLES AND PRACTICE), third edition, sapringer press (SPRINGER VERLAG), 1994). Such methods typically involve immobilizing an agent (e.g., an antibody) and contacting it with a labeled target (in the case of an antibody, an antigen). After washing to remove non-specifically bound proteins, the amount of label, and thus the bound antigen, is detected. Of course, the antigen binding site may be labeled and the antigen immobilized. Panning type assays may also be used. Alternatively or additionally, surface plasmon resonance assays may 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 the small molecule SLC-0111 (CAS 178606-66-1), SLC-149 (as described in EP 3317255 B1, incorporated herein by reference), SLC-0121 or SLC-101.

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

In any embodiment, the moiety for binding to CAIX is a small molecule sulfonamide-nitroimidazole (as described in Rami et al, (2013), "journal of pharmaceutical chemistry (j. Med. Chem), 56:8512-8520, which is incorporated herein by reference).

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

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

In any embodiment, the moiety for binding to CAIX is small molecule ^99m Tc-HEHEHE-Z09781 (Kim et al, (2017) advanced science (ADVANCED SCIENCE), 4:1600471; gebauer and Skerra (2009) recent views of chemical biology (Current Opin in Chem Biol), 13 (3): 245-55; schardt et al, (2017) molecular pharmaceutical (Mol Pharmaceutics), 14 (4): 1047-56; tolmachev et al, (2008) bioconjugate chemistry (Bioconjugate Chem), 19 (8): 1579-87; liu et al, (2022) analytical and biological analytical chemistry (ANALYTICAL AND Bioanalytical Chemistry), 414:1095-1104; grindel et al, (2022) journal of chemistry of the United states society of chemical Biol (ACS Chem Biol), 17 (6): 1543-55, which is incorporated herein by reference), ^99mTc-MIP-1486、^99m Tc-1490 (2-bis- (1, 5-dicarboxyiethyl) -2- (2-dicarboxyiethyl) -2-amino-2-methylsulfonylamino-ethyl) -2-imidazole-sulfonyl-2-amino-ethyl-2-sulfonyl-amino-2-ethyl-2-sulfonyl-amino-2-methyl-amine, wherein x=ethyl) or ^99m Tc-MIP-1504 (4- (2-bis ((1- (2- ((1, 5-dicarboxy-3- (2-carboxyethyl) pentan-3-yl) amino) -2-oxoethyl) -1H-imidazol-2-yl) methyl) amino-X) benzenesulfonamide, wherein x=n-butoxy) (Hillier et al, (2012) journal of nuclear medicine (Journal of Nuclear Medicine), 53 (s 1) 217, which is incorporated herein by reference).

In any embodiment, the moiety for binding to CAIX is a small molecule PHC-102 (as described in WO 2015/114171 A1;WO 2018154517A1;US2014/0357650 A1;WO 2015/114171 A1, incorporated herein by reference).

Peptides

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

In any embodiment, the moiety for binding to CAIX is peptide 3B-301 (also known as Debio 0228; queen et al, (2018) International journal of biological macromolecules (Int J of Biol Macromol), 106:840-850; eldehna et al, (2019) biological chemistry (Bioorganic Chem), 90:103102; lavecchia et al, (2011) Carbohydrate research (Carbohydrate Res), 346 (3) 442-48; krymov et al, (2022) European journal of medical chemistry (Eur J of MEDICINAL CHEM), 228:113997; supuran (2008) BJI International journal of medicine chemistry (BJI Int), 101 (s 4): 39-40; koyuncu et al, (2019) enzyme inhibition and pharmaceutical chemistry journal (J of Enzyme Inhibition AND MEDICINAL CHEM), 34 (1) 703-11): mar et al, (2017) medical chemistry journal 136-62) and incorporated herein by reference in the text of either J.3-52 or 302.

In any embodiment, the moiety for binding to CAIX is the peptide CAIX-P1, having the amino acid sequence YNTNHVPLSPKY (as described in Askoxylakis et al, (2010), "public science library-complex (PLoS One)," 5 (12): e 15972), optionally wherein the peptide is labeled with ¹²⁵ I or ¹³¹ I for detection thereof (although it will be appreciated that any suitable radiolabel or other detectable moiety may be used).

Polypeptides and antibodies

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

The reference herein to an antibody or antigen binding fragment thereof that "binds to Carbonic Anhydrase IX (CAIX)" provides literal support for an antibody or fragment thereof that "specifically binds to CAIX (binds specifically to)" or "specifically binds to CAIX (SPECIFICALLY BINDS TO)".

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. Antibodies may be multivalent, or multivalent and multispecific.

In a particularly preferred embodiment, the antibody is a whole antibody comprising at least one antigen binding domain (Fab) of the antibody and at least one Fc region of the antibody. Antibodies may include human constant regions of IgG1, igG2a, igG3, or IgG 4.

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

In any of the embodiments, the moiety for binding to CAIX is in the form of antibody BAY-794620 or an antigen binding fragment thereof (as described in WO 2003/100029 A2;WO 2003/033674 A2;Theiner et al, (2021) veterinarian practice G edition: large animal-farm animal (Tierarztl Prax Ausg G Grosstiere Nutztiere), 49 (6) 392-402; kimani et al, (2011) photochemical and photobiological (Photochemistry and Photobiology), 88 (1): 175-87; NCT01065623 (v 24,2014, 9, 30, and NCT01028755 (v 30,2015, 1, 19), 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 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, antigen binding proteins that bind or specifically bind CAIX are as described in WO 2002/062972 A2(US2004/0219633 A1)、WO 2004/002526 A1(US 7,632,496 B2)、WO 2006/002889 A2(US 7,691,375B2)、WO 2009/056342 A1(US2014/0017252 A1)、WO 2011/032973 A1(US 2012/0207672A1) and any of WO 2014/128258 A1 (US10,620,208B2) or WO 2021/000017 A1, the entire contents of each of these publications are incorporated herein by reference.

Antibodies for use in the present invention may be produced by any suitable method known in the art, including but not limited to the methods described in PCT/EP 02/0182 and PCT/EP 02/01083, which are incorporated herein by reference.

A particularly preferred antibody is cG250, preferably gemtuximab (INN). Another particularly preferred embodiment is monoclonal antibody G250 produced by the hybridoma cell line DSMACC 2526. Antibody cG250 is an IgG1 kappa light chain chimeric version of the orthomurine monoclonal antibody mg250.

Variants of the original chimeric G250 (cG 250) antibodies, including WX-G250 and WX-G250RIT (131 iodine), are known (Janssen Global SERVICES LLC).

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

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

(a) Heavy chain variable domain (VH) comprising three Complementarity Determining Regions (CDRs) of the amino acid sequences as 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 sequences as set forth in SEQ ID NOs 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) and comprises:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4-linker-FR 1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a

Wherein:

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

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

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

CDR1a, CDR2a and CDR3a are each complementarity determining regions;

Wherein the sequence of any of the complementarity determining regions has an amino acid sequence as described in table 1 below. Preferably, the framework regions have amino acid sequences, including amino acid variations at specific residues, as also described in table 1 below, which can be determined by aligning the various framework regions derived from each antibody. The CDR1, CDR2, and CDR3 may be sequences from the VH, CDR1a, CDR2a, and CDR3a may be sequences from the VL, or the CDR1, CDR2, and CDR3 may be sequences from the VL, CDR1a, CDR2a, and CDR3a may be sequences from the VH.

