Combination therapy

Abstract

The present invention relates to methods and compositions for use in combined radiotherapy immunotherapy.

Description

Technical Field

[0001] The present invention relates to checkpoint inhibitors and radiolabeling agents for binding to CAIX, in particular agents such as antibodies conjugated to radioisotopes for use in radioimmunotherapy, and to combination therapies of kits and compositions for use therewith.

[0002] Related Applications This application claims priority from Australian Provisional Application Nos. 2022 / 901582 and 2022 / 902973 (the entire contents of which are incorporated herein by reference).

Background Art

[0003] Radiation therapy is an important form of cancer therapy. Various methods of radiation therapy for treating tumors have been developed. Among them, radioimmunotherapy (RAIT or RIT), also called molecular targeted radiation (MTR) or targeted radionuclide therapy (TRT), is one of the new approaches to the delivery of radiation therapy. This uses antibodies, antibody fragments or peptides that can bind to target cells to direct radioisotopes to specific tissues and cells, thus increasing the specificity of cancer treatment and reducing toxicity.

[0004] Radiation damage to healthy tissues and organs is a major problem associated with radiation therapy. Such damage is mainly due to reactive oxygen species generated by radiation that oxidize functionally important biological molecules such as nucleic acids, carbohydrates, lipids and lipoproteins, damaging tissues and cells. They are involved in various biological processes such as antibacterial defense, inflammation, carcinogenesis and aging. Decreases in white blood cell (WBC) and platelet counts, and myelosuppression and blood cell loss such as hematotoxicity, as reflected in weight loss, are the most prominent consequences of radiation damage. Toxicity significantly limits the radiation dose of RAIT and reduces the effectiveness of cancer treatment.

[0005] Several methods have been developed to attempt to reduce the hematotoxicity of radiation. Stem cell transplantation (SCT) and bone marrow transplantation (BMT) are the most frequently used methods. However, these approaches are invasive and expensive and can contribute to an extended hospital stay for the individual receiving treatment.

[0006] As other methods, using cytokines to stimulate the immune system and using blood regulatory proteins such as HP5b to arrest hematopoiesis during the period of radiation exposure can be mentioned. These methods have achieved varying degrees of success in suppressing hematotoxicity in small-scale studies, but on the other hand, patients need to be exposed to further dosing and treatment, and therefore remain little used.

[0007] Immunotherapy has been shown to be promising for cancer treatment by its ability to slow the growth and spread of cancer cells and by helping the immune system destroy existing cancer cells. Immunotherapy can assist the immune system, for example, by enhancing the adaptive immune response or the innate immune response, by regulating inhibitory pathways, by reducing immunosuppression in the tumor microenvironment, and by priming and enhancing the immune system through the stimulation of antigen-presenting cells, T cells, or innate cells.

[0008] An example of a class of immunotherapy agents is checkpoint inhibitors. Checkpoint inhibitors currently approved by the US Food and Drug Administration (FDA) target cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), or LAG3. Such checkpoint inhibitors act by blocking T cell inhibitory signals. Ipilimumab, the first approved drug, received FDA marketing approval for metastatic melanoma in 2011. Since ipilimumab, at least five or more checkpoint inhibitor drugs have been approved for at least 14 different indications.

[0009] Immune checkpoint inhibition (ICI) has substantially changed cancer treatment, but (long-term) responses have not yet been seen in the majority of patients. Important determinants for successful ICI treatment are high tumor mutational burden and existing tumor-infiltrating lymphocytes (TIL).

[0010] There remains a need for new methods to reduce the toxicity associated with radioimmunotherapy.

[0011] There also remains a need for improved immunotherapy for the treatment of cancer.

[0012] Any reference in this specification to prior art is not an admission, or suggestion, that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art is regarded as relevant, and / or reasonably foreseeable to be combined with other prior art by a person skilled in the art. SUMMARY OF THE INVENTION

[0013] The present invention provides combined immune checkpoint inhibition (ICI) and targeted radionuclide therapy (TRT). Surprisingly, the combination of ICI and TRT showed improved outcomes compared to either treatment type alone, enabled reduction of the TRT dose, and in certain embodiments, enabled immunological memory of the subject's tumor.

[0014] Accordingly, the present invention is a method of treating, preventing, or minimizing the progression of cancer in a subject, - administering to the subject a radiolabeled agent that binds to, or specifically binds to, carbonic anhydrase IX (CAIX) in combination with immune checkpoint inhibitor therapy, thereby treating, preventing, or minimizing the progression of cancer in the subject, and includes a method.

[0015] In another aspect, the present invention is a method of treating, preventing, or minimizing the progression of cancer in a subject, - Administer to a subject a radiopharmaceutical that binds or specifically binds to carbonic anhydrase IX (CAIX), - Administer to the subject an immune checkpoint inhibitor, Thereby providing a method comprising treating, preventing, or minimizing the progression of cancer in the subject.

[0016] In a further aspect, the present invention is a method of treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving checkpoint inhibitor therapy, comprising: - Administer to the subject a radiopharmaceutical that binds or specifically binds to carbonic anhydrase IX (CAIX), Thereby providing a method comprising treating, preventing, or minimizing the progression of cancer in the subject.

[0017] In any aspect or embodiment, the radiopharmaceutical can be a small molecule, peptide, or protein conjugated to a radionuclide and capable of binding to CAIX. Preferably, the agent can bind specifically and / or selectively to CAIX.

[0018] In any embodiment, the agent for binding to CAIX is optionally selected from the group consisting of SLC-0111, SLC-149, SLC-0121, SLC-101, PMI-05, sulfamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, -MIP-1486, MIP-1490, MIP-1504 (especially 99m Tc-HEHEHE-Z09781, 99m Tc-MIP-1486, 99m Tc-MIP-1490 or 99m Tc-MIP-1504 / 5), and PHC-102.

[0019] In any embodiment, the agent for binding to CAIX is optionally a peptide selected from the group consisting of 3B-301, 3B-302, or CAIX-P1.

[0020] In particularly preferred embodiments and aspects, the agent for binding to CAIX is a polypeptide comprising an antibody or an antigen-binding fragment thereof.

[0021] In one aspect, the agent is a peptide or protein, and as a result, the present invention provides a method for treating, preventing, or minimizing the progression of cancer in a subject, comprising: - administering to the subject a radiolabeled peptide or radiolabeled complex protein that binds or specifically binds to carbonic anhydrase IX (CAIX) in combination with an immune checkpoint inhibitor therapy, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0022] In another aspect, the present invention provides a method for treating, preventing, or minimizing the progression of cancer in a subject, comprising: - administering to the subject a radiolabeled peptide or radiolabeled complex protein that binds or specifically binds to carbonic anhydrase IX (CAIX), and administering to the subject an immune checkpoint inhibitor, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0023] In another aspect, the agent is a radiolabeled antibody or an antigen-binding fragment thereof, and as a result, the present invention provides a method for treating, preventing, or minimizing the progression of cancer in a subject, comprising: - administering to the subject an antibody or an antigen-binding fragment thereof for binding to CAIX, which is conjugated to a radionuclide, in combination with an immune checkpoint inhibitor therapy, Provided is a method comprising treating, preventing, or minimizing the progression of cancer in a subject.

[0024] In another aspect, the invention is a method of treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving checkpoint inhibitor therapy, comprising: - administering to the subject an antigen-binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX) and is conjugated to a radionuclide, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0025] In another aspect, the invention is a method of treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving checkpoint inhibitor therapy, comprising: - administering to the subject an antibody or antigen-binding fragment thereof for binding to CAIX, which is conjugated to a radionuclide, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0026] In any aspect of the invention, the radiolabeled agent (preferably a radiolabeled antigen-binding protein such as an antibody) that binds or specifically binds to carbonic anhydrase IX (CAIX) and the checkpoint inhibitor may be administered simultaneously. Alternatively, they may be administered sequentially. For example, the checkpoint inhibitor may be administered before the radiolabeled agent (preferably the radiolabeled antigen-binding protein), or the radiolabeled agent (preferably the radiolabeled antigen-binding protein) may be administered before the checkpoint inhibitor. Alternatively, the treatments with the checkpoint inhibitor and / or the radiolabeled agent (preferably the radiolabeled antigen-binding protein) may be staggered.

[0027] In any aspect or embodiment, the immune checkpoint inhibitor therapy targets any one or more of PD-1, PD-L1, and CTLA-4 and is a PD-1, PD-L1, and / or CTLA-4 checkpoint inhibitor. The checkpoint inhibitor can be an antibody or an antigen-binding fragment thereof, a protein, a peptide, or a small molecule. In any aspect or embodiment, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 in the form of an antibody or an antigen-binding fragment thereof. In any aspect or embodiment, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 in the form of a peptide. In any aspect or embodiment, the checkpoint inhibitor is an inhibitor of PD-1. In any aspect or embodiment, the checkpoint inhibitor is an inhibitor of CTLA-4. In any aspect or embodiment, the immune checkpoint inhibitor therapy involves administering inhibitors of PD-1 and CTLA-4. In any aspect or embodiment, the immune checkpoint inhibitor therapy involves administering inhibitors of PD-L1 and CTLA-4.

[0028] In any aspect or embodiment described herein, an agent that binds or specifically binds to carbonic anhydrase IX (CAIX) may be an antibody or an antigen-binding fragment thereof that binds or specifically binds to CAIX. In some embodiments, the antibody is gilentuximab, or a variant or antigen-binding fragment thereof, that retains the ability to bind to CAIX.

[0029] A radiolabeling agent (preferably a radiolabeled antibody or a fragment thereof) is actinium-225 ( 225 Ac), astatine-211 ( 211 At), bismuth-212 and bismuth-213 ( 212 Bi, 213 Bi), copper-64 and copper-67 ( 64 Cu, 67 Cu), iodine-123, -124, -125 or -131 ( 123 I, 124 I, 125 I, 131 I) (123 I), lead-212 ( 212 Pb), lutetium-177 ( 177 Lu), radium-223 and radium-224 ( 223 Ra, 224 Ra), rhenium-186 and rhenium-188 ( 186 Re and 188 Re), samarium-153 ( 153 Sm), scandium-47 ( 47 Sc), strontium-90 ( 90 Sr), terbium-149 and terbium-161 ( 149 Tb and 161 Tb), zirconium ( 89 Zr), and yttrium-90 ( 90 Y), may be conjugated to any suitable radionuclide for therapeutic use. In some embodiments, the agent is an antibody or an antigen-binding fragment thereof, and the radionuclide conjugated to the antibody or fragment thereof is lutetium- 177 is.

[0030] As used herein, the terms radiolabeled complex and radiolabel may be used interchangeably. These terms are understood to refer to an agent conjugated to a radionuclide (e.g., a small molecule, peptide, or polypeptide such as an antibody or antigen-binding fragment thereof). It will also be understood that the terms radionuclide and radioisotope may be used interchangeably herein.

[0031] In another aspect, the present invention provides a method of inducing an immune response, preferably a T cell immune response, against cancer in a subject, the method comprising administering to the subject a radiolabeled agent for binding to CAIX in combination with an immune checkpoint inhibitor, thereby inducing a T cell response against cancer in the subject. Preferably, the radiolabeled agent is a radiolabeled peptide, or a radiolabeled antibody or an antigen-binding fragment thereof.

[0032] In another aspect, the present invention provides a method of inducing an adaptive immune response against cancer in a subject, the method comprising administering to the subject, in combination with an immune checkpoint inhibitor, a radiolabeled agent that binds to or specifically binds to CAIX, thereby inducing a T cell response against cancer in the subject. Preferably, the radiolabeled agent is a radiolabeled peptide, or a radiolabeled antibody or antigen-binding fragment thereof.

[0033] In any aspect or embodiment of the present invention, the methods described herein further comprise identifying a subject having cancer. In one embodiment, the cancer may be premalignant or non-metastatic. In another embodiment, the cancer may be malignant or metastatic.