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

(i) VH comprising Complementarity Determining Region (CDR) 1 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO1, CDR2 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO1, and CDR3 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 92%, at least 91%, at least 98%, at least 95%, or at least 99% identical to the sequence set forth in SEQ ID NO 3;

(ii) VH comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in any one of SEQ ID nos. 4, 20, 36, 52 or 68;

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

(iv) A VL comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID No. 84, 100, 116, 132, 148 or 164;

(v) A VH comprising a CDR1, the CDR1 comprising the sequence shown in SEQ ID No.1, a CDR2, the CDR2 comprising the sequence shown in SEQ ID No. 2, and a CDR3, the CDR3 comprising the sequence shown in SEQ ID No. 3;

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

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

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

(ix) A VH comprising a CDR1 comprising the sequence shown in SEQ ID NO:1, a CDR2 comprising the sequence shown in SEQ ID NO:2, and a CDR3 comprising the sequence shown in SEQ ID NO:3, and a VL comprising a CDR1 comprising the sequence shown in SEQ ID NO:81, a CDR2 comprising the sequence shown in SEQ ID NO:82, and a CDR3 comprising the sequence shown in SEQ ID NO:83, or

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

In further embodiments, the antibody or antigen binding fragment thereof comprises:

(i) VH comprising a Framework Region (FR) 1, the FR1 comprising or consisting of a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence as set forth in any one of SEQ ID nos. 9, 25, 41, 57 or 73; FR2 comprising or consisting of a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence as shown in any of SEQ ID nos. 10, 26, 42, 58 or 74, FR3 comprising or consisting of a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence as shown in any of SEQ ID nos. 11, 27, 43, 59 or 75, FR4 comprising at least about 80%, at least 82%, at least 83%, at least 81% of the sequence as shown in any of SEQ ID nos. 12, 28, 44, 60 or 76 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 sequence or consist of the same sequence, and

(Ii) VL comprising a Framework Region (FR) 1, the FR1 comprising or consisting of a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence as set forth in any one of SEQ ID nos. 89, 105, 121, 137, 153 or 169; FR2 comprising or consisting of at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence as set forth in any one of SEQ ID No. 90, 106, 122, 138, 154 or 170, FR3 comprising or consisting of at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence as set forth in any one of SEQ ID No. 90, 108, 123, 139, 155 or 171, or 172, FR4 comprising at least about 80%, at least 81% of a sequence as set forth in any one of SEQ ID No. 91, 108, 124, 140, 156 or 172, or 172 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 sequence or consist thereof.

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

(Ii) VL comprising a Framework Region (FR) 1, said FR1 comprising or consisting of a sequence as set forth in SEQ ID No:89, 105, 121, 137, 153 or 169, FR2 comprising or consisting of a sequence as set forth in any one of SEQ ID No:90, 106, 122, 138, 154 or 170, FR3 comprising or consisting of a sequence as set forth in SEQ ID No:91, 107, 123, 139, 155 or 171, FR4 comprising or consisting of a sequence as 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 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 order from N to C-terminus or C to N-terminus).

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

(a) A heavy chain variable domain (VH) comprising or consisting of a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence as set forth in SEQ ID NO 4, 20, 36, 52 or 68, and/or

(B) A light chain variable domain (VL) comprising or consisting of a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence as 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 fragments (scFv);

(ii) Dimeric scFv (di-scFv), or

(Iii) One of (i) or (ii) linked to the constant region of an antibody, fc or heavy chain constant domain (CH) 2 and/or CH 3.

(i) A bifunctional antibody;

(ii) A trifunctional antibody;

(iii) A four-functional antibody;

(iv)Fab;

(v)F(ab')2;

(vi) Fv or

(Vii) One of (i) to (vi) linked to a constant region of an antibody, fc or heavy chain constant domain (CH) 2 and/or CH 3.

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

Antigen binding fragments, immunoglobulin variable domains, antibodies, dabs, bis-scFv, scFv, fab, fab ', F (ab') 2, fv fragments, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, linear antibodies, single chain antibody molecules or multispecific antibodies, fusion proteins or conjugates as described herein may be obtained by expressing a nucleic acid encoding the same.

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. Where the antibody or protein comprises 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 stable heavy chain constant region comprising a mixture of sequences with or without a C-terminal lysine residue, either fully or partially.

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

The functional properties of the antigen binding fragments thereof as described herein will be deemed suitable for use with antibodies as described herein, mutatis mutandis.

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

TABLE 1 overview of amino acid and nucleotide sequences of preferred CAIX binding antibodies

In further embodiments, the moiety for binding to CAIX may comprise BCA-356, a bispecific antibody comprising an affinity matured humanized anti-CAIX antibody linked to an attenuated subunit of IL-12, which is fused at the C-terminus to each of the heavy chains of the anti-CAIX antibody by a linker, thereby forming a "knob-to-socket" form (as described in Nair et al, journal of cancer immunotherapy (Journal for ImmunoTherapy of Cancer), 10:s2).

In particularly preferred embodiments of the methods and uses described herein, the agent for binding to CAIX is ⁸⁹ Zr-gemtuximab, ¹²⁴ I-gemtuximab, ¹⁷⁷ Lu-gemtuximab or ¹¹¹ In-gemtuximab-IRDye 800CW (e.g., as described In Stroet et al, (2022), "cancer (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-gemtuzumab. ⁸⁹ Zr-gemtuximab is a chimeric monoclonal antibody (INN name: gemtuximab (GTX), also known as cG250 and TLX 250) specific for the CAIX antigen, radiolabeled with positron-emitting radiometal zirconium-89, which is linked to the lysine residue of GTX by NSuc-DFO-TFP-ester (DFO-TFP) to yield ⁸⁹ Zr DFO-TFP-GTX.

Constant region

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

Sequences of constant regions useful for producing antibodies or antigen binding fragments thereof as described herein can be obtained from a number of different sources. In some examples, the constant region of the protein, or a portion thereof, is derived from a human antibody. The constant region or portion thereof may be derived from any antibody class, including IgM, igG, igD, igA and IgE, as well as any antibody isotype, including IgG1, igG2, igG3, and IgG4. In one example, the constant region is a human isotype IgG4 or a stable IgG4 constant region.

Neonatal Fc receptors (FcRn) are important for the metabolic fate of IgG class antibodies in vivo. FcRn functions to salvage IgG from lysosomal degradation pathways, resulting in reduced clearance and prolonged half-life. It is a heterodimeric protein consisting of two polypeptides, a 50kDa class I major histocompatibility complex-like protein (a-FcRn) and a 15kDa p 2-microglobulin (β2ηiota). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG class antibodies. The interaction between IgG class antibodies and FcRn is pH dependent and occurs in a 1:2 stoichiometry, i.e. one IgG antibody molecule can interact with two FcRn molecules through its two heavy chain Fc region polypeptides (see, e.g., huber, a.h. et al, journal of molecular biology 230 (1993) 1077-1083).

Thus, the in vitro FcRn binding/properties of IgG are indicative of its in vivo pharmacokinetic properties in the blood circulation. In the interaction between FcRn and the Fc region of IgG class antibodies, the different amino acid residues of the heavy chain CH 2-and CH 3-domains are involved.

Different mutations that affect FcRn binding and half-life in the blood circulation are known. Residues of the Fc region that are critical for mouse Fc region-mouse FcRn interactions 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 interactions (Medesan, C et al, (European J. Immunol.)) 26 (1996) 2533-2536; finan, M. Et al, (International Immunol.)) 13 (2001) 993-1002; kim, J. K. Et al, (European J.Immunol.)) 24 (1994) 542-548. (Using the Kabat system, the relevant residues are Ile266, his329, his464, asn465 and His 466). Residues Ile253, his310 and His435 were found to be critical for the interaction of the human Fc region with murine FcRn (Kim, j.k. Et al, journal of immunology 29 (1999) 2819-2885).

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

Preferred modifications

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

In one embodiment, the one or more amino acid substitutions are selected from substitutions in heavy chain constant region 2 (CH 2) of an IgG molecule that reduce affinity of the IgG molecule for FcRn. Alternatively, the one or more amino acid substitutions may be in the heavy chain constant region 3 (CH 3) of the IgG molecule, thereby reducing the affinity of the IgG molecule for FcRn. Still further, the amino acid substitutions may include at least one substitution in the CH2 region and at least one substitution in the CH3 region of the IgG molecule, whereby the substitution reduces affinity of IgG 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 the IgG. Preferably, the amino acid substitution comprises a substitution in the heavy chain constant region at position His310 or His 435. More preferably, the amino acid substitution that reduces the affinity of the antibody for FcRn is at both His310 and His 435.

In other preferred embodiments, the antibody and/or antigen binding fragment thereof has a constant region that is substantially identical to a naturally occurring IgG class antibody constant region, wherein at least one amino acid residue selected from the group consisting of residues His310, his435, and Ile253 is different from the amino acid residues present in a naturally occurring IgG class antibody, thereby altering the FcRn binding affinity and/or serum half-life of the antibody relative to the naturally occurring antibody. In preferred embodiments, the naturally occurring IgG class antibody comprises the heavy chain constant region of a human IgG1, igG2M3, igG3 or IgG4 molecule.

Also in a preferred embodiment, amino acid residue 310 and/or residue 435 of the heavy chain constant region from an antibody having substantially the same constant region as a naturally occurring IgG class antibody is any amino acid that is not histidine and reduces the affinity of the constant region for FcRn. For example, the amino acid 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 from histidine residues to alanine, glutamine, glutamic acid or aspartic acid. Preferably, the amino acid substitution at His310 is a substitution for alanine. Preferably, the amino acid substitution at His435 is a substitution for glutamine. Preferably, the amino acid substitution at Ile253 is alanine.

In preferred embodiments of the invention, the binding affinity and/or serum half-life of the modified antibody for 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 preferred embodiments of the 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%.

In addition, the antibodies or fragments thereof used in accordance with the present invention may be modified to include one or more mutations that alter the affinity of the antibody for any one or more fcγ receptors. For example, the one or more amino acid modifications alter the affinity of the antibody constant domain, fc region, or fcγ receptor binding fragment to any one or more fcγ receptors.

In certain embodiments, the modified antibodies or antigen binding fragments thereof retain the ability to bind to one or more Fc-gamma receptors, and thus in certain embodiments, the modified antibodies retain the ability to stimulate an effector response (including ADCC). In one example, the Fc region of the constant region 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, e.g., as 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 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 of 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 for arginine, proline, glutamic acid or aspartic acid, more preferably alanine.

In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4 constant region), such as a human IgG4 Fc region. Suitable IgG4 Fc region sequences will be apparent to the skilled artisan and/or available from public databases (e.g., available from national center for biotechnology information (National Center for Biotechnology Information)).

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

In one example, the stable IgG4 constant region comprises proline at position 241 of the hinge region according to the system of Kabat (Kabat et al, sequence of proteins with immunological significance (Sequences of Proteins of Immunological Interest) U.S. health and public Services, columbia, washington, 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. After serine substitution for proline, the IgG4 hinge region comprises the sequence CPPC. In this regard, the skilled artisan will appreciate that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that links the Fc and Fab regions, which imparts fluidity to the two Fab arms of the antibody. The hinge region includes cysteine residues involved in the disulfide bond between the heavy chains. According to the numbering system of Kabat, it is generally defined as extending from Glu226 to Pro243 of human IgG1 (or from Glu216 to Pro230 using the EU index). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues forming the inter-heavy chain disulfide bond (S-S) in the same position (see, e.g., WO 2010/080538).

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

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

In any embodiment, the heavy chain constant region of the antibody or antigen binding protein comprises amino acid substitutions at both His310 and His 435. The antibody may further comprise amino acid substitutions at residues corresponding to Ser228 and Leu235 of the constant heavy chain region.

In any embodiment, the antibody or antigen binding fragment thereof comprises mutations at Ser228, leu235, his310, and His 435. Preferably, the amino acid modifications are Ser228Pro, leu235Glu, his310Ala and His435Gln.

Further examples of stable IgG4 antibodies are antibodies in which arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) is replaced with lysine, threonine, methionine or leucine (e.g., as described in WO 2006/033386). The Fc region of the constant region may additionally or alternatively comprise residues selected from the group consisting of alanine, valine, glycine, isoleucine and leucine at a position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline (i.e., CPPC sequence) at position 241 (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 substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region comprising deletions of one or more of the following changes E233P, L234V, L A and G236 and/or one or more of the following changes A327G, A S and P331S (Armour et al, J.European.Immunol.29:2613-2624, 1999; shields et al, J.Biol.chem.) (276 (9): 6591-604, 2001). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall' Acqua et al, J.Immunol.177:1129-1138 2006, and/or Hezareh, J.Virol.75:12161-12168,2001.

In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, wherein the Fc region comprises substitutions 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 WO 2010/085682). 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 as set forth in any one of SEQ ID NOS: 177 to 180, preferably as set forth in SEQ ID NO: 178.

In still further embodiments, the antibody or antigen binding fragment thereof preferably comprises a heavy chain comprising the sequence set forth in any one of SEQ ID NOS: 182 to 185, preferably as 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 as 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 at least about 95%, or 96%, or 97%, or 98% or 99% identical to or comprising the sequence set forth in SEQ ID NO:36 or 52, and a VL comprising a sequence at least about 95%, or 96%, or 97%, or 98% or 99% identical to or comprising the sequence set forth in SEQ ID NO:116, 132 or 148.

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

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

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

Detectable moiety

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

In any embodiment of the invention, the small molecule, peptide, protein, or antibody used to bind to CAIX and as described herein may be directly or indirectly linked to a detectable moiety (e.g., a radioisotope, dye, or fluorescent moiety).

In any embodiment, the detectable moiety is a radioisotope. Examples of suitable isotopes include gallium-67 and gallium-68 (⁶⁷ Ga and ⁶⁸ Ga), indium-111 (¹¹¹ In), iodine-123, iodine-124 or iodine 131 (¹²³I、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 the radioisotope may be conjugated to the polypeptide (e.g., antibody) directly (via a chelator or prosthetic group or linker) or indirectly through binding to a single or multiple amino acid residues in the protein (e.g., halogenation of tyrosine residues).

In alternative embodiments, chelators or linkers may be used to conjugate the determinable time moiety to a peptide or protein for binding to CAIX. In one example, a peptide or protein (e.g., an antibody) may be conjugated to a chelating moiety selected from the group consisting of: TMT (6, 6 "-bis [ N, N", N '"-tetrakis (carboxymethyl) aminomethyl) -4' - (3-amino-4-methoxyphenyl) -2,2':6',2" -terpyridine), DOTA (1, 4,7, 10-tetraazacyclododecane-NN ', N "(N'" -tetraacetic acid, also known as tetan-stam (tetraxetan)), TCMC (tetraprimary amine of DOTA), DO3A (1, 4,7, 10-tetraazacyclododecane-1, 4, 7-tris (acetic acid) -10- (2-thioethyl) acetamide), CB-DO2A (4, 10-bis (carboxymethyl) -1,4,7, 10-tetraazabicyclo [5.5.2] tetradecane), NOTA (1, 4, 7-triazacyclononane-triacetic acid), diamsar (3,6,10,13,16,19-hexaazabicyclo [6, 6] eicosane-1, 8-diamine), DTPA (pentic acid or diethylenetriamine pentaacetic acid), CHX-a "-DTPA ([ (R) -2-amino-3- (4-isothiocyanatophenyl) propyl ] -trans- (S, S) -cyclohexane-1, 2-diamine), t-1, 4, 7-tetraazacyclopentaacetic acid (4, 7-tetraacetic acid), tetra-azacyclopentaacetic acid (1, 8-tetraacetic acid) Te2A (4, 11-bis (carboxymethyl) -1,4,8, 11-tetraazabicyclo [6.6.2] hexadecane), HBED, DFO (deferoxamine), DFOsq (DFO-squaramide), and HOPO (3, 4,3- (LI-1, 2-HOPO)) or other chelators as described herein. Other known chelating moieties include 3p-C-NETA ({ 4- [2- (bis-carboxy-methylamino) -5- (4-nitrophenyl) pentyl ] -7-carboxymethyl- [1,4,7] triazanonen-1-yl } acetic acid), 5p-C-NETA (2- ({ 1- [4, 7-bis (carboxymethyl) -1,4, 7-triazanonen-1-yl ] -7- (4-nitrophenyl) hept-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, chelating groups can be used to attach ¹⁸ F or ¹⁹ F that are complexed to metals (e.g., aluminum) to provide alternative modalities for imaging, detection, and/or diagnosis. It is contemplated that the fluorescently labeled molecules may be used more in intraoperative procedures, while ¹⁸ F-labeled molecules may be used more in pre-or post-operative imaging, detection, and/or diagnosis of diseased tissue.