[0034] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising: - identifying a subject having cancer who has received or is receiving a checkpoint inhibitor for the treatment of cancer; - evaluating whether the subject is responsive to the checkpoint inhibitor; if the subject is not responsive to the checkpoint inhibitor, - administering to the subject a radiolabeled agent that binds to or specifically binds to CAIX (preferably, a radiolabeled antigen-binding protein), thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0035] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising: - identifying a subject having cancer who is not responsive to treatment with a checkpoint inhibitor; - administering to the subject a radiolabeled agent comprising, consisting of, or essentially consisting of a radiolabeled antigen-binding protein that binds to or specifically binds to CAIX, Thereby providing a method comprising the step of treating, preventing, or minimizing the progression of cancer in a subject.

[0036] In another aspect, the present invention is a method of treating, preventing, or minimizing the progression of cancer in a subject, - identifying a subject having cancer; - administering to the subject a radiolabeled agent (preferably a radiolabeled antigen-binding protein) that binds to or specifically binds to CAIX and a checkpoint inhibitor, Thereby providing a method comprising the step of treating, preventing, or minimizing the progression of cancer in the subject.

[0037] In another aspect, the present invention is a method of treating, preventing, or minimizing the progression of cancer in a subject, - identifying a subject having cancer; - administering to the subject a radiolabeled agent (preferably a radiolabeled antibody or an antigen-binding fragment thereof) for binding to CAIX and a checkpoint inhibitor, Thereby providing a method comprising the step of treating, preventing, or minimizing the progression of cancer in the subject.

[0038] In another aspect, the present invention is a method of increasing the survival rate of a subject having cancer, comprising administering to the subject a radiolabeled agent (preferably a radiolabeled antigen-binding protein) that binds to or specifically binds to CAIX and a checkpoint inhibitor, thereby increasing the survival rate of the subject having cancer.

[0039] In another aspect, the present invention is a method of increasing the survival rate of a subject having cancer, comprising administering to the subject a radiolabeled agent (preferably a radiolabeled antibody or an antigen-binding fragment thereof) for binding to CAIX and a checkpoint inhibitor, thereby increasing the survival rate of the subject having cancer.

[0040] In another aspect, the present invention provides a method for minimizing, reducing, or preventing tumor growth in a subject having cancer, the method comprising administering to the subject a radiolabeling agent that binds to or specifically binds to CAIX (preferably a radiolabeled antigen-binding protein) in combination with an immune checkpoint inhibitor therapy, thereby minimizing, reducing, or preventing tumor growth in the subject having cancer.

[0041] In another aspect, the present invention provides a method for minimizing, reducing, or preventing tumor growth in a subject having cancer, the method comprising administering to the subject a radiolabeling agent (preferably a radiolabeled antibody or an antigen-binding fragment thereof) for binding to CAIX in combination with an immune checkpoint inhibitor therapy, thereby minimizing, reducing, or preventing tumor growth in the subject having cancer.

[0042] In another aspect, the present invention provides a method for minimizing, reducing, or preventing metastasis in a subject having cancer, the method comprising administering to the subject a radiolabeling agent that binds to or specifically binds to CAIX (preferably a radiolabeled antigen-binding protein) in combination with an immune checkpoint inhibitor therapy, thereby minimizing, reducing, or preventing metastasis in the subject having cancer.

[0043] In any aspect or embodiment, the present invention provides a method for minimizing, reducing, or preventing metastasis in a subject having cancer, the method comprising: - identifying a subject having a primary tumor; - removing the primary tumor from the subject; - administering to the subject a radiolabeling agent that binds to or specifically binds to CAIX (such as a radiolabeled antigen-binding protein) in combination with an immune checkpoint inhibitor therapy; Further provided is a method comprising minimizing, reducing, or preventing metastasis in a subject having cancer.

[0044] In another aspect, the present invention provides a method of minimizing, reducing, or preventing tumor growth at at least one site distant from the site of another tumor (e.g., a primary tumor) in a subject, the method comprising administering to the subject a radiolabeled agent (preferably a radiolabeled antigen-binding protein) that binds or specifically binds to CAIX, in combination with an immune checkpoint inhibitor therapy, thereby minimizing, reducing, or preventing tumor growth at at least one site distant from the site of the primary tumor in the subject.

[0045] In any aspect or embodiment of the present invention, the immune checkpoint inhibitor therapy may comprise administering one or more immune checkpoint inhibitors.

[0046] In any aspect or embodiment of the present invention, the method may further comprise conjugating a radionuclide to an antigen-binding protein that binds or specifically binds to CAIX to provide a radiolabeled antigen-binding protein that binds or specifically binds to CAIX.

[0047] In another aspect, the present invention further provides the use of an agent comprising, consisting of, or essentially consisting of a small molecule, peptide, or antigen-binding protein that binds or specifically binds to CAIX, and / or a checkpoint inhibitor, in the manufacture of a medicament for treating, preventing, or minimizing cancer progression in a subject. Preferably, the agent is conjugated or can be conjugated to a radionuclide.

[0048] In another aspect, the present invention relates to the use of an agent (a small molecule, peptide, or antigen-binding protein, preferably an antigen-binding protein, etc.) that binds to or specifically binds to CAIX in the manufacture of a first pharmaceutical, and a checkpoint inhibitor in the manufacture of a second pharmaceutical, wherein the first and second pharmaceuticals are · for treating, preventing, or minimizing the progression of cancer in a subject, · for minimizing, reducing, or preventing the growth of a tumor in a subject, · for minimizing, reducing, or preventing metastasis in a subject, or · for increasing the survival time of a subject, Preferably, the present invention further provides a use wherein the agent is conjugated to a radionuclide.

[0049] Alternatively, the first and second pharmaceuticals are for any other method or use of the present invention described herein.

[0050] In another aspect, the present invention provides an agent (a small molecule, peptide, or antigen-binding protein, preferably an antigen-binding protein, etc.) that binds to or specifically binds to CAIX, wherein the agent is conjugated to a radionuclide or can be conjugated to a radionuclide for use in combination with a checkpoint inhibitor therapy for treating, preventing, or preventing the progression of cancer in a subject.

[0051] In another aspect, the present invention further provides the use of an agent (a small molecule, peptide, or antigen-binding protein, preferably an antigen-binding protein, etc.) that binds to or specifically binds to CAIX and is conjugated to a radionuclide, and a checkpoint inhibitor, for treating, preventing, or preventing the progression of cancer in a subject.

[0052] In another aspect, the present invention further provides an agent (a small molecule, a peptide, or an antigen-binding protein, preferably an antigen-binding protein, etc.) that binds to or specifically binds to CAIX and is conjugated to a radionuclide, for use in treating, preventing, or preventing the progression of cancer in a subject in combination with an immune checkpoint inhibitor therapy. Alternatively, an agent that binds to or specifically binds to CAIX and a checkpoint inhibitor are for use in any other method or use of the present invention described herein, including treating, preventing, or minimizing the progression of cancer in a subject, minimizing, reducing, or preventing tumor growth in a subject, minimizing, reducing, or preventing metastasis in a subject, or increasing the survival rate of a subject.

[0053] In another aspect, the present invention · Treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving a checkpoint inhibitor, · Minimizing, reducing, or preventing the growth of tumors in a subject who has received or is receiving a checkpoint inhibitor, · Minimizing, reducing, or preventing metastasis in a subject who has received or is receiving a checkpoint inhibitor, or · Use of an agent (a small molecule, a peptide, or an antigen-binding protein, preferably an antigen-binding protein, etc.) that binds to or specifically binds to CAIX in the manufacture of a medicament for increasing the survival rate of a subject who has received or is receiving a checkpoint inhibitor, Preferably, the agent is conjugated to or can be conjugated to a radionuclide, and further provides the use.

[0054] In any aspect or embodiment of the present invention, any pharmaceutical described herein is suitable for administration intraperitoneally, intratumorally, locally, orally, intravenously, to the airway (preferably by inhalation or intranasally), subcutaneously, or intramuscularly. Typically, any pharmaceutical described herein is suitable for intravenous administration.

[0055] In another aspect, the present invention provides an antigen-binding protein that binds to or specifically binds to CAIX for use in treating, preventing, or minimizing cancer progression in a subject that has received or is receiving a checkpoint inhibitor, preferably, the antigen-binding protein is conjugated to a radionuclide. Alternatively, the antigen-binding protein that binds to or specifically binds to CAIX is for use in any other method or use of the present invention described herein. In one aspect of the present invention, the antigen-binding protein that binds to or specifically binds to CAIX and is conjugated to a radionuclide is preferably suitable for administration intraperitoneally, intratumorally, locally, orally, to the airway (preferably by inhalation or intranasally), intravenously, subcutaneously, or intramuscularly. Preferably, the antigen-binding protein that binds to or specifically binds to CAIX is suitable for intravenous administration.

[0056] In another aspect, the present invention provides the use of an antigen-binding protein that binds to or specifically binds to CAIX for treating, preventing, or minimizing cancer progression in a subject that has received or is receiving a checkpoint inhibitor, preferably, the antigen-binding protein is conjugated to a radionuclide. Alternatively, the use of the antigen-binding protein that binds to or specifically binds to CAIX is any other method or use of the present invention described herein.

[0057] In any of the uses described herein, a peptide or antigen-binding protein that binds to or specifically binds to CAIX may be conjugated to a radionuclide via a linker or chelating agent. The radionuclide may be actinium-225 ( 225 Ac), astatine-211 ( 211 At), bismuth-212 and bismuth-213 ( 212 Bi, 213 Bi), copper-64 and copper-67 ( 64 Cu, 67 Cu), iodine-123, -124, -125 or -131 ( 123 I, 124 I, 125 I, 131 I)( 123 I), lead-212 ( 212 Pb), lutetium-177 ( 177 Lu), radium-223 and radium-224 ( 223 Ra, 224 Ra), samarium-153 ( 153 Sm), scandium-47 ( 47 Sc), strontium-90 ( 90 Sr), and yttrium-90 ( 90 Y). In some embodiments, the radionuclide is lutetium-177.

[0058] In any embodiment of the invention, the therapeutic effect of any radiocompound (such as an antigen-binding protein) that binds to or specifically binds to CAIX described herein and a checkpoint inhibitor may be significant compared to the effect of the radiocompound or checkpoint inhibitor alone (i.e., compared to monotherapy with a radiocompound that binds to or specifically binds to CAIX or monotherapy with a checkpoint inhibitor). In one aspect, the effect may be additive or synergistic. Preferably, the effect is synergistic.

[0059] In another aspect, when a radioactive conjugate (preferably a radiolabeled antigen-binding protein) that binds to or specifically binds to CAIX and a checkpoint inhibitor are administered to a subject, the radioactive conjugate improves the effectiveness of the checkpoint inhibitor. Optionally, the radiolabeled agent (preferably an antigen-binding protein) improves the effectiveness of the checkpoint inhibitor to such an extent that the total dose of the checkpoint inhibitor administered to the subject is lower than the dose required to achieve the same effect when the checkpoint inhibitor is administered alone (i.e., monotherapy). Preferably, the improvement is such that the dose of the checkpoint inhibitor can be reduced by at least 10%, at least 25%, at least 35%, at least 50%, at least 75%, or more compared to the dose required for monotherapy with the checkpoint inhibitor. More preferably, the improvement is such that the dose of the checkpoint inhibitor can be at least one quarter, at least one third, at least half, or at least two thirds of the dose required for monotherapy with the checkpoint inhibitor.

[0060] In another aspect, when a radiolabeled conjugate (preferably, an antigen-binding protein) that binds to or specifically binds to CAIX and a checkpoint inhibitor described herein are administered to a subject, the checkpoint inhibitor can improve the efficacy of the radiolabeled conjugate (preferably, an antigen-binding protein). Optionally, the checkpoint inhibitor can improve the efficacy of the agent (preferably, an antigen-binding protein) to such an extent that the total dose of the agent administered to the subject can be a lower dose than the dose required to achieve the same effect when the agent is administered alone. Preferably, the improvement is such that the dose of the radiolabeled conjugate (preferably, an antigen-binding protein) that specifically binds to CAIX can be reduced by at least 10%, at least 25%, at least 35%, at least 50%, at least 75%, or more compared to the dose required for monotherapy with the radiolabeled conjugate (preferably, an antigen-binding protein). More preferably, the improvement is such that the dose of the radiolabeled conjugate (preferably, an antigen-binding protein) can be at least one-fourth, at least one-third, at least one-half, or at least two-thirds of the dose required for monotherapy with the radiolabeled conjugate (antigen-binding protein).