The agent may be modified to contain a thiol group for attachment to a maleimide-modified fluorescent probe. Alternatively, bifunctional cross-linkers or fluorescent dyes conjugated with other reactive species may be used to link the fluorescent probes to different groups on the agent for binding to CAIX. For example, the number of the cells to be processed,488 And the like800 Are available as amine reactive dyes derivatized with NHS esters for labeling primary amines (product numbers 46402 and 46421, sammer Electric, rockford, ill.). The skilled artisan will recognize that the fluorescent probes used are not limiting, and othersDyes 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 may be conjugated with a fluorescent probe (to form an immunoconjugate). Methods for covalently conjugating fluorescent probes and other functional groups are known in the art, and any such known methods may be utilized. For example, the fluorescent probe may be attached at the hinge region of the reduced antibody component by disulfide bond formation or thiol-maleimide interactions. Alternatively, such agents may be linked using heterobifunctional cross-linking agents such as N-succinyl 3- (2-pyridyldithio) propionate (SPDP). Yu et al, J.cancer International journal (int.J.cancer) 56:244 (1994). General techniques for such conjugation are well known in the art. See, e.g., wong, protein conjugation and crosslinking chemistry (CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING) (CRC Press 1991), upeslacis et al, "modification of antibodies by chemical means (Modification of Antibodies by Chemical Methods)", monoclonal antibodies: principle and use (MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS), birch et al (edit), pages 187-230 (Wiley-Liss, inc.) 1995), price, "production and characterization of synthetic peptide-derived antibodies (Production and Characterization of SYNTHETIC PEPTIDE-Derived Antibodies)", monoclonal antibodies: production, engineering and clinical use (MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION), ritter et al (edit), pages 60-84 (Cambridge university Press Cambridge University Press 1995).

Alternatively, the fluorescent probe may be conjugated through a carbohydrate moiety in the Fc region of the antibody. See, for example, shih et al, international journal of cancer 41:832 (1988), shih et al, international journal of cancer 46:1101 (1990), and Shih et al, U.S. Pat. No. 5,057,313, examples of which are incorporated herein by reference. The general method involves reacting an antibody component having an oxidized carbohydrate moiety with a fluorescent probe having at least one free amine function. This reaction produces an initial schiff base (imine) bond, 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 not be present. However, it is possible to introduce carbohydrate moieties into the light chain variable region of a full length antibody or antibody fragment. See, for example, leung et al, J.Immunol.) "154:5919 (1995), U.S. Pat. Nos. 5,443,953 and 6,254,868, the examples of which are incorporated herein by reference. The engineered carbohydrate moiety is used to attach a functional group to the antibody fragment.

An alternative method for attaching fluorescent probes or other functional groups to targeting molecules involves the use of click chemistry. Click chemistry methods were originally conceived as methods for rapidly producing complex substances by connecting small subunits together in a modular fashion. Various forms of click chemistry reactions are known in the art (see, e.g., kolb et al, 2004, international English edition of applied chemistry (ANGEW CHEM INT ED), 40:3004-31; evans,2007, australian journal of chemistry (Aust J Chem), 60:384-95.), such as Huisgen 1, 3-dipolar cycloaddition copper catalyzed reactions (Tornoe et al, 2002, journal of Organic chemistry, 67:3057-64), which are commonly referred to as "click reactions". Other alternatives include cycloaddition reactions such as Diels-Alder, nucleophilic substitution reactions (especially small strained rings such as epoxy and aziridine compounds), carbonyl chemical formation of urea compounds, and reactions involving carbon-carbon double bonds such as alkynes in thiol-alkyne reactions.

Copper-free click reactions for covalent modification of biomolecules have been proposed. (see, e.g., agard et al, 2004, journal of american society of chemistry (J Am Chem Soc) 126: 15046-47.) the copper-free reaction uses a ring strain instead of a copper catalyst to promote the [3+2] azide-alkyne cycloaddition reaction. For example, cyclooctyne is an 8-carbocyclic ring structure comprising internal alkyne bonds. The closed-loop structure causes significant bond angle distortion of acetylene, which highly reacts with azido groups to form triazoles. Thus, cyclooctyne derivatives can be used for copper-free click reactions.

Ning et al (2010, international English edition of applied chemistry, 49:3065-68) report another type of copper-free click reaction involving a strain-promoted alkyne-nitrone cycloaddition reaction. To address the slow rate of initial cyclooctyne reaction, electron withdrawing groups are attached adjacent to the triple bond. Examples of such substituted cyclooctynes include cyclooctynes difluoride, 4-dibenzocyclooctynol, and azacyclooctyne. An alternative copper-free reaction involves a strain-promoted alkyne-nitrone cycloaddition reaction to produce an N-alkylated isoxazolidine. The reaction is reported to have exceptionally fast reaction kinetics and is used in a one-pot three-step protocol for site-specific modification of peptides and proteins. Nitrones are prepared by condensation of the appropriate aldehyde with N-methylhydroxylamine, and the cycloaddition reaction is carried out 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 coupled to a radioisotope ¹²⁴ l. The isotope is a positron emitter that can be linked to an antibody, for example, as described in Larsson et al (journal of Nuclear medicine 33 (1992), 2020-2023) or US 5,185,142, the contents of which are incorporated herein by reference.

In any embodiment, the radiolabeling of the protein or antibody is accomplished by covalent iodination, in particular with the use of a chloroglycoluril reagent (1, 3,4, 6-tetrachloro-3 a,6 a-diphenyl glycoluril). Although the chloroglycoluril label is a solid phase oxidation process similar to the chloramine-T process, it is generally considered milder because the reaction proceeds on the surface of the oxidizing agent, thereby minimizing exposure of the substrate (Salacinzki, P.R.P. et al, analytical biochemistry (Anal. Biochem.)) 117:136 (1981)).

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

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

The skilled artisan will be familiar with standard methods for conjugating chelators to proteins, including antibodies and derivatives or fragments thereof. In addition, the skilled artisan will be familiar with methods for selecting relevant chelators for pairing with radioactive metals, e.g., as described in the chemistry society review (chem. Soc. Rev.), 2014,43,260, which is incorporated herein by reference.

In any of the embodiments, the determinable time moiety may be a fluorescent dye, such as but not limited to the fluorescent dye described in US20150086482, 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' -dimethoxyfluorescein, 5-carboxy-2 ',4',5',7' -Tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylamino, cascade Blue (Cascade Blue), cy2, cy3, cy5, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1, 3-diazole), oreg green 488, oreg green 500, oreg green 514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, pacific acid cresyl violet (CRESYL FAST violet), cresyl Blue violet (cresyl Blue violet), leucinblue (brilliant cresyl Blue), para-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tris bipyridyl diamine europium (europium trisbipyridine diamine), europium cryptate or chelate, diamine, bisanthocyanin, la Jolla Blue dye (La Jolla Blue dye), allophycocyanin (allopycocyanin), allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiool), tetramethyl rhodamine, and texas red.

Administration of pharmaceutical agents

The skilled person will appreciate that the dosage of the medicament for use in the method according to the invention will depend on a variety of factors including the age, sex, height and weight of the subject to whom the medicament is to be administered, and on the medicament.

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

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

Antibodies are typically administered as pharmaceutical compositions with a pharmaceutically acceptable carrier (e.g., physiological saline solution), optionally containing a protein stabilizing agent, such as Human Serum Albumin (HSA). The antibody is preferably administered by infusion.

CAIX-binding agent (preferably gemtuximab) 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 about 15 minutes. Of course, CAIX inhibitors may also be administered intraperitoneally or intramuscularly.