[0061] In certain embodiments, the effect of a radiolabeled conjugate (preferably, an antigen-binding protein) that binds to or specifically binds to CAIX and a checkpoint inhibitor on the survival of a subject can be significantly greater than the effects of the agent and the checkpoint inhibitor when administered alone. In a further embodiment, the effect of any radiolabeled conjugate that binds to or specifically binds to CAIX and a checkpoint inhibitor described herein on tumor growth in a subject can be significantly greater than the effects of the agent and the checkpoint inhibitor when administered alone.

[0062] In any aspect of the present invention, a radiolabeled conjugate (preferably, an antigen-binding protein) that binds to or specifically binds to CAIX and / or a checkpoint inhibitor is administered once. In another embodiment, the agent (preferably, an antigen-binding protein) and / or a checkpoint inhibitor is administered to the subject two, three, four, or more times.

[0063] In any aspect of the present invention, a radiolabeled conjugate (antigen-binding protein) that binds to or specifically binds to CAIX and / or a checkpoint inhibitor may be administered in the same composition or in separate compositions. In another aspect, the agent (antigen-binding protein) and / or a checkpoint inhibitor may thus be administered together or sequentially. Alternatively, the administrations may be staggered. The agent (antigen-binding protein) and / or a checkpoint inhibitor may also be administered at the same frequency or at different frequencies.

[0064] Optionally, a radiolabeled conjugate (antigen-binding protein) that binds to or specifically binds to CAIX is administered to the subject prior to administration of the checkpoint inhibitor. In some examples, the radiolabeled conjugate that binds to or specifically binds to CAIX is administered to the subject at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days prior to administration of the checkpoint inhibitor. Preferably, the radiolabeled conjugate (antigen-binding protein) that binds to or specifically binds to CAIX is administered at least 1 day prior to administration of the checkpoint inhibitor.

[0065] In any aspect of the present invention, an antigen-binding protein and a checkpoint inhibitor that bind to or specifically bind to CAIX may be administered by any known route of administration in the art, including intraperitoneally, intratumorally, locally, orally, to the airway (by inhalation or intranasally), intravenously, subcutaneously, or intramuscularly. Preferably, an antigen-binding protein that binds to or specifically binds to CAIX, conjugated to a radionuclide, and / or a checkpoint inhibitor is administered intravenously.

[0066] In any aspect of the present invention, the amount of the antigen-binding protein that binds to or specifically binds to CAIX, conjugated with a radionuclide, administered may be in the range of about 250 nanomoles / kg body weight / dose to 0.005 nanomoles / kg body weight / dose.

[0067] In any aspect of the present invention, the amount of the antigen-binding protein that binds to or specifically binds to CAIX, conjugated with a radionuclide, administered may be in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μg / kg, or more.

[0068] In any aspect of the present invention, the amount of the checkpoint inhibitor administered may be in the range of about 0.001 to about 100 mg / kg, about 0.001 to about 75 mg / kg, about 0.001 to about 50 mg / kg, about 0.001 to about 25 mg / kg, about 0.001 to about 20 mg / kg, about 0.005 to about 20 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 20 mg / kg, about 0.1 to about 10 mg / kg, about 0.1 to about 5 mg / kg, about 1 to about 5 mg / kg, about 2 to about 5 mg / g, about 7.5 to about 12.5 mg / kg, or about 0.1 to about 30 mg / kg of the subject's body weight. For example, the dosage may be about 0.01, about 0.1, about 0.3, about 1, about 2, about 3, about 5, or about 10 mg / kg body weight, or about 0.3, about 1, about 2, about 3, about 5 mg / kg, or about 10 mg / kg body weight.

[0069] In any aspect of the present invention, the cancer can include any cancer associated with or caused by the expression of CAIX. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, adenocarcinoma, mesothelioma, bladder cancer, prostate cancer, germ cell cancer, liver tumor / cholangiocarcinoma, neuroendocrine cancer, pituitary tumor, small round cell tumor, squamous cell carcinoma, melanoma, atypical fibroxanthoma, spermatoma, nonseminomatous tumor, interstitial Leydig cell tumor, Sertoli cell tumor, skin tumor, kidney tumor, testicular tumor, brain tumor, ovarian tumor, stomach tumor, oral tumor, bladder tumor, bone tumor, cervical tumor, esophageal tumor, laryngeal tumor, liver tumor, lung tumor, fibrosarcoma, vaginal tumor, blood cancer, or Wilms tumor. In preferred embodiments, the cancer is melanoma, breast cancer, or colon cancer. In some embodiments, the cancer is colorectal cancer or kidney cancer, for example, renal cortical adenocarcinoma or clear cell renal cell carcinoma (ccRCC).

[0070] In any aspect of the present invention, the checkpoint inhibitor can be a PD-1, PD-L1, or CTLA-4 checkpoint inhibitor. In one aspect, the checkpoint inhibitor is an antibody. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 in the form of an antibody. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor therapy involves administering inhibitors of PD-1 and CTLA-4. Examples of such inhibitors are further described herein.

[0071] An antigen-binding protein that binds or specifically binds to CAIX The present invention involves administering an antigen-binding protein that binds or specifically binds to CAIX, and the antigen-binding protein is conjugated to a radionuclide.

[0072] In any embodiment, the radiolabeled antigen-binding protein is an antibody or an antigen-binding fragment thereof for binding to CAIX. Any suitable antibody or antigen-binding fragment thereof for binding to CAIX may be used. In some embodiments, the radiolabeled antigen-binding protein that binds or specifically binds to CAIX is radiolabeled cilengitide that retains the ability to bind to CAIX, or a radiolabeled functional variant or fragment thereof. In some embodiments, the radiolabeled antigen-binding protein that binds or specifically binds to CAIX is radiolabeled G250. In some embodiments, the radiolabeled antigen-binding protein that binds or specifically binds to CAIX is a radiolabeled chimeric antibody or an antigen-binding fragment thereof. In some embodiments, the radiolabeled antigen-binding protein that binds or specifically binds to CAIX is a radiolabeled humanized antibody or a radiolabeled antigen-binding fragment thereof. Optionally, the radiolabeled antigen-binding protein is radiolabeled humanized G250 (hG250).

[0073] In any embodiment, the antigen-binding protein that binds or specifically binds to CAIX is as described in any of WO2002 / 062972A2 (US2004 / 0219633A1), WO2004 / 002526A1 (US7,632,496B2), WO2006 / 002889A2 (US7,691,375B2), WO2009 / 056342A1 (US2014 / 0017252A1), WO2011 / 032973A1 (US2012 / 0207672A1), WO2014 / 128258A1 (US10,620,208B2), or US11,629,199. One antibody for binding to CAIX described in these publications is referred to herein as TLX250, which is further described herein in the Examples. In any embodiment, the antibody or antigen-binding fragment for binding to CAIX is as described in WO2021 / 000017A1. The entire contents of each of these publications are incorporated herein by reference.

[0074] In any embodiment, the antigen-binding protein is (a) Three CDRs of an antigen-binding domain having a heavy-chain variable domain (VH) set forth in SEQ ID NO: 4, 20, 36, 52, or 68, and / or (b) Three CDRs of an antigen-binding domain having a light-chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0075] One of ordinary skill in the art will be familiar with various methods for determining the CDRs of any given heavy or light variable chain, including using methods conventionally used in the art and described elsewhere in this specification. Further, one of ordinary skill in the art will understand that the definition of the CDR boundaries of any given variable chain depends on the particular nomenclature system used (such as the IMGT, Chothia, or Kabat numbering systems).

[0076] In any embodiment, the antigen-binding protein (a) A heavy-chain variable domain (VH) comprising three complementarity-determining regions (CDRs) comprising the amino acid sequence set forth in SEQ ID NO: 4, 20, 36, 52, or 68, and / or (b) A light-chain variable domain (VL) comprising three complementarity-determining regions (CDRs) comprising the amino acid sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0077] In a preferred embodiment, the antigen-binding protein - SEQ ID NO: 4 and SEQ ID NO: 84 - SEQ ID NO: 36 and SEQ ID NO: 132 - SEQ ID NO: 52 and SEQ ID NO: 132 - The amino acid sequences of VH and VL set forth in SEQ ID NO: 52 and SEQ ID NO: 148, or a variant thereof, wherein VH and VL contain 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 11 or fewer, 12 or fewer, 13 or fewer, 14 or fewer, 15 or fewer, 16 or fewer, 17 or fewer, 18 or fewer, 19 or fewer, or 20 or fewer amino acid residue substitutions, deletions, or additions compared to the amino acid sequences described above, the amino acid substitutions, deletions, or additions are not within the CDRs, and the antigen-binding protein retains the ability to bind to CAIX.

[0078] In any embodiment, the antigen-binding protein specifically binds to carbonic anhydrase IX (CAIX) and contains an antigen-binding domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, wherein FR1, FR2, FR3, and FR4 are each a framework region, CDR1, CDR2, and CDR3 are each a complementarity-determining region, FR1a, FR2a, FR3a, and FR4a are each a framework region, CDR1a, CDR2a, and CDR3a are each a complementarity-determining region, The sequence of any of the complementarity-determining regions has the amino acid sequence set forth in Table 1. Preferably, the framework region has the amino acid sequence also set forth in Table 1 below, which contains amino acid variations at specific residues that can be determined by aligning various framework regions from each antibody. CDR1, CDR2, and CDR3 may be sequences from VH, CDR1a, CDR2a, and CDR3a may be sequences from VL, or CDR1, CDR2, and CDR3 may be sequences from VL, and CDR1a, CDR2a, and CDR3a may be sequences from VH.

[0079] References herein to proteins or antibodies that "bind to" carbonic anhydrase IX (CAIX) provide literal support for proteins or antibodies that "bind specifically to" or "specifically binds to" CAIX.

[0080] In any embodiment, the antigen-binding protein is (i) a variable heavy chain (VH) comprising complementarity-determining region (CDR) 1 having a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 1, CDR 2 having a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 2, and CDR 3 having a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 3; (ii) an VH comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any of SEQ ID NO: 4, 20, 36, 52, or 68 (iii) a CDR1 comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 81, a CDR2 comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 83; and a VL (iv) 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, VL, (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3, (vi) a VH comprising the sequence set forth in any of SEQ ID NO: 4, 20, 36, 52, or 68, (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 81, a CDR2 comprising the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising the sequence set forth in SEQ ID NO: 83, (viii) a VL comprising the sequence set forth in any of SEQ ID NO: 84, 100, 116, 132, 148, or 164, (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3, and a VL comprising a CDR1 comprising the sequence set of SEQ ID NO: 81, a CDR2 comprising the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising the sequence set forth in SEQ ID NO: 83, or (x) a VH comprising the sequence set forth in any of SEQ ID NO: 4, 20, 36, 52, or 68, and a VL comprising the sequence set forth in any of SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0081] In a further embodiment, the antigen-binding protein is (i) A VH comprising a framework region (FR) 1 that contains or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any of SEQ ID NO: 9, 25, 41, 57, or 73; an FR2 that contains or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any of SEQ ID NO: 10, 26, 42, 58, or 74; an FR3 that contains or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any of SEQ ID NO: 11, 27, 43, 59, or 75; and an FR4 that contains or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least(ii) a framework region (FR) 1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in any of SEQ ID NOs: 89, 105, 121, 137, 153, or 169; an FR2 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in any of SEQ ID NOs: 90, 106, 122, 138, 154, or 170; an FR3 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in any of SEQ ID NOs: 91, 107, 123, 139, 155, or 171; an FR4 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in any of SEQ ID NOs: 92, 108, 124, 140, 156, or 172.Comprising VL, which comprises FR4 that contains or consists of a sequence that is at least 99% identical.