Detection method

It will be appreciated that the method used to detect or image an agent for use in accordance with the present invention will depend on the nature of the detectable portion 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 in a tumor include the use of in vivo/partial or whole body imaging techniques, such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging. immune-PET and immune-SPECT imaging can include the use of CAIX-binding molecules conjugated to radioisotopes to achieve non-invasive imaging of CAIX-expressing tissues and tumors.

Where the detectable moiety is a radioisotope, the method will therefore involve determining radiation for the subject to whom the agent is administered.

The in vivo detection step in the methods described above may be whole body imaging or local imaging of a specific site, such as, but not limited to, the intended or likely site of growth of a solid tumor.

In the case of SPECT, the agent for binding to CAIX typically comprises a detectable agent in the form of a gamma emitting radioisotope (radionuclide), usually by injection into the bloodstream. Typically, gamma-emitting radioisotopes for 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 comprises PET/CT imaging or PET/MRI scanning.

After administration (preferably infusion) of the agent, it may be practical to wait for 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 is at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days. Preferably, the period of time between administration of the agent and detection of the agent (e.g., by PET or other 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.

In the case of imaging or detecting cancer using PET, PET imaging may preferably be performed within 7±2 days of infusion of the radiolabeled agent, particularly within 5±2 days after infusion, in order to obtain optimal imaging results, including accumulation of the agent at the site where CAIX is present.

In the context of the detectable moiety being a fluorescent probe or dye, fluorescent imaging may be used to detect the moiety, including during intraoperative, intravascular, or endoscopic procedures, as described in U.S. patent nos. 4,932,412, 6,096,289, 6,387,350, 7,201,890, the examples of each of which are incorporated herein by reference. Such imaging methods may be used, for example, to image the distribution of tumor tissue in order to facilitate removal of the tumor tissue. Fluorescence imaging can also be used for diagnostic purposes, for example, to distinguish malignant, benign and proliferative tissues.

Cancer to be detected or diagnosed

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

Thus, in a preferred embodiment, the methods of the present invention are capable of diagnosing any of the cancers listed herein as the sole, primary or primary mode of cancer diagnosis, and preferably without the need for additional invasive diagnostic methods, including biopsy-related methods.

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

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

The methods of the invention find particular use in imaging, detecting and/or diagnosing cancers that have not been previously identified using in vivo imaging techniques that utilize agents for binding to CAIX.

A subject for whom cancer diagnosis or detection or imaging as 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 have one or more genetic markers indicative of risk or likelihood of developing cancer. A subject considered at risk for cancer may exhibit one or more symptoms of cancer, may have a family history of cancer, or may have one or more genetic markers indicative of risk or likelihood of developing cancer.

In any embodiment, the cancer detected, imaged, or diagnosed is breast cancer. High levels of CAIX in breast cancer have been previously reported, and CAIX expression has also been reported as an indicator of resistance to chemotherapy or treatment success. These observations can be traced back to decades ago, and before that, this situation of diagnosis of this patient group using CAIX binding imaging agents has not been previously reported.

Breast cancer may be a so-called "triple negative breast cancer" (TNBC), a invasive, metastatic and drug resistant form of breast cancer, with limited treatment options, and 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 (HER 2 positive breast cancer).

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

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

In any embodiment, the cancer detected, imaged, or diagnosed is cervical cancer. Cervical cancer may be squamous cell carcinoma or adenocarcinoma. In any embodiment, a subject diagnosed or imaged for cervical cancer may exhibit one or more cervical cancer symptoms, such as abnormal vaginal bleeding, including contact bleeding, or pelvic pain. A subject considered to be at risk for developing cervical cancer may have previously been infected with HPV 16 or 18 strain or have one or more genetic markers indicative of being at risk for cervical cancer.

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

In any embodiment, the cancer detected, imaged or diagnosed is esophageal cancer (including esophageal Squamous Cell Carcinoma (SCC) and esophageal/esophageal-gastric junction adenocarcinoma). Subjects diagnosed with or imaged or detected for esophageal cancer may exhibit one or more symptoms selected from dysphagia, unexplained weight loss, chest pain, stress or burning, worsening dyspepsia or heartburn, or cough or hoarseness. A subject considered to be at risk of having esophageal cancer may have a family history of the disease or have one or more genetic markers considered to be associated with an increased risk of colorectal cancer or may have previously been diagnosed with Barrett's esophagus.

In any embodiment, the cancer that is detected, imaged, or diagnosed is gastric cancer (including gastric adenocarcinoma). Subjects diagnosed with or imaged or detected for gastric cancer may exhibit one or more symptoms selected from dysphagia, gastric pain, feeling bloated after a small meal, loss of appetite, dyspepsia, nausea and vomiting, fatigue, and darkening of stool. A subject considered at risk for developing gastric cancer may have a family history of the disease or have one or more genetic markers considered to be associated with an increased risk for gastric cancer.

In any embodiment, the cancer detected, imaged or diagnosed is glioblastoma multiforme. Subjects diagnosed with, or imaged or detected by, glioblastoma may exhibit one or more symptoms including visual, auditory, equilibrium, coordination, strength, and reflex symptoms, nausea, vomiting, seizures, or other neurological symptoms.

In any embodiment, the cancer detected, imaged, or diagnosed is a head and neck cancer (including head and neck squamous cell carcinoma and nasopharyngeal and hypopharyngeal carcinoma). Subjects diagnosed with or 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, unexplained weight loss. A subject considered at risk for developing head and neck cancer may have a family history of the disease, have one or more genetic markers considered to be associated with an increased risk for head and neck cancer, may have been previously infected with HPV or Epstein-Barr virus (Epstein-Barr virus), have a weakened immune system, have poor oral hygiene (including gum disease), smoke or chewing betel nut (betel nut), betel nut (areca nut), vicat (gutka) or pan an (pan), or have a genetic condition such as fanconi anemia or Li Famei ni syndrome (Li-Fraumeni syndrome).

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

As used herein, one or more bile duct cancerous conditions include abdominal pain, yellowing of the skin (jaundice), weight loss, systemic pruritus, fever, light stool, or dark urine. The skilled artisan will be familiar with various risk factors for cholangiocarcinomas, including primary sclerosing cholangitis (inflammatory disease of the bile duct), ulcerative colitis, cirrhosis, hepatitis c, hepatitis b, certain fasciola hepatica infections, and some congenital hepatic malformations. However, most people have no identifiable risk factors.

In any embodiment, the cancer that is detected, imaged, or diagnosed is lung cancer (including epithelial non-small cell cancer and small cell cancer). As used herein, the term "lung cancer" includes, but is not limited to, all types of lung cancer in all stages of progression, such as lung cancer, metastatic lung cancer, non-small cell lung cancer (NSCLC) such as lung adenocarcinoma, squamous cell carcinoma, or Small Cell Lung Cancer (SCLC). In some embodiments, the subject has non-small cell lung cancer (NSCLC).

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

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. Bladder cancer may be non-muscle invasive bladder cancer (NMIBC).

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

Imaging or diagnosis of cancer will typically be assessed after administration of the agent and detection thereof by qualitatively assessing detection of the agent as compared to conventional imaging. Quantitative assessment can be based on each lesion, including normalized uptake value (SUV) (SUV maximum and SUV mean), SUV corrected for lean body mass (SUL), metabolic Tumor Volume (MTV), and tumor To Background Ratio (TBR).

Tumor To Background Ratio (TBR) will typically be defined as the ratio of lesion normalized uptake value (SUV max) to reference area SUV (liver, blood pool, etc.). The type of lesion and the number, size and other characteristics indicative of the detected lesions will be compared through PET scanning and standard imaging modalities, including high resolution CT/MRI and other potential imaging per patient (depending on the tumor type).

Qualitative visual analysis of imaging (the presence or absence of local agent uptake associated with a tumor, as seen on contrast-enhanced CT, MRI or FDG PET/CT) can be used to assess the consistency of tumor lesion detection between a particular agent PET/CT and conventional imaging. The RECIST 1.1 standard for conventional imaging can be used as the primary tool for consistency comparisons with PET.