[0082] In a further embodiment, the antigen-binding protein (i) comprises or consists of a framework region (FR)1 that contains or consists of the sequence set forth in any one of SEQ ID NO: 9, 25, 41, 57, or 73, an FR2 that contains or consists of the sequence set forth in any one of SEQ ID NO: 10, 26, 42, 58, or 74, an FR3 that contains or consists of the sequence set forth in any one of SEQ ID NO: 11, 27, 43, 59, or 75, an FR4 that contains or consists of the sequence set forth in any one of SEQ ID NO: 12, 28, 44, 60, or 76, and (ii) comprises VL that comprises or consists of a framework region (FR)1 that contains or consists of the sequence set forth in any one of SEQ ID NO: 89, 105, 121, 137, 153, or 169, an FR2 that contains or consists of the sequence set forth in any one of SEQ ID NO: 90, 106, 122, 138, 154, or 170, an FR3 that contains or consists of the sequence set forth in any one of SEQ ID NO: 91, 107, 123, 139, 155, or 171, an FR4 that contains or consists of the sequence set forth in any one of SEQ ID NO: 92, 108, 124, 140, 156, or 172.

[0083] In any embodiment, an antibody that specifically binds to CAIX comprises an antigen-binding protein that essentially consists of or consists of the amino acid sequence of any one of SEQ ID NO: 4, 20, 36, 52, or 68 and / or any one of SEQ ID NO: 84, 100, 116, 132, 148, 164 (in the order from N-terminus to C-terminus or C-terminus to N-terminus).

[0084] In any embodiment, the antigen-binding protein (a) a heavy chain variable domain (VH) that comprises, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 4, 20, 36, 52, or 68, and / or (b) a light chain variable domain (VL) that comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164. In some embodiments, the antigen-binding protein is a modified IgG antibody that comprises a heavy chain constant region having one or more amino acid substitutions as compared to the wild-type antibody of class IgG, and the one or more amino acid substitutions decrease the affinity of the antibody for the neonatal Fc receptor (FcRn), thereby decreasing the serum half-life of the modified antibody as compared to the wild-type antibody of class IgG.

[0085] In one embodiment, the one or more amino acid substitutions are selected from substitutions in the heavy chain constant region 2 (CH2) of the IgG molecule and decrease the affinity of the IgG molecule for FcRn. Alternatively, the one or more amino acid substitutions can be within the heavy chain constant region 3 (CH3) of the IgG molecule, thereby decreasing the affinity of the IgG molecule for FcRn. Further, the amino acid substitutions can include at least one substitution in the CH2 region and at least one substitution in the CH3 region of the IgG molecule, thereby the substitutions decreasing the affinity of the IgG for FcRn.

[0086] In certain preferred embodiments, one or more amino acid substitutions may be at one or more of the IgG residues His310, His433, His435, His436, or Ile253. Preferably, the amino acid substitution comprises a substitution in the heavy chain constant region at positions His310 or His435. More preferably, the amino acid substitutions that decrease the affinity of the antibody for FcRn are at both His310 and His435.

[0087] In certain embodiments, the modified antibody retains the ability to bind to one or more Fc-gamma receptors and, thus, in certain embodiments, the modified antibody retains the ability to stimulate effector responses (including ADCC).

[0088] In alternative embodiments, one or more amino acid modifications that decrease the affinity for the FcRn receptor also decrease the affinity for Fc gamma receptors. The modified antibody may further comprise one or more amino acid substitutions compared to the wild-type antibody of class IgG, and the amino acid substitutions further decrease the affinity of the antibody for one or more Fc gamma receptors.

[0089] In further embodiments, the modified antibody further comprises one or more amino acid substitutions compared to the wild-type antibody of class IgG, and the amino acid substitutions increase the stability of the CH1-CH2 hinge region in the modified antibody compared to the wild-type antibody of class IgG.

[0090] In any embodiment, the antibody for binding to CAIX is conjugated to a therapeutic agent. The therapeutic agent can be conjugated directly or indirectly to the antibody, for example, by halogenation of amino acid residues. Preferably, the therapeutic agent is conjugated indirectly to the antibody via a linker or chelator moiety. In one example, the antibody is conjugated to a chelating moiety selected from the group consisting of TMT (6,6’’-bis[N,N’’,N’’’-tetra(carboxymethyl)aminomethyl)-4’-(3-amino-4-methoxyphenyl)-2,2’:6’,2’’-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN’,N’’(N’’’-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, DiamSar, DTPA, CHX-A’’-DTPA, TETE, Te2A, HBED, DFO, DFOsq, DFO-NCS, HOPO, or WO2022 / 133537 (incorporated herein by reference), or other chelating agents described herein.

[0091] In another example, the antibody is conjugated using a bifunctional linker, such as bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, or any amine or thiol modification chemistry known in the art.

[0092] Preferably, the therapeutic agent is a radioisotope. Examples of suitable isotopes include actinium-225 ( 225 Ac), astatine-211 ( 211 At), bismuth-212 and bismuth-213 ( 212 Bi, 213 Bi), copper-64 and copper-67 ( 64 Cu, 67 Cu), iodine-123, -124, -125 or -131 ( 123 I, 124 I, 125 I, 131 I)( 123 I), lead-212 ( 212 Pb), lutetium-177 (177 Lutetium), radium-223, and radium-224( 223 Ra, 224 Ra), samarium-153( 153 Sm), scandium-47( 47 Sc), strontium-90( 90 Sr), and yttrium-90( 90 Y) are included.

[0093] In any embodiment, the heavy chain constant region of the antibody contains amino acid substitutions at both His310 and His435. The antibody may also contain amino acid substitutions at residues corresponding to Ser228 and Leu235 of the constant heavy chain region.

[0094] Preferably, the antibody contains a heavy chain constant region having the sequence set forth in any one of SEQ ID NOs: 177-180, preferably the sequence set forth in SEQ ID NO: 178.

[0095] In a still further embodiment, the antibody preferably contains a heavy chain having the sequence set forth in any one of SEQ ID NOs: 182-185, preferably the sequence set forth in SEQ ID NO: 183.

[0096] In any embodiment, the antibody contains a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 181. Preferably, the antibody contains a light chain having the amino acid sequence set forth in SEQ ID NO: 186.

[0097] In any embodiment, the antibody contains the sequence set forth in SEQ ID NO: 183 and the sequence set forth in SEQ ID NO: 186.

[0098] The present invention also relates to the use of a molecule comprising an immunoglobulin moiety and a non-protein agent conjugated thereto, The immunoglobulin portion specifically binds to CAIX and comprises an antigen-binding protein that consists essentially of, or consists of, any one of SEQ ID NO: 4, 20, 36, 52, or 68 and any one of SEQ ID NO: 84, 100, 116, 132, 148, 164 in order from the N-terminus to the C-terminus or from the C-terminus to the N-terminus, Optionally, the immunoglobulin portion has a reduced or abolished affinity for the FcRn receptor as compared to wild-type immunoglobulin, Provided is the use of a non-protein agent that comprises a therapeutic moiety such as a radioactive element.

[0099] Preferably, the immunoglobulin portion has an amino acid substitution at a residue corresponding to His310 and / or His435 in the constant heavy chain region. The immunoglobulin portion may also include an amino acid substitution at a residue corresponding to Ser228 and Leu235 in the constant heavy chain region. Preferably, the non-protein agent comprises a radioactive element.

[0100] The present invention also provides the use of a molecule comprising an immunoglobulin portion and a non-protein agent conjugated thereto, optionally, the immunoglobulin portion having a reduced or abolished affinity for the FcRn receptor as compared to wild-type immunoglobulin, The immunoglobulin portion specifically binds to CAIX and (i) a complementarity-determining region (CDR) 1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 1, 17, 33, 49, or 65, a CDR2 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 2, 18, 34, 50, or 66, and a CDR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 3, 19, 35, 51, or 67, of VH, (ii) a VH comprising a sequence that is at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4, 20, 36, 52, or 68, (iii) a VL comprising a CDR1 that is at least about 80%, at least 85%, at least 9O%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 81, 9L, 113, 129, 145, or 161, a CDR2 that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 82, 98, 114, 130, 146, or 162, and a CDR3 that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 83, 99, 115, 131, 147, or 163, (iv) a VL comprising a sequence that is at least about 95% identical to the sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164, (v) a VH comprising a CDR1 that is the sequence set forth in SEQ ID NO: 1, 17, 33, 49, or 65, a CDR2 that is the sequence set forth between SEQ ID NO: 2, 18, 34, 50, or 66, and a CDR3 that is the sequence set forth in SEQ ID NO: 3, 19, 35, 51, or 67, (vi) a VH comprising the sequence set forth in SEQ ID NO: 4, 20, 36, 52, or 68, (vii) a VL comprising a CDR1 that is the sequence set forth in SEQ ID NO: 81, 97, 113, 129, 145, or 161, a CDR2 that is the sequence set forth in SEQ ID NO: 82, 98, 114, 130, 146, or 162, and a CDR3 that is the sequence set forth in SEQ ID NO: 83, 99, 115, 131, 147, or 163, (viii) a VL comprising the sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164, (ix) A VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, 17, 33, 49, or 65, a CDR2 comprising the sequence set forth between SEQ ID NO: 2, 18, 34, 50, or 66, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3, 19, 35, 51, or 67, and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 81, 97, 113, 129, 145, or 161, a CDR2 comprising the sequence set forth in SEQ ID NO: 82, 98, 114, 130, 146, or 162, and a CDR3 comprising the sequence set forth in SEQ ID NO: 83, 99, 115, 131, 147, or 163, or (x) Provided for use is at least one of a VH comprising the sequence set forth in SEQ ID NO: 4, 20, 36, 52, or 68 and a VL comprising the sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0101] Preferably, the non - protein agent comprises a radioactive element. Preferably, the immunoglobulin moiety has an amino acid substitution at a residue corresponding to His310 and / or His435 in the constant heavy - chain region. The immunoglobulin moiety may also include an amino acid substitution at a residue corresponding to Ser228 and Leu235 in the constant heavy - chain region. Preferably, the non - protein agent comprises a radioactive element.

[0102] In any embodiment, the immunoglobulin comprises a heavy - chain constant region comprising the sequence set forth in any one of SEQ ID NOs: 177 - 180, preferably the sequence set forth in SEQ ID NO: 178.

[0103] In a still further embodiment, the immunoglobulin comprises a heavy chain comprising the sequence set forth in any one of SEQ ID NOs: 182 - 185, preferably the sequence set forth in SEQ ID NO: 183.

[0104] In any embodiment, the immunoglobulin comprises a light - chain constant region comprising the sequence set forth in SEQ ID NO: 181. In one embodiment, the light chain comprises the sequence of SEQ ID NO: 186.

[0105] In certain preferred embodiments, the immunoglobulin comprises the amino acid sequences set forth in SEQ ID NOs: 183 and 186.

[0106] In certain embodiments, the antibody is an antibody as described herein having any of the complementarity determining regions, framework regions, variable light or variable heavy regions set forth in Table 1 below. Preferably, the modified antibody also comprises amino acid substitutions at residues corresponding to Ser228 and Leu235 of the constant heavy chain region. Preferably, the radioactive element is conjugated to the modified antibody using a chelating agent, such as DOTA.

[0107] In any embodiment, the modified antibody for use as described herein has reduced toxicity compared to the unmodified antibody. Reduced toxicity includes reducing some of the toxic effects (including hematological toxicity, bone uptake, and bone marrow irradiation) that would otherwise result from the long-term retention of the radioisotope in circulation.

[0108] In any embodiment, the toxicity of the radiolabeled antibody or radioimmunoconjugate described herein is evaluated by determining the tumor:blood ratio of the antibody or immunoconjugate after administration to an individual.

[0109] In any embodiment of the invention, the tumor:blood ratio of the modified antibody of the invention is at least 2-fold, at least 3-fold, at least 4-fold, at least 6-fold, at least 8-fold, or at least 10-fold, or more, greater than that of an unmodified antibody having no modification to the constant heavy chain region as described herein when this ratio is determined at least 8 hours after administration of the antibody. Alternatively, this ratio is determined at least 24, 48, 72, or 120 hours after administration of the antibody to the individual. In certain embodiments, the tumor:blood ratio of the modified antibody of the invention is at least 50-fold, at least 100-fold, at least 200-fold, or at least 300-fold greater than that of an unmodified antibody having no modification to the constant heavy chain region as described herein when this ratio is determined at least 120 hours after administration of the antibody.

[0110] In any embodiment of the present invention, a modified antibody described herein having a reduced or altered serum half-life compared to an unmodified antibody has a serum clearance rate that is at least 2-fold, at least 3-fold, or more rapid than that of the unmodified antibody.

[0111] In certain embodiments of the present invention, the CAIX-binding antibodies described herein are suitable for use in a theranostic pair, the theranostic pair comprising 1) an antibody conjugated to a contrast agent and 2) an antibody conjugated to a therapeutic agent. For example, the antibody can first be used as a diagnostic agent when conjugated to a radioisotope suitable for use in radiation imaging. Second, the antibody can be used as a therapeutic agent when conjugated to a radioisotope or cytotoxic agent suitable for use in therapy.

[0112] In a further embodiment, the method of the invention may also include diagnosing, monitoring, or prognosticating a disease, disorder, or infection in a subject, (a) administering to the subject an antibody described herein, wherein the antibody specifically binds to an antigen associated with the disease, disorder, or infection, (b) concentrating the antibody at a site in the subject where the antigen is found, (c) detecting the antibody, wherein detection of the antibody above background or a standard level indicates that the subject has the disease, disorder, or infection.

[0113] In any aspect of the present invention, the antigen-binding domain further comprises at least one of the following: (i) A framework region (FR) 1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 9, 25, 41, 57, or 76; an FR2 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 10, 26, 42, 58, or 74; an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 11, 27, 43, 59, or 75; and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 12, 28, 44, 60, or 76, VH, (ii) An FR1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 89, 105, 121, 137, 153, or 169; an FR2 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 90, 106, 122, 138, 154, or 170; an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 91, 107, 123, 139, 155, or 171; and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 92, 108, 124, 140, 156, or 172, VL, (iii) An VH comprising FR1 containing the sequence set forth in SEQ ID NO: 9, 25, 41, 57, or 76, FR2 containing the sequence set forth between SEQ ID NO: 10, 26, 42, 58, or 74, FR3 containing the sequence set forth in SEQ ID NO: 11, 27, 43, 59, or 75, and FR4 containing the sequence set forth in SEQ ID NO: 12, 28, 44, 60, or 76. (iv) A VL, or an FR1 containing the sequence set forth in SEQ ID NO: 89, 105, 121, 137, 153, or 169, FR2 containing the sequence set forth between SEQ ID NO: 90, 106, 122, 138, 154, or 170, FR3 containing the sequence set forth in SEQ ID NO: 91, 107, 123, 139, 155, or 171, and FR4 containing the sequence set forth in SEQ ID NO: 92, 108, 124, 140, 156, or 172. (v) An VH comprising FR1 containing the sequence set forth in SEQ ID NO: 9, 25, 41, 57, or 76, FR2 containing the sequence set forth between SEQ ID NO: 10, 26, 42, 58, or 74, FR3 containing the sequence set forth in SEQ ID NO: 11, 27, 43, 59, or 75, and FR4 containing the sequence set forth in SEQ ID NO: 12, 28, 44, 60, or 76, and a VL comprising FR1 containing the sequence set forth in SEQ ID NO: 89, 105, 121, 137, 153, or 169, FR2 containing the sequence set forth between SEQ ID NO: 90, 106, 122, 138, 154, or 170, FR3 containing the sequence set forth in SEQ ID NO: 91, 107, 123, 139, 155, or 171, and FR4 containing the sequence set forth in SEQ ID NO: 92, 108, 124, 140, 156, or 172.

[0114] In one embodiment, VH comprises a sequence that is 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 in SEQ ID NO: 36 or 52, and VL comprises a sequence that is at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 116, 132, or 148, or comprises the sequence set forth in SEQ ID NO: 116, 132, or 148. Preferably, VH comprises a sequence that is 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 in SEQ ID NO: 36 or 52, and VL comprises a sequence that is 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 in SEQ ID NO: 132 or 148. More preferably, VH comprises a sequence that is 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 the sequence set forth in SEQ ID NO: 36, and VL comprises a sequence that is 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 the sequence set forth in SEQ ID NO: 148. Alternatively, VH comprises a sequence that is at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 52, or comprises the sequence set forth in SEQ ID NO: 52, and VL comprises a sequence that is 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, preferably the sequence set forth in SEQ ID NO: 148, or comprises the sequence set forth in SEQ ID NO: 132 or 148, preferably the sequence set forth in SEQ ID NO: 148.

[0115] In any of those embodiments, the antigen-binding protein may be referred to as the antigen-binding domain of an antibody.

[0116] Preferably, the antigen-binding protein described herein is an antibody or an antigen-binding fragment thereof. Typically, the antigen-binding protein is an antibody or an antigen-binding fragment thereof, for example, a monoclonal antibody.

[0117] As described herein, the antigen-binding protein may be (i) a single-chain Fv fragment (scFv), (ii) a dimeric scFv (di-scFv), (iii) one of (i) or (ii) linked to the constant region of the antibody, Fc, or heavy-chain constant domain (CH)2 and / or CH3, or (iv) one of (i) or (ii) linked to a protein that binds to immune effector cells.

[0118] Furthermore, as described herein, the antigen-binding protein may be (i) a diabody, (ii) a triabody, (iii) a tetrabody, (iv) a Fab, (v) an F(ab’)2, (vi) an Fv, (vii) one of (i) to (vi) linked to the constant region of the antibody, Fc, or heavy-chain constant domain (CH)2 and / or CH3, or (viii) one of (i) to (vi) linked to a protein that binds to immune effector cells.

[0119] In any aspect or embodiment, the antibody is a naked antibody. Specifically, the antibody is in a non-conjugated form and is not adapted to form a conjugate.

[0120] In any embodiment, an antigen-binding protein for use according to the invention may be an antigen-binding protein as described herein, an immunoglobulin variable domain, an antibody, a dab (single-domain antibody), a di-scFv, a scFv, a Fab, a Fab’, an F(ab’)2, an Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single-chain antibody molecule, or a fusion protein comprising a multispecific antibody.

[0121] In any embodiment, an antigen-binding protein for use according to the invention may be a conjugate in the form of an antigen-binding protein as described herein, an immunoglobulin variable domain, an antibody, a dab, a di-scFv, a scFv, a Fab, a Fab’, an F(ab’)2, an Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody or fusion protein as described herein conjugated to a label or a cytotoxic agent.

[0122] In a preferred embodiment, the antigen-binding protein is an IgG immunoglobulin comprising one or more amino acid substitutions within the antibody constant domain, CH2-CH3 region, which modifies the binding of the antibody to the neonatal Fc receptor (FcRn) compared to the wild-type antibody Fc region. The one or more amino acid modifications alter the affinity of the antibody constant domain, Fc region, or its FcRn-binding fragment for FcRn, thereby modifying the serum half-life of the antigen-binding protein.

[0123] Preferably, the substitution modifies the binding affinity for FcRn and / or the serum half-life of the modified antibody compared to the unmodified wild-type antibody. The invention further provides a modified antibody having a reduced binding affinity for FcRn and / or a reduced serum half-life compared to an unmodified antibody, wherein any one or more amino acid residues at position Ile253 or His310 from the CH2 domain and / or residue His435 from the CH3 domain are substituted with another amino acid different from the amino acid present in the unmodified antibody or unmodified IgG.

[0124] In one example, one or more amino acid modifications are selected from amino acid substitutions at residues corresponding to H310 and H435. In a further example, the antibody comprises amino acid substitutions at both the His310 residue and the His435 residue.

[0125] In a further embodiment, the antibody may comprise an amino acid substitution at residue Lys322. Preferably, the substitution is K322A. In a particularly preferred embodiment, the antibody comprises the substitutions K322A, H310A, and H435Q.

[0126] Amino acid substitutions may include substitutions from a histidine residue to alanine, glutamine, glutamic acid, or aspartic acid. Preferably, the amino acid substitution at His310 is a substitution to alanine. Preferably, the amino acid substitution at His435 is a substitution to glutamine. Preferably, the amino acid substitution at Ile253 is alanine.

[0127] In a further embodiment, the antigen-binding protein is an antibody or an antigen-binding fragment thereof that comprises one or more amino acid substitutions that modify the binding of the antibody to the activating Fc gamma receptor. The one or more amino acid modifications alter the affinity of the antibody constant domain, Fc region, or Fc gamma receptor-binding fragment for any one or more Fc gamma receptors. Preferably, the amino acid modification is at the residue corresponding to Leu235. More preferably, the amino acid modification is from Leu235 to glutamic acid.

[0128] In one embodiment, the amino acid modification is a hinge-stabilizing mutation at Ser228. Preferably, the amino acid modification at Ser228 is to proline.

[0129] In one embodiment of the invention, the antibody comprises mutations at Ser228, Leu235, His310, and His435. Preferably, the amino acid modifications are Ser228Pro, Leu235Glu, His310Ala and His435Gln.

[0130] Amino acid modification is preferably performed in an antibody having an IgG1 isotype or in an IgG4 isotype.

[0131] In a preferred embodiment, the antibody comprises a heavy chain constant region as set forth in any one of SEQ ID NOs: 177 to 180.

[0132] An antigen-binding protein, immunoglobulin variable domain, antibody, dab, di-scFv, scFv, Fab, Fab’, F(ab’)2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate described herein can be obtained by expressing a nucleic acid encoding them.

[0133] In one example, such a nucleic acid is contained in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct can be in a vector, such as a plasmid. In an example targeting a single polypeptide chain antigen-binding protein, the expression construct can comprise a promoter linked to a nucleic acid encoding the polypeptide chain. In an example targeting multiple polypeptide chains forming an antigen-binding protein, the expression construct can comprise, for example, a nucleic acid encoding a polypeptide comprising VH operably linked to a promoter and a nucleic acid encoding a polypeptide comprising VL operably linked to a promoter.

[0134] In another example, the expression construct is, for example, the following operably linked components: (i) a promoter (ii) a nucleic acid encoding a first polypeptide, (iii) an internal ribosome entry site, and (iv) a nucleic acid encoding a second polypeptide, and is a bicistronic expression construct containing them in the order from 5’ to 3’, wherein the first polypeptide comprises VH and the second polypeptide comprises VL or vice versa.

[0135] The present invention also contemplates the use of separate expression constructs, one of which encodes a first polypeptide comprising VH and another of which encodes a second polypeptide comprising VL. For example, the present invention also (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising VL operably linked to a promoter, the composition comprising.

[0136] The antigen-binding proteins or antibodies or antigen-binding fragments thereof described herein may comprise human constant regions, such as IgG constant regions such as IgG1, IgG2, IgG3 or IgG4 constant regions, or mixtures thereof. In the case of antibodies or proteins comprising VH and VL, VH can be linked to the heavy chain constant region and VL can be linked to the light chain constant region.

[0137] In one example, the proteins or antibodies described herein, or compositions of the proteins or antibodies described herein, comprise a stabilized heavy chain constant region that completely or partially comprises a mixture of sequences, regardless of the presence or absence of a C-terminal lysine residue.

[0138] In one example, an antibody for use according to the present invention comprises VH disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (as discussed above), and VL is linked or fused to a kappa light chain constant region.

[0139] The functional characteristics of the antigen-binding proteins for use according to the present invention shall apply mutatis mutandis to the antibodies of the present invention.

[0140] The antigen-binding proteins as described herein may be purified, substantially purified, isolated, and / or recombinant.

[0141] The present invention also provides a method for treating or preventing cancer in a subject, the method comprising administering to the subject an antigen-binding protein as described herein. In this regard, the antigen-binding protein can be used to prevent recurrence of the condition, which is considered prevention of the condition.

[0142] Other exemplary cancers include kidney cancer. It will be understood that the antibodies of the invention having affinity for CAIX are useful for this purpose.

[0143] The present invention also includes steps for in vivo methods of diagnosing, monitoring, or prognosticating a disease, disorder, or infection in a subject, before or in conjunction with administering the treatment described herein.

[0144] As used herein, unless the context requires otherwise, the term "comprise" and variations such as "comprising," "comprises," and "comprised" are not intended to exclude further additives, components, integers, or steps.

[0145] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description given by way of example and with reference to the accompanying drawings.

Brief Description of the Drawings

[0146]

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[0147] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0148] 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.

[0149] ​Here, a particular embodiment of the present invention will be described in detail. Although the present invention is described in conjunction with embodiments, it will be understood that the intention is not to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims.

[0150] The present invention relates to the combination of immune checkpoint inhibitor (ICI) therapy and targeted radionuclide therapy (TRT). Surprisingly, the inventors have found that the combination of these distinct treatment approaches results in improved treatment outcomes compared to either immune checkpoint inhibitor therapy or targeted radionuclide therapy alone.