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

Examples

Example 1 clinical trial protocol

The assay involved using ⁸⁹ Zr labeled gemtuximab deferoxamine PET/CT imaging to assess CAIX expression in a subset of solid tumors.

Main objective

For non-invasive assessment of the PET/CT imaging of ⁸⁹ Zr-gemtuximab expressed in CAIX tumors in different solid tumors. Formal imaging studies have not been performed on the uptake ⁸⁹ Zr-gemtuximab for these tumor types.

The main endpoint is qualitative (yes/no) and quantitative assessment of 89 Zr-gemtuximab uptake compared to conventional imaging. Descriptive statistics are reported for each tumor type. Each lesion analysis included SUV maximum, SUV mean, SUL (SUV corrected for lean body mass), tumor To Background Ratio (TBR), and Metabolic Tumor Volume (MTV).

Secondary target

To assess the tolerability and safety of ⁸⁹ Zr-gemtuximab administration in patients with different tumor types.

Secondary endpoint patient safety is assessed based on the incidence and nature of Adverse Events (AEs) and Severe Adverse Events (SAE) as well as clinically significant changes in laboratory test values, vital signs, or physical examination results. Laboratory abnormalities were assessed according to NCI CTCAE v.5.0. Patients were notified that abnormal physical signs occurred within 24 hours after the examination, they had to report to the main investigator and enroll. Reference was made to version 5.0 using the NCI co-toxicity standard.

Three-level target

To assess the correlation between normalized uptake value (SUV) of ⁸⁹ Zr-gemtuximab and CAIX histological expression in patients undergoing biopsies or surgery within 90 days of ⁸⁹ Zr-gemtuximab imaging.

Tertiary endpoint if a biopsy or surgical sample is available (and its tissue sample is available) 90 days prior to administration or 90 days after ⁸⁹ Zr-gemtuximab imaging, the correlation between normalized uptake value (SUV) of ⁸⁹ Zr-gemtuximab and CAIX histological expression is assessed by comparing 89 Zr-gemtuximab semi-quantitative data with immunohistochemical results (IHC) of biopsied/resected tumor at the site.

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

General study design

Open-label, non-randomized studies were performed to assess the expression of CAIX by ⁸⁹ Zr-gemtuximab PET/CT imaging in different tumor types and to assess the feasibility of targeting CAIX for potential diagnostic and therapeutic applications.

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

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

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

Qualitative visual analysis (presence or absence of tumor-associated local ⁸⁹ Zr-gemtuzumab uptake, as seen on contrast-enhanced CT, MRI or FDG PET/CT) was used to assess the consistency of tumor lesion detection between 89 Zr-gemtuximab PET/CT and conventional imaging. Lesions shown by 89 Zr-gemtuximab alone are described.

Tissue samples (biopsies or surgery from patients) were collected as much as possible and sent to a central laboratory for CAIX expression analysis.

Study evaluation was performed as shown in the following table:

¹ Can be obtained within 28 days prior to day 0 outside of the study

² Only in the case of clinical indications

³ Remote visit (telephone follow-up visit)

⁴ Pre-study procedure to be performed within 30 days after day 0

⁵ CT is contraindicated if the patient cannot experience PET/CT, or PET/MRI may be the preferred option for the patient, instead of PET/CT, PET/MRI may be performed.

Administration of drugs

The ⁸⁹ Zr-gemtuzumab dose used herein (37 MBq [1mCi ] + -10%, containing gemtuximab at a mass dose of 10 mg) was consistent with the dosing regimen of 89 Zr-gemtuximab in the ongoing phase 3 clinical trial, and Merkx et al, (2021) showed that PET imaging was allowed 4-7 days after administration.

⁸⁹ Zr-gemtuximab is a radiolabeled chimeric monoclonal antibody (INN name: zr ⁸⁹ gemtuximab deferoxamine). Gemtuximab is a chimeric monoclonal antibody (INN: gemtuximab, synonym: cG250, TLX 250) specific for CAIX (carbonic anhydrase 9) antigen, radiolabeled with positron-emitting radiometal zirconium-89, which is linked to the lysine residue of gemtuximab via NSuc-DFO-TFP-ester (DFO-TFP) to produce ⁸⁹ Zr-DFO-gemtuximab.

⁸⁹ Zr-gemtuzumab was formulated as a solution containing a total of 10mg of gemtuximab for single intravenous use for intravenous administration at a nominal dose intensity of 37MBq (+ -10%) (1 mci+ -10%). ⁸⁹ Zr-gemtuximab solution for intravenous administration is supplied in a glass vial or syringe (depending on the geographical area) in a suitable package (lead shielded container with a radioactive warning symbol according to the radiopharmaceutical requirements).

The compound was administered as a single dose of ⁸⁹ Zr-gemtuzumab (37 mega Bei Keer ±10%, [1mci±0.1mCi ], containing a mass dose of 10mg of gemtuximab) by slow intravenous injection over 3 minutes via a single peripherally placed intravenous cannula. The injection volume was about 10ml, depending on the activity administered.

Inclusion criteria

All participants will meet the following criteria:

1. Written and voluntarily given informed consent.

2. Men or women with an age of 18 years or more at the time of consent.

3. Is capable of learning about the study and is willing and able to follow all protocol requirements.

4. The participants must have histologically or cytologically proven solid tumors of the following types, but are not limited to:

● Cervical cancer

● Colorectal cancer

● Esophageal cancer (esophageal SCC and esophageal/esophageal gastric junction adenocarcinoma)

● Stomach cancer (stomach gland cancer)

● Glioblastoma multiforme

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

● Liver cancer (bile duct cancer and hepatocellular carcinoma)

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

● Ovarian cancer (epithelial ovarian cancer)

● Pancreatic cancer (pancreatic duct adenocarcinoma)

● Sarcoma of soft tissue

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

6. Participants agreed to participate in the study while not participating in another intervention study, defined as signing an Informed Consent Form (ICF) until the last study visit was completed.

7. Negative serum pregnancy test for female patients with fertility potential when negative pregnancy test results were screened and confirmed from urine within 24 hours prior to receiving study product. Female patients without fertility potential must be screened for evidence by meeting one of the following criteria:

● Postmenopausal is defined as all exogenous hormonal treatments that are older than 50 years of age and amenorrhea for at least 12 months after cessation of treatment.

● Women under 50 years old will be considered postmenopausal if they have amenorrhea for 12 months or more after cessation of exogenous hormone therapy and Luteinizing Hormone (LH) and follicular hormone (FSH) levels are within institutionally specified postmenopausal ranges.

● Recording of irreversible sterilization surgery by hysterectomy, bilateral ovariectomy or bilateral tubectomy instead of tubal ligation.

8. For all participants, dual barrier contraception was agreed to be performed until a minimum of 42 days after ⁸⁹ Zr-gemtuximab administration.

Exclusion criteria

Patients were excluded from participation in the trial if one or more of the following criteria were met:

1. Murine or chimeric antibodies have been contacted in the last 5 years.

2. Any radionuclide was previously administered within 10 half-lives (of the radionuclide) (i.e., within 10 half-lives after day 0) before the ⁸⁹ Zr-gemtuximab was expected to be administered.

3. Any CAIX targeting compound was contacted (diagnosis/treatment) within the last 3 months.

4. Serious non-malignant diseases (e.g., psychosis, infectious diseases, autoimmune diseases, or metabolic diseases) that may interfere with safety or compliance of a study target or subject at the discretion of the researcher.

5. Any clinically significant abnormalities detected during screening laboratory tests or physical examinations, researchers believe that these abnormalities will adversely affect the ability of the participants to participate in the study. The main investigator evaluates whether the patient is suitable for inclusion based on pathology and tumor type.

6. Mental impairment of the ability to give informed consent and to follow the requirements of the study may be compromised.

7. Any anti-tumor treatment was contacted within 14 days from the day of planned administration of ⁸⁹ Zr-gemtuximab (i.e., within 14 days after day 0).

8. Pregnant or lactating females.

9. It is known to be allergic, hypersensitive or intolerant to gemtuximab, DFO (deferoxamine) or any component of the study agent.

10. Renal insufficiency, glomerular Filtration Rate (GFR) of less than or equal to 45 ml/min/1.73 m2.

11. Patients who are vulnerable (e.g., are arrested).

Efficacy assessment

Imaging is based on the ability to non-invasively assess CAIX tumor expression in patients using PET/CT imaging of ⁸⁹ Zr-gemtuximab. After a single administration of ⁸⁹ Zr-gemtuximab on day 0, a whole body PET/CT scan was performed on day 5±2 post administration according to the table and imaging manual above. Patients with metastatic (suspected or diagnosed) disease may receive an optional additional whole-body PET/CT scan if clinically indicated (e.g., if the tumor to background ratio makes the tumor lesions difficult to identify and hopefully improve).

The ⁸⁹ Zr-gemtuzumab tumor uptake (yes/no) will be qualitatively assessed compared to conventional imaging, with up to 10 most active lesions in individual patients. Quantitative assessment will be based on each lesion (including SUV maximum, SUV mean, SUL, MTV and TBR).

For patients who are not CT-scanned for any reason and/or patients with CT scanning contraindications, PET/MRI may be performed instead of PET/CT if available at the study site. PET/MRI may also be performed in patients where the disease condition may be better visualized by MRI (e.g., GBMR).

Tumor To Background Ratio (TBR) will be defined as the ratio of lesion SUV maximum to reference area SUV (liver, blood pool, etc.). The PET scan through ⁸⁹ Zr-gemtuximab and standard imaging modalities (including high resolution CT/MRI and other potential imaging per patient (depending on tumor type)), the type of lesion and the number, size and other characteristics indicative of the detected lesions will be compared.

Qualitative visual analysis (presence or absence of tumor-associated local ⁸⁹ Zr-gemtuzumab uptake, as seen on contrast-enhanced CT, MRI or FDG PET/CT) was used to assess consistency of tumor lesion detection between ⁸⁹ Zr-gemtuximab PET/CT and conventional imaging. The RECIST 1.1 standard for conventional imaging must be used as much as possible as the primary tool for consistency comparisons with PET. These guidelines should also be followed for tumor radiation assessment using different recommendations according to the scientific oncology guidelines (Clinical Guidelines of Scientific Oncology Societies). In addition to the above, all visible tumor lesions in conventional imaging can also be compared to PET imaging results.

For each patient, the SUV maximum, SUV mean, SUL, MTV, TBR and conventional imaging compliance rates will be calculated locally by the nuclear medicine specialist at each site and incorporated into the eCRF. The detailed information will be included in the imaging manual.

Example 2: radiolabeled Ji preparation of toximab

Radiolabeled gemtuximab was prepared as described previously (see e.g. WO 2021/000017). Briefly, bioconjugated gemtuximab was prepared using standard techniques to obtain DOTA-gemtuximab or DFO-gemtuximab, followed by labeling with a radioisotope (e.g., ⁸⁹ Zr) that can be used for imaging.

Example 3 in vitro and in vivo binding of radiolabeled gemtuximab to various cancers

Imaging studies were performed using radiolabeled DOTA-gemtuximab to assess the ability of imaging reagents to detect non-RCC cancer types.

First, the ability of radiolabeled DOTA-gemtuximab to bind to various cell lines. These data, shown in figure 1, demonstrate the ability of antibodies to bind to various cell types expressing CAIX, but the extent of binding in vitro is variable.

Subsequently, three groups of mice were tested, each group carrying a different tumor xenograft. These groups are as follows:

group 1 mice carrying an AsPc-1 cell xenograft (pancreatic cancer cell line); n=4

Group 2 mice carrying FaDu cell xenografts (squamous carcinoma pharyngeal/hypopharynx carcinoma cell line); n=4

Group 3 mice carrying HT-29 xenografts (colorectal cancer cell line) n=4

Radiolabeled gemtuximab was administered intravenously and imaged at 24 hour and 72 hour time points. Biodistribution was assessed 72 hours after administration. The radioactivity and dose of the administered antibodies are summarized in the following table:

representative images of mice from each of the three groups are shown in fig. 2.

Figure 3 shows the quantification of the percentage of injected doses in tumors (24 hours and 72 hours after injection). The results confirm observations obtained using flow cytometry, demonstrating the ability of radiolabeled antibodies to bind to each respective cancer cell line. In other words, the results demonstrate (except for FaDu cells-see further comments below) that antibodies can bind to target tumor cells with similar affinity as in vitro in an in vivo background.

The results also show that the radiolabeled antibody remains detectable in the circulation and spleen 72 hours after administration.

The in vitro biodistribution of radiolabeled DOTA-GmAb was compared to the in vivo biodistribution. Briefly, the ex vivo biodistribution corresponds to the distribution of radiolabeled DOTA-GmAb in the mouse organ, as assessed after necropsy. The in vivo biodistribution corresponds to that seen in whole mouse imaging experiments (e.g., as shown in figure 1).

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

The results show that positive in vitro binding results (e.g., good binding to HT-29 and AsPc-1 cells) can be summarized as positive binding observed in vivo.

Interestingly, the inventors observed that although radiolabeled antibodies bound very poorly to FaDu cells in vitro, antibodies were able to bind to tumor cells in vivo. These results indicate that negative findings of in vitro binding may not be predictive of in vivo binding and thus radiolabeled DOTA-GmAb has potential utility for imaging of specific cancer types.

Example 4 imaging of alternative cancer types

An experiment similar to the experiment performed in example 2 was performed using ⁸⁹ Zr-DFO-GmAb to detect the presence of tumor xenografts in the following mice:

Group 1 mice carrying xenografts of A-549 cell line (lung cancer)

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

Group 3 mice carrying HeLa cell line xenograft (cervical carcinoma)

Group 4 mice carrying AGS cell line xenograft (gastric cancer)

Group 5 mice carrying xenografts of the HepG2 cell line (liver cancer)

Group 6 mice bearing xenografts of the A2780 cell line (ovarian cancer)

Group 7 mice bearing the SK-LMS1 cell line xenograft (soft tissue sarcoma-vulvar leiomyosarcoma)

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

The results will show that the radiolabeled antibody is able to bind to tumor xenografts. In other words, the results will demonstrate that the antibodies are able to bind to target tumor cells in an in vivo background and with similar affinity as in vitro.

These results indicate that radiolabeled GmAb is suitable for producing images of different cancer types in vivo and is therefore useful as a non-invasive diagnostic reagent for diagnosing and detecting cancers other than renal cell carcinoma.

Example 4 imaging of triple negative breast cancer

Triple Negative Breast Cancer (TNBC) is an invasive, metastatic and drug resistant cancer with limited treatment options.

The inventors believe that CAIX, a hypoxia-mediated breast tumor growth regulator, may be important for maintaining breast cancer stem cells within a hypoxic region. Thus, the inventors evaluated imaging of TNBC using PET/CT imaging using ⁸⁹ Zr-labeled gemtuximab in 12 metastatic TNBC patients.

Patients received fluorodeoxyglucose F18 (FDG) and ⁸⁹ Zr-gemtuximab PET-CT and CT imaging. The patient received a single slow intravenous administration of 37.+ -. 10% MBq ⁸⁹ Zr-gemtuximab (10 mg). On day 3 post-administration, PET/CT was acquired from the skull to the middle of the thigh, with 10 minutes per bed acquisition time. Gold standard was determined by FDG PET/CT, CT and follow-up, lesions detected by at least 2 morphologies were considered true positive. Tumor SUV _{[ Maximum value , Average value of} ], total Lesion Glycolysis (TLG) and Metabolic Tumor Volume (MTV) were measured. Immunohistochemistry (IHC) was performed with Bond RX full-automatic study staining with anti-CAIX antibody (Leica, clone TH 22). Staining was assessed by semi-quantitative analysis (percentage and intensity of tumor cell expression) and SUV values were compared to the extent of CAIX expression assessed by IHC.