[0151] The present invention is, in part, directed to the identification of a new approach for reducing the toxicity of radioimmunoconjugates for use in radioimmunotherapy. In particular, the methods of the present invention enable the use of relatively low doses of radioimmunoconjugates to achieve equivalent, and in some cases superior, treatment outcomes.

[0152] Furthermore, the present invention is, in part, directed to improved immune checkpoint inhibitor therapy.

[0153] As outlined above, radiation damage to healthy tissues and cells is a major problem associated with radioimmunotherapy. Toxicity significantly limits the radiation dose in RAIT and reduces the effectiveness of tumor treatment. However, the inventors recognize that it is possible to achieve at least equivalent (and in favorable situations, improved) outcomes using lower doses of radionuclides.

[0154] Overview Throughout this specification, unless otherwise specified or the context otherwise requires, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be construed to include one and more (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter. Accordingly, as used in this specification, the singular forms "a", "an", and "the" include plural aspects unless the context clearly dictates otherwise, and vice versa. For example, a reference to "a" includes not only a single one but also two or more, a reference to "an" includes not only a single one but also two or more, a reference to "the" includes not only a single one but also two or more, and so on.

[0155] One of ordinary skill in the art will appreciate that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or shown in this specification, as well as any and all combinations, or any two or more of such steps or features.

[0156] One of ordinary skill in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0157] All patents and publications referred to in this specification are hereby incorporated by reference in their entirety.

[0158] The present invention should not be limited in scope by the specific examples described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention.

[0159] Any example or embodiment of the present invention in this specification shall be construed as applicable mutatis mutandis to any other example or embodiment of the present invention unless otherwise specified.

[0160] Unless otherwise specifically defined, all technical and scientific terms used herein shall be construed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0161] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to one of ordinary skill in the art. Such techniques are described and explained throughout the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0162] The description and definition of variable regions and portions thereof, immunoglobulins, antibodies, and fragments thereof in this specification can be further clarified by the considerations in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989 and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0163] The term "and / or", e.g., "X and / or Y", is to be understood to mean either "X and Y" or "X or Y", and is to be interpreted as explicitly supporting both meanings or either meaning.

[0164] As used herein, the term "derived from" is to be interpreted to indicate that the specified integer can be obtained from the specified source, although not necessarily directly from that source.

[0165] CAIX As used herein, carbonic anhydrase is also known as CA-IX, CA9, CAIX, carbonic anhydrase IX, carbonic anhydrase 9, carbonic anhydrase IX, carbonic dehydrogenase, G250, membrane antigen MN, P54 / 58N, pMW1, RCC-related antigen G250, RCC-related protein G250, and renal cell carcinoma-related antigen G250.

[0166] Cancer cells mainly express plasma membrane-associated CA isoforms such as CAIX and CAXII, and intracellular CAs such as CAI and CAII. The expression of these isoforms in healthy tissues is limited to epithelial cells in the stomach and intestine, but in renal cancer, it is strongly upregulated. Therefore, among cancer-related CAs, CAIX has received the most attention.

[0167] CAIX expression is under the control of hypoxia-inducible factor 1 (HIF-1) and is mainly located in chronically hypoxic tumor regions. However, since CAIX expression can be activated by components of the mitogen-activated protein kinase (MAPK) pathway, CAIX can also be seen in mildly hypoxic or even normoxic regions.

[0168] Agents for binding to CAIX Agents for binding to CAIX and for use in the methods of the present invention may be any compound that specifically recognizes CAIX or binds to CAIX, mediates its activity by binding to CAIX or its fragment or splice variant, binds irreversibly at the entrance to the active site, and / or inhibits CAIX by coordinating to zinc ions at the active site of CAIX.

[0169] Preferably, the agent for binding to CAIX specifically interacts with the CAIX polypeptide. Specifically interacting (e.g., recognizing or binding) means that the agent, e.g., an antibody, has a higher affinity for CAIX compared to other polypeptides. In one embodiment, the agent interacts with (i.e., binds to or recognizes) the CAIX polypeptide, or modulates its activity, and / or mediates antibody-dependent cell cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). Thus, according to one embodiment, the agent is a CAIX inhibitor. The CAIX inhibitor can act at the protein level or the nucleic acid level. Examples of CAIX inhibitors acting at the protein level include, but are not limited to, peptides and anti-CAIX antibodies, and functional fragments of those antibodies, or small organic molecules (preferably having a molecular weight of less than 500 g / mol).

[0170] Examples of anti-CAIX antibodies or antibodies for binding to CAIX are described in EP637336, WO93 / 18152, WO95 / 34650, WO00 / 24913, WO02 / 063010, WO04 / 025302, WO05 / 037083, WO2011 / 139375, US11,629,199, Murri-Plesko et al., Eur J Pharmacol 2011, 657: 173-183.

[0171] Examples of small organic molecules for binding to CAIX include, but are not limited to, sulfonamides, heteroaromatic sulfonamides, sulfamates, coumarins and thiocoumarins, and BAY-79-4620. Examples of inhibitors acting at the nucleic acid level are siRNA molecules, ribozymes and / or antisense molecules.

[0172] As used herein, the terms "specifically binds" or "binds specifically" are to be construed to mean that an agent for use in accordance with the present invention reacts or associates with CAIX or cells expressing it more frequently, more rapidly, for a longer duration, and / or with a higher affinity than in the case of alternative antigens or cells. For example, an antigen-binding protein that binds to CAIX with a substantially higher affinity (e.g., 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than to other antigens.

[0173] Methods for assessing binding to a protein (e.g., CAIX) are known in the art, for example, as described in Scopes (In Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve immobilizing an agent (e.g., an antibody) and contacting it with a labeled target (an antigen in the case of an antibody). After washing to remove non-specifically bound protein, the amount of label, and as a result the amount of bound antigen, is detected. Of course, the antigen-binding site can be labeled and the antigen immobilized. Panning-type assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.

[0174] Other standard methods for assessing binding to a target such as CAIX are also known in the art.

[0175] In any embodiment, the agent for binding to CAIX is the small molecule SLC-0111 (CAS 178606-66-1), SLC-149 (described in EP3317255B1, which is incorporated herein by reference), SLC-0121 or SLC-101.

[0176] In any embodiment, the agent for binding to CAIX is the small molecule / contrast agent PMI-05 (described in US2019 / 0192699A1, which is incorporated herein by reference).

[0177] In any embodiment, the agent for binding to CAIX is the small molecule sulfamide-nitroimidazole (described in Rami et al., (2013), J. Med. Chem, 56:8512 - 8520, which is incorporated herein by reference).

[0178] In any embodiment, the agent for binding to CAIX is the small molecule JS-403 (described in WO2010 / 089752A1, which is incorporated herein by reference).

[0179] In any embodiment, the agent for binding to CAIX is the small molecule UB-TT220 (described in WO2022 / 015955A1, which is incorporated herein by reference).

[0180] In any embodiment, the agent for binding to CAIX is the small molecule 99m Tc-HEHEHE-Z09781 (Kim et al., (2017) Advanced Science, 4:1600471, Gebauer and Skerra (2009) Current Opin in Chem Biol, 13(3):245 - 55, Schardt et al., (2017) Mol Pharmaceutics, 14(4):1047 - 56, Tolmachev et al., (2008) Bioconjugate Chem, 19(8):1579 - 87, Liu et al., (2022) Analytical and Bioanalytical Chemistry, 414:1095 - 1104, Grindel et al., (2022) ACS Chem Biol, 17(6):1543 - 55, which are incorporated herein by reference), 99m Tc-MIP-1486, 99mTc-MIP-1490 (4-(2-bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide, wherein X = ethyl), or 99m Tc-MIP-1504 (4-(2-bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide, wherein X = n-butyloxy) (Hillier et al., (2012) Journal of Nuclear Medicine, 53(s1):217 (incorporated herein by reference)).

[0181] In any embodiment, the agent for binding to CAIX is the small molecule PHC-102 (described in WO2015 / 114171A1, WO2018 / 154517A1, US2014 / 0357650A1, WO2015 / 114171A1 (incorporated herein by reference)).

[0182] In any embodiment, the agent for binding to CAIX is a peptide. As used herein, a peptide is understood to comprise a chain of more than one amino acid residue. Typically, a peptide may comprise about 2 to 30 amino acids, such as about 5 to 30, about 10 to 30, about 2 to 25, about 5 to 25, about 10 to 25, or about 10 to 20 amino acids. A peptide may have a length of at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 amino acids. Typically, a peptide is about 40 amino acids or less, such as about 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids or less.

[0183] In any embodiment, the agent for binding to CAIX is peptide 3B-301 (also known as Debio 0228; Queen et al., (2018) Int J of Biol Macromol, 106:840-850, Eldehna et al., (2019) Bioorganic Chem, 90:103102, Lavavecchia et al., (2011) Carbohydrate Res, 346(3):442-48, Krymov et al., (2022) Eur J of Medicinal Chem, 228:113997, Supuran (2008) BJI Int, 101(s4):39-40, Koyuncu et al., (2019) J of Enzyme Inhibition and Medicinal Chem, 34(1):703-11, Kumar et al., (2017) Eur J of Medicinal Chem, 136:52-62, incorporated herein by reference), or 3B-302.

[0184] In any embodiment, the agent for binding to CAIX is the peptide CAIX-P1 having the amino acid sequence YNTNHVPLSPKY (described in Askoxylakis et al., (2010), PLoS One, 5(12):e15962), and optionally, the peptide is labeled with 125I or 131I to enable its detection (it should be understood, however, that any suitable radioactive label or other detectable moiety may be used). As used herein, the term "seranostic" refers to the ability of a compound / material used for diagnosis and therapy. The term "seranostic reagent" relates to any reagent suitable for both the detection, diagnosis, and / or treatment of a patient's disease or condition. The purpose of seranostic compounds / materials is to overcome undesirable differences in biodistribution and selectivity that may exist between different diagnostic and therapeutic agents. Using a seranostic pair, a seranostic compound containing a radionuclide for imaging is first administered to a patient to identify a disease or find a diseased site in the body. Once identified / found, since the biodistribution of the radionuclides for imaging and therapy is the same, the disease can be treated by administering a seranostic compound containing a radionuclide for therapy in a target-specific manner.

[0185] Antibody for binding to CAIX According to a particularly preferred embodiment, the agent for binding to CAIX is preferably an anti-CAIX antibody and / or a functional fragment of such an antibody. A fragment of an anti-CAIX antibody may have essentially the same CAIX binding and / or inhibitory activity as the full-length anti-CAIX antibody and / or may be an epitope-binding fragment of the anti-CAIX antibody.

[0186] 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 "binds specifically to" or "specifically binds to" CAIX.

[0187] The antibody and / or its antibody fragment may be selected from the group consisting of polyclonal antibodies, monoclonal antibodies, F(ab’)2, Fab’, scFv, dsFv, and antigen-binding fragments thereof such as chimerized, humanized, and fully human variants thereof. The antibody may be multivalent and may be multivalent and multispecific. The antibody may comprise a human constant region of IgG1, IgG2a, IgG3, or IgG4.

[0188] According to a further preferred embodiment, the anti-CAIX antibody or its epitope-binding fragment for use according to the invention binds to the amino acid sequences LSTAFARV and / or ALGPGREYRAL.

[0189] In any embodiment, the agent for binding to CAIX is in the form of the antibody BAY-794620, or an antigen-binding fragment thereof (WO2003 / 100029A2, WO2003 / 033674A2, Theiner et al., (2021) Tierarztl Prax Ausg G Grosstiere Nutztiere, 49(6): 392-402, Kimani et al., (2011) Photochemistry and Photobiology, 88(1):175-87, NCT01065623 (v24, 30 September 2014), NCT01028755 (v30, 19 January 2015), which are incorporated herein by reference).

[0190] In any embodiment, the agent for binding to CAIX is in the form of the antibody SLC-0131, or an antigen-binding fragment thereof.

[0191] In any embodiment, the agent for binding to CAIX is in the form of an antibody or an antigen-binding fragment thereof as described in US11,629,199 (incorporated herein by reference).

[0192] According to an even more particularly preferred embodiment, the agent for binding to CAIX is an anti-G250 antibody and / or an antigen-binding fragment thereof. Anti-G250 antibodies are described, for example, in EP-B-0637336. The antibody or fragment thereof may be a chimeric or humanized G250 antibody. In some embodiments, the antigen-binding protein that binds or specifically binds to CAIX is as described in any of WO2002 / 062972A2 (US2004 / 0219633A1), WO2004 / 002526A1 (US7,632,496B2), WO2006 / 002889A2 (US7,691,375B2), WO2009 / 056342A1 (US2014 / 0017252A1), WO2011 / 032973A1 (US2012 / 0207672A1), and WO2014 / 128258A1 (US10,620,208B2) or WO2021 / 000017A1 (the entire contents of each of these publications are incorporated herein by reference).

[0193] Antibodies for use in the present invention can be produced by any suitable method known in the art, including but not limited to the methods described in PCT / EP02 / 01282 and PCT / EP02 / 01283, which are incorporated herein by reference.

[0194] Particularly preferred antibodies are cG250, preferably girentuximab (INN). Another particularly preferred embodiment is the monoclonal antibody G250 produced by the hybridoma cell line DSM ACC 2526. Antibody cG250 is an IgG1 kappa light chain chimeric form of the original mouse monoclonal antibody mG250.

[0195] Variants of the original chimeric G250 (cG250) antibody, including WX-G250 and WX-G250RIT (iodine-131) (Janssen Global Services LLC), are known.

[0196] In a particularly preferred embodiment, the antibody is 89 Zr-girentuximab, i.e.,89 Zr-cG250), 123 I−, 124 I−, or 131 I-girentuximab, or 177 Lu-girentuximab.

[0197] In the context of the present invention, the antibody for use according to the present invention is particularly useful for inclusion in a theranostic pair, for example, where the antibody is conjugated to a radioisotope for imaging or diagnostic purposes and the same antibody is conjugated to a different radioisotope or a cytotoxic agent suitable for therapy. The antigen-binding domain of the antibody directs or targets a diagnostic radioisotope to the site of the tumor (including tumor distribution, tumor size, tumor density) to facilitate diagnosis, while the same antigen-binding domain of the antibody directs a radioisotope to the tumor for therapy.

[0198] As used herein, the term "Fc region", which may be referred to as "Fc" or "Fc domain", refers to the portion of the IgG molecule that correlates with the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region consists of the C-terminal halves of the two heavy chains of the IgG molecule linked by disulfide bonds. It has no antigen-binding activity but contains a carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor). The Fc region includes the entire second constant domain CH2 (also defined as residues 231-340 of human IgG1 according to the EU index numbering system, residues 244-360 in the Kabat system) and the third constant domain CH3 (residues 341-447 (EU index) / 361-478 (Kabat)) (for example, for the sequence of CH2 and SEQ ID NO: 2, see SEQ ID NO: 1 or FIG. 1C of WO2015 / 175874; for the sequence of CH3, see FIG. 1D, which is incorporated herein by reference). Also see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs for a comparison of the numbering conventions used for various residues in the Fc region of immunoglobulins).

[0199] As used herein, "EU index" or "EU numbering scheme" refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, which is incorporated herein by reference in its entirety). As used herein, "Kabat system" refers to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991. One of ordinary skill in the art will be able to readily determine whether a given amino acid sequence is numbered according to either the EU or Kabat system.

[0200] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by virtue of its origin or source of derivation, is not associated with the naturally associated components in its natural state and is substantially free of other proteins from the same source. The protein may be made substantially free of naturally associated components using protein purification techniques known in the art, or may be substantially purified by isolation. "Substantially purified" means that the protein is substantially free of contaminants, e.g., contains at most about 30% or 25% or 20% or 15% or 10% or 5% or 4% or 3% or 2% or 1% contaminants.

[0201] The term "recombinant" is understood to mean the product of artificial genetic recombination. Thus, in the context of a recombinant protein containing an antibody antigen-binding domain, this term does not include antibodies that occur naturally in the subject, which are products of natural recombination that occur during B cell maturation. However, if such an antibody is isolated, it should be considered an isolated protein containing an antibody antigen-binding domain. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody antigen-binding domain. Recombinant proteins also include proteins expressed by artificial recombinant means when they are within a cell, tissue or subject, e.g., when they are expressed.

[0202] The term "protein" is to be interpreted as including a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently bound to each other (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently bound using suitable chemical or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0203] Note: There seems to be an error in the original text where it says "at most about 30% or 25% or 20% or 15% or 10% or 5% or 4% or 3% or 2% or 1% contaminants" which is likely meant to be something like "at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminants" as it was in the first paragraph. The translation has been adjusted accordingly.The term "polypeptide" or "polypeptide chain" will be understood from the foregoing paragraphs to mean a series of contiguous amino acids linked by peptide bonds.

[0204] As used herein, the term "antigen-binding site" may be used interchangeably with "antigen-binding domain" and is to be construed to mean the region of an antibody that specifically binds to an antigen, i.e., an antigen that can specifically bind to VH or VL, or Fv containing both VH and VL. The antigen-binding domain need not be in the context of the whole antibody and can be, for example, isolated (e.g., domain antibody), or in another form (e.g., scFv) as described herein.

[0205] For the purposes of the present disclosure, the term "antibody" includes proteins that can specifically bind to one or several closely related antigens by an antigen-binding domain contained within the Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant antibodies or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synhumanized antibodies, half antibodies, bispecific antibodies). Antibodies generally include a constant domain that can be arranged in a constant region or a constant fragment or a crystallizable fragment (Fc). Exemplary forms of antibodies include a four-chain structure as their basic unit. Full-length antibodies include two covalently linked heavy chains (about 50-70 kD) and two light chains (each about 23 kDa). Light chains generally include a variable region (if present) and a constant domain, and in mammals, are either κ light chains or λ light chains. Heavy chains generally include a variable region and one or two constant domains linked by a hinge region to additional constant domains. Mammalian heavy chains are of one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently bound to one of the heavy chains. For example, the two heavy chains as well as the heavy and light chains are held together by interchain disulfide bonds and non-covalent interaction. The number of interchain disulfide bonds can vary between different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each about 110 amino acids in length) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL, about 110 amino acids in length) is aligned and disulfide-bonded with the first constant domain of the heavy chain (CH1, about 330-440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can include two or more additional CH domains (CH2, CH3, etc.) and can include a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a mouse (mouse or rat) antibody or a primate (e.g., human) antibody.In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is humanized, synthetically humanized, chimeric, CDR-grafted, or deimmunized.

[0206] The terms "full-length antibody", "intact antibody", or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form as contrasted with an antigen-binding fragment of the antibody. Specifically, whole antibodies include those having heavy and light chains that include an Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0207] As used herein, "variable region" refers to the portion of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes the amino acid sequences of the complementarity determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, and the framework regions (FRs). For example, the variable region includes three CDRs together with three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0208] As used herein, the term "complementary determining region" (synonyms: CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable region whose presence contributes significantly to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2, and CDR3. The CDRs of VH are herein also referred to as CDR H1, CDR H2, and CDR H3, respectively, where CDR H1 corresponds to CDR1 of VH, CDR H2 corresponds to CDR2 of VH, and CDR H3 corresponds to CDR3 of VH. Similarly, the CDRs of VL are herein referred to as CDR L1, CDR L2, and CDR L3, respectively, where CDR L1 corresponds to CDR1 of VL, CDR L2 corresponds to CDR2 of VL, and CDR L3 corresponds to CDR3 of VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html).The present invention is not limited to FRs and CDRs defined by the Kabat numbering system, but includes all numbering systems, including standard numbering systems, or those of Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987, Chothia et al., Nature 342: 877-883, 1989, and / or Al-Lazikani et al., J. Mol. Biol. 273:927-948, 1997; the numbering system of Honnegher and Plukthun, J. Mol. Biol. 309:657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25:206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include the 5 C-terminal amino acids listed herein, or one or more of those amino acids are substituted with another natural amino acid. In this regard, Padlan et al., FASEB J., 9:133-139, 1995 demonstrated that the 5 C-terminal amino acids of the heavy chain CDR2 are generally not involved in antigen binding.

[0209] The "framework region" (FR) is the variable region residue other than the CDR residue. The FRs of VH are herein also referred to as FR H1, FR H2, FR H3, and FR H4, respectively, where FR H1 corresponds to FR1 of VH, FR H2 corresponds to FR2 of VH, FR H3 corresponds to FR3 of VH, and FR H4 corresponds to FR4 of VH. Similarly, the FRs of VL are herein referred to as FR L1, FR L2, FR L3, and FR L4, respectively, where FR L1 corresponds to FR1 of VL, FR L2 corresponds to FR2 of VL, FR L3 corresponds to FR3 of VL, and FR L4 corresponds to FR4 of VL.

[0210] As used herein, the term "Fv" is to be construed to mean any protein that forms a complex in which VL and VH associate, whether composed of multiple polypeptides or a single polypeptide, and that has an antigen-binding domain (i.e., is capable of specifically binding to an antigen). The VH and VL that form the antigen-binding domain may be on a single polypeptide chain or on different polypeptide chains. Further, the Fv of the present invention (and any protein of the present invention) may have multiple antigen-binding domains that may or may not bind to the same antigen. This term is understood to encompass fragments directly derived from an antibody and proteins corresponding to such fragments produced using recombinant means. In some examples, VH is not linked to the heavy-chain constant domain (CH)1 and / or VL is not linked to the light-chain constant domain (CL). Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetra-bodies or higher-order complexes, or any of the foregoing linked to their constant regions or domains (e.g., CH2 or CH3 domains) (e.g., minibodies). A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of an intact antibody with the enzyme papain to obtain a fragment consisting of an intact light chain and a portion of the heavy chain, or may be produced using recombinant means. The "Fab' fragment" of an antibody can be obtained by treating the intact antibody with pepsin followed by reduction to obtain a molecule consisting of an intact light chain and a portion of the heavy chain containing VH and a single constant domain. Two Fab' fragments are obtained for each antibody treated in this manner. Fab' fragments can also be produced by recombinant means. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and is obtained by treating the intact antibody molecule with the enzyme pepsin without subsequent reduction. A "Fab2" fragment is a recombinant fragment containing two Fab fragments linked using, for example, a leucine zipper or a CH3 domain."Single-chain Fv" or "scFv" is a recombinant molecule containing a variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable and flexible polypeptide linker.

[0211] As used herein, the term "binds" with respect to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody generally recognizes and binds not to a protein in general, but to a specific protein structure. When an antibody binds to epitope "A", in a reaction containing labeled "A" and a protein, the presence of a molecule containing epitope "A" (or free, unlabeled "A") reduces the amount of labeled "A" bound to the antibody.

[0212] As used herein, the terms "specifically binds" or "binds specifically" are to be construed to mean that an antigen-binding protein for use according to the invention reacts or associates with a particular antigen or a cell expressing it more frequently, more rapidly, for a longer duration, and / or with a higher affinity than with alternative antigens or cells. For example, an antigen-binding protein binds to CAIX with a substantially higher affinity (e.g., 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than to other antigens.

[0213] As used herein, the term "epitope" (synonym: "antigenic determinant") is understood to mean the region of a cell surface protein (such as CAIX) to which an antigen-binding protein containing the antigen-binding domain of an antibody binds.

[0214] As used herein, the term "condition" refers to a disruption or interference with normal function and is not limited to any particular condition, including diseases or disorders.

[0215] As used herein, the terms "prevent," "preventing," or "prevention" include administering an active agent such as an antigen-binding protein or checkpoint inhibitor described herein, thereby preventing or impeding the occurrence of at least one symptom of a condition. The term also encompasses treating a subject in remission to prevent or impede recurrence.

[0216] As used herein, the terms "treat," "treating," or "treatment" include administering an active agent such as an antigen-binding protein or checkpoint inhibitor described herein, thereby reducing or eliminating at least one symptom of a particular disease or condition.

[0217] As used herein, the term "subject" is to be construed to mean any animal, including a human, such as a mammal. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0218] Modified antibody The present invention relates in part to the use of antibodies that include modification of an IgG antibody, which includes one or more amino acid substitutions to a region of the antibody that decreases or abrogates the antibody's affinity for FcRn, thereby decreasing the serum half-life of the antibody.