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

⁸⁹ The overall sensitivity of Zr-gemtuximab PET/CT was 93.2%, with sensitivity of bone, lung, breast, skin 100% and sensitivity of lymph node 87.5%. The overall sensitivity of both CT and FDG-PET/CT was 82.7%. For ⁸⁹ Zr-and ⁸⁹ Zr-gemtuximab FDG, the median tumor SUV _{Maximum value} was 3.45[ IQ:2.03-4.69] and 4.68[ IQ:3.27-10.71], respectively. IHC shows that two patients present two CAIX-high expression lesions [100%,20% ], whereas two patients present a correspondingly low profile [3%,0% ]. IHC CAIX cell status and ⁸⁹ Zr-gemtuximab SUV _{Average value of} exhibited weak correlation (rho=0.80; p=0.20). No safety problem was reported for ⁸⁹ Zr-gemtuximab.

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

Example 6 imaging of bladder cancer

Patients with non-muscle invasive bladder cancer (NMIBC) are often treated with cystectomy. Thus, new treatment options that can preserve the bladder are needed.

CAIX is expressed on the luminal surface of the papillary structures in direct contact with the bladder lumen. The present inventors conducted a pilot prospective study aimed at ensuring intravesical radioactivity limitation and tumor targeting following intravesical instillation of ⁸⁹ Zr-gemtuximab.

The patient received one intravesical instillation of ⁸⁹ Zr-gemtuzumab (10 mg) with 37±10% MBq and remained in urine for 2 hours. Then 4 PET/CT scans were performed, 3 one scan at pelvis (h+2, day 1 and day 2) and one scan at h+4 from skull to mid thigh to observe changes in radioactivity in the bladder over time.

Blood samples were collected on day 1 to determine the number of radiolikely blood vessels. For all acquisitions, an acquisition time of 10 minutes was used per bed. The gold standard was determined by a second cystoscopy and transurethral resection of the bladder (TURB) at ⁸⁹ Zr-gemtuximab PET/CT positive sites. Immunochemistry (IHC) was performed with an anti-CAIX antibody (lycra, clone TH 22). Staining was assessed by semi-quantitative analysis (percentage and intensity of tumor cell expression) and ⁸⁹ Zr-gemtuximab PET/CT bladder pattern was compared to the extent of CAIX expression assessed by IHC.

Results from 4/6 patients were obtained. Although prior intravesical perfusion BCG (BCG, a common intravesical immunotherapy for treating bladder cancer) was previously performed multiple times, each patient was identified as recurrent pTaG3.

⁸⁹ Zr-gemtuximab PET/CT showed no extra-bladder leakage. Of 2/4 patients with positive IHC, TURB demonstrated uptake spots on the bladder wall of one patient with corresponding recurrent lesions and the other patient had an inflammatory scar response. For the other two patients, no uptake consistent with negative IHC was observed. No adverse radiation contamination was observed during the process and no specific worker contact was observed.

The results (as shown in fig. 5 and 6) indicate that intravesical instillation ⁸⁹ Zr-gemtuximab showed radioactivity localized in the bladder, and that ⁸⁹ Zr-gemtuximab was useful for detection and imaging of tumors in this patient group in patients with positive IHC.

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

Claims

1. A method for in vivo imaging or detection of cancer in a subject of need, wherein the method comprises:

- Administer to the subject a drug agent for binding to CAIX expressed by the cancer, wherein the drug agent comprises a detectable portion for in vivo detection of the drug agent in the subject's body.

- Detect the drug in the subject's body.

The cancers mentioned are selected from:

Bladder cancer

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

●Cervical cancer

●Colorectal cancer

●Esophageal cancer (including esophageal squamous cell carcinoma (SCC) and adenocarcinoma of the esophageal/gastric junction)

● Stomach cancer (including gastric adenocarcinoma)

● Glioblastoma multiforme

● Head and neck cancer (including squamous cell carcinoma of the head and neck and nasopharyngeal carcinoma)

● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

● Lung cancer (including non-small cell lung cancer and small cell lung cancer)

● Ovarian cancer (including epithelial ovarian cancer)

● Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

● Soft tissue sarcoma

The presence of cancer is indicated by the detection of the drug at levels higher than the background or standard levels, thereby enabling imaging or detection of the cancer in the subject.

2. A method for diagnosing cancer in a subject of need, wherein the method comprises:

- Determine the presence or absence of the drug in the subject's body.

The cancers mentioned are selected from:

Bladder cancer

●Cervical cancer

●Colorectal cancer

● Stomach cancer (including gastric adenocarcinoma)

● Glioblastoma multiforme

● Head and neck cancers (including squamous cell carcinoma of the head and neck, hypopharyngeal carcinoma, and nasopharyngeal carcinoma)

● Liver cancer (including bile duct cancer and hepatocellular carcinoma)

● Ovarian cancer (including epithelial ovarian cancer)

● Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

● Soft tissue sarcoma

The detection of the drug at levels higher than the background or standard levels indicates that the subject has the cancer, thereby diagnosing the subject's cancer.

3. A method for generating images of cancer, the method comprising:

- Administer an effective amount of a drug agent for binding to CAIX expressed by the cancer to a subject suspected of having the cancer, wherein the drug agent comprises a detectable portion for in vivo detection of the drug in the subject.

- Detect the drug in the subject's body.

The cancers mentioned are selected from:

Bladder cancer

●Cervical cancer

●Colorectal cancer

● Stomach cancer (including gastric adenocarcinoma)

● Glioblastoma multiforme

● Liver cancer (including cholangiocarcinoma and hepatocellular carcinoma)

● Ovarian cancer (including epithelial ovarian cancer)

● Pancreatic cancer (including pancreatic ductal adenocarcinoma) and

● Soft tissue sarcoma

This produces an image of the cancer.

4. The method according to any one of claims 1 to 3, wherein the method further comprises, prior to detecting the agent or determining the presence or absence of the agent in the subject, allowing the agent to be concentrated in the subject at the site and/or tissue in which the CAIX antigen is found in the subject.

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

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

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

8. The method according to any one of claims 1 to 7, wherein the agent for binding with CAIX is a small molecule, said small molecule optionally selected from the group consisting of: SLC-0111, SLC-149, SLC-0121, SLC-101, PMI-05, sulfonamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, MIP-1486, MIP-1490, MIP-1504 (especially ^99m Tc-HEHEHE-Z09781, ^99m Tc-MIP-1486, ^99m Tc-MIP-1490 or ^99m Tc-MIP-1504/5) and PHC-102.

9. The method according to any one of claims 1 to 7, wherein the agent for binding with CAIX is a peptide, said peptide optionally selected from the group consisting of 3B-301, 3B-302 or CAIX-P1.

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

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

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

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

14. The method according to any one of the preceding claims, wherein the detectable portion of the agent is conjugated to the agent directly or via a chelating agent or a connector.

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

16. The method according to any one of claims 1 to 14, wherein the detectable portion is a radioactive isotope.

17. The method of claim 16, wherein the radioactive isotope is selected from: gallium-67 and gallium-68 ( ⁶⁷ Ga and ⁶⁸ Ga), indium-111 ( ¹¹¹ In), iodine-123, iodine-124 or iodine-131 ( ¹²³ I, ¹²⁴ I or ¹³¹ I), lutetium-177 ( ¹⁷⁷ Lu), technetium-99 ( ^99m Tc), yttrium-90 ( ⁹⁰ Y), and zirconium-89 ( ⁸⁹ Zr).

18. The method according to claims 1 to 14, 16 or 17, wherein the detectable portion is a radioactive isotope, and the detection of the agent or the detection of the presence or absence of the agent comprises determining radiation emitted by the radioactive isotope or detecting the presence or absence of the radiation.

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

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

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

22. The 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

48. The method according to any one of claims 1 to 47, wherein the agent is a radiolabeled gemtuximab, the radiolabeled gemtuximab containing one or more amino acid substitutions in the Fc region of the antibody, the one or more amino acid substitutions reducing the serum half-life of the antibody.

49. The method according to any one of claims 35 to 48, wherein the radiolabeled gemutuximab comprises gemutuximab conjugated with 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).