[0219] It will be understood that any antibody for which a decrease in serum half-life is desired can be modified according to the methods described herein in accordance with the present invention.

[0220] In certain embodiments, antibodies suitable for modification according to the present invention to decrease affinity for FcRn are antibodies having one or more of the sequences set forth in Table 1.

[0221] The present invention also provides for the use of antigen binding proteins which are at least 80% identical to a sequence disclosed herein. In one example, the antigen binding protein for use according to the present invention comprises a sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a sequence disclosed herein.

[0222] Alternatively, or additionally, the antigen binding protein may be a V protein according to any of the examples described herein. H or V L 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical CDRs (e.g., three CDRs) to the CDRs of

[0223] The present invention also contemplates variants of the antigen binding proteins for use according to the present invention which comprise one or more conservative amino acid substitutions compared to the sequences set forth herein. In some examples, the antigen binding protein comprises 10 or fewer conservative amino acid substitutions, for example 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydrophilicity and / or hydrophilicity.

[0224] Families of amino acid residues with similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydrophobicity indices are described, for example, in Kyte and Doolittle J.Mol.Biol., 157:105-132, 1982, and hydrophilicity indices are described, for example, in US4554101.

[0225] In some embodiments, the antigen-binding protein contains non-conservative amino acid changes. For example, of particular interest are substitutions of one charged amino acid for another charged amino acid and for neutral or positively charged amino acids. In some examples, the antigen-binding protein contains 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0226] In one example, the mutation occurs within the FR of the antigen-binding domain of the antigen-binding protein. In another example, the mutation occurs within the CDR of the antigen-binding protein.

[0227] Exemplary methods for generating variant forms of the antigen-binding protein include the following: ·DNA mutagenesis (Thie et al., Methods Mol.Biol. 525:309-322, 2009) or RNA mutagenesis (Kopsidas et al., Immunol.Lett. 107:163-168, 2006, Kopsidas et al. BMC Biotechnology, 7:18, 2007, and WO1999 / 058661), · Introducing a nucleic acid encoding a polypeptide into mutator cells, such as XL-1 Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene). · DNA shuffling (e.g., as disclosed in Stemmer, Nature 370:389-91, 1994), and · Site-directed mutagenesis (e.g., as described in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0228] Exemplary methods for determining the biological activity of an antigen-binding protein for use according to the present invention will be apparent to those skilled in the art and / or are described herein (e.g., antigen binding). For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein.

[0229] Constant region The present invention also provides for the use of the antigen-binding proteins and / or antibodies described herein that include the constant region of an antibody. This includes antigen-binding fragments of antibodies fused to Fc.

[0230] The sequences of constant regions useful for the production of the proteins of the present invention can be obtained from several different sources. In some examples, the constant region of a protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof can be derived from any antibody class including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype including IgG1, IgG2, IgG3, and IgG4. In one example, the constant region is the human isotype IgG4 or a stabilized IgG4 constant region.

[0231] Preferred modifications The present invention also contemplates modifications to antibodies or antigen-binding proteins for use according to the present invention that include an Fc region or constant region.

[0232] The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG-class antibodies in vivo. FcRn functions to salvage IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. It is a heterodimeric protein consisting of two polypeptides, a 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG-class antibodies. The interaction between IgG-class antibodies and FcRn is pH-dependent and occurs with a 1:2 stoichiometry, i.e., one IgG antibody molecule can interact with two FcRn molecules via its two heavy-chain Fc-region polypeptides (see, for example, Huber, A. H., et al., J. Mol. Biol. 230 (1993) 1077-1083).

[0233] Thus, the in vitro FcRn-binding properties / characteristics of IgG indicate its in vivo pharmacokinetic properties in the bloodstream. In the interaction between the FcRn and the Fc region of IgG-class antibodies, different amino acid residues of the heavy-chain CH2- and CH3-domains are involved.

[0234] Different mutations are known that affect FcRn binding and the associated half-life in the blood circulation. Fc region residues important for mouse Fc region - mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall’Acqua, W.F., et al. J. Immunol 169 (2002) 5171 - 5180). Residues Ile253, His310, His433, Asn434 and His435 (numbering according to the EU index numbering system) are involved in the interaction (Medesan, C, et al., Eur. J. Immunol. 26 (1996) 2533 - 2536, Firan, M., et al., Int. Immunol. 13 (2001) 993 - 1002, Kim, J.K., et al, Eur. J. Immunol. 24 (1994) 542 - 548). (Using the Kabat system, the relevant residues are Ile266, His329, His464, Asn465 and His466). Residues Ile253, His310, and His435 have been found to be important for the interaction between the human Fc region and mouse FcRn (Kim, J.K., et al, Eur. J. Immunol. 29 (1999) 2819 - 2885).

[0235] More specifically, the antibody can comprise one or more amino acid substitutions that decrease the half-life of the protein. For example, the antibody comprises an Fc region that comprises one or more amino acid substitutions that decrease the affinity of the Fc region for the neonatal Fc region (FcRn).

[0236] The invention also provides for the use of an antibody having a constant region that is substantially identical to a naturally occurring class IgG antibody constant region, wherein at least one amino acid residue selected from the group consisting of His310, His435, and Ile253 is different from that present in a naturally occurring class IgG antibody, whereby the FcRn binding affinity and / or serum half-life of the antibody is altered compared to a naturally occurring antibody. In a preferred embodiment, the naturally occurring class IgG antibody comprises the heavy chain constant region of a human IgG1, IgG2, IgG2M3, IgG3 or IgG4 molecule.

[0237] Also, in a preferred embodiment, amino acid residue 310 or residue 435 from the heavy chain constant region of an antibody having a constant region substantially identical to a naturally occurring class IgG antibody is not histidine, but any amino acid that reduces the affinity of the constant region for FcRn. For example, the amino acid at residue 310 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.

[0238] Preferably, the residue at position 310 is selected from alanine, or glutamic acid or glutamine, or the amino acid residue 435 from the heavy chain constant region is selected from arginine, glutamine, or alanine. In other preferred embodiments, an antibody having a constant region substantially identical to a naturally occurring class IgG antibody has an alanine residue at position 310 and a glutamine residue at position 435.

[0239] In a preferred embodiment of the present invention, the binding affinity for FcRn and / or the serum half-life of the modified antibody is reduced by at least about 30%, 50%, 80%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold. In a preferred embodiment of the present invention, the binding affinity for FcRn and / or the serum half-life of the modified antibody is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0240] In addition, the antibody for use according to the present invention may comprise one or more mutations that modify the affinity of the antibody for any one or more Fc gamma receptors.

[0241] In some embodiments, the Fc region of the constant region retains the ability to induce effector functions. In one example, the Fc region of the constant region includes one or more amino acid substitutions that modulate effector functions, including increasing effector functions as compared to wild-type IgG.

[0242] In one example, the Fc region of the constant region has a reduced ability to induce effector functions as compared to, for example, the Fc region of native or wild-type human IgG1 or IgG3. In one example, effector functions are 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 functions of Fc region-containing proteins are known in the art and / or described herein.

[0243] In one example, the amino acid substitution that modifies the ability of the antibody to induce effector functions is an amino acid substitution at residue Ile253 from the heavy chain constant region. In one example, the substitution is a substitution to any amino acid selected from alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine, and the substitution decreases the ability of the antibody to induce effector functions. In a preferred embodiment, the substitution from Ile at residue 253 is a substitution to arginine, proline, or aspartate, more preferably alanine.

[0244] In one example, the Fc region is an IgG4 Fc region (i.e., from the IgG4 constant region), for example, a human IgG4 Fc region. Suitable sequences of the IgG4 Fc region will be apparent to those skilled in the art and / or are available in publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0245] In one example, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region modified to reduce the tendency to undergo Fab arm exchange or to be susceptible to Fab arm exchange, or the formation or tendency to form half antibodies. "Fab arm exchange" refers to a type of protein modification to human IgG4 in which the IgG4 heavy chain and attached light chain (half molecule) are exchanged for a heavy chain-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by reducing agents such as purified blood cells or reduced glutathione. A "half antibody" is formed when an IgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

[0246] In one example, the stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to the Kabat system (see Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system. In human IgG4, this residue is generally serine. After substitution of serine for proline, the IgG4 hinge region contains the sequence CPPC. In this regard, one of ordinary skill in the art will recognize that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that connects the Fc and Fab regions, imparting mobility to the two Fab arms of the antibody. The hinge region contains cysteine residues involved in inter-heavy chain disulfide bonds. Generally, it is defined as extending from Glu226 to Pro24 in human IgG1 according to the Kabat numbering system (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 that form the inter-heavy chain disulfide (S-S) bonds at the same positions (see, for example, WO2010 / 080538).

[0247] Additional examples of stabilized IgG4 antibodies are antibodies in which arginine at position 409 within the heavy chain constant region of human IgG4 (according to the EU numbering system) is substituted with lysine, threonine, methionine, or leucine (as described, for example, in WO2006 / 033386). The Fc region of the constant region can additionally or alternatively contain a residue 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 contains a proline at position 241 (i.e., the CPPC sequence) (as described above).

[0248] In another example, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region". For example, the Fc region is an IgG1 Fc region containing substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region comprising one or more of the following changes: one or more of E233P, L234V, L235A and deletion of G236, and / or one or more of the following changes: A327G, A330S and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999, Shields et al. J Biol Chem. 276(9):6591-604, 2001). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall’Acqua et al., J Immunol. 177:1129-1138 2006, and / or Hezareh J Virol, 75:12161-12168, 2001).

[0249] In another example, the Fc region is, for example, at least one C H 2 domain and at least one C H 3 domain-containing chimeric Fc region, and the Fc region contains 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) (as described, for example, in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0250] Antibody production Preferably, the antigen-binding protein described herein according to any embodiment is recombinant.

[0251] In the case of a recombinant protein, the nucleic acid encoding it can be cloned into an expression construct or vector, which is then transfected into host cells such as E. coli cells, yeast cells, insect cells, or mammalian cells such as simian COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or in other cases myeloma cells that do not produce the protein. Exemplary cells used to express the protein are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques for achieving these purposes are known in the art and are described, for example, in Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art; see, for example, US4816567 or US5530101.

[0252] After isolation, the nucleic acid is inserted operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of DNA) or for expression in a cell-free system or in cells.

[0253] As used herein, the term "promoter" should be understood in its broadest context and includes, for example, additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers and silencers) that change the expression of nucleic acids in response to developmental or and / or external stimuli, or in a tissue-specific manner, and the TATA box or initiator element necessary for accurate transcription initiation, whether or not present, of the transcriptional regulatory sequences of genomic genes. In this context, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative, that confers, activates or enhances the expression of an operably linked nucleic acid. Exemplary promoters can contain additional copies of one or more specific regulatory elements for further enhancing the expression of the nucleic acid and / or for changing spatial and / or temporal expression.

[0254] As used herein, the term "operably linked to" means positioning a promoter relative to a nucleic acid such that the expression of the nucleic acid is controlled by the promoter.

[0255] Many vectors are available for expression in cells. Vector components generally include, but are not limited to, one or more of a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. One of ordinary skill in the art will recognize sequences suitable for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, IPP, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, α-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0256] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-α promoter (EF1), the small nuclear RNA promoters (U1a and U1b), the α-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the β-actin promoter, the CMV enhancer / β-actin promoter or hybrid regulatory elements comprising its immunoglobulin promoter or active fragments. Examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), the human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture), the baby hamster kidney cells (BHK, ATCC CCL 10), or the Chinese hamster ovary cells (CHO).

[0257] Typical promoters suitable for expression in yeast cells such as yeast cells selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae, and S. pombe include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0258] Means for introducing into a cell a nucleic acid isolated for expression or an expression construct containing the same are known to those skilled in the art. The technique used for a given cell depends on known successful techniques. Means for introducing recombinant DNA into a cell include, inter alia, microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes, for example, transfection by using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), DNA uptake mediated by PEG, electroporation, and microparticle bombardment, for example, by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0259] Host cells used for producing a protein can be cultured in various media depending on the cell type used. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle’s Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0260] Isolation of the protein Methods for isolating a protein are known in the art and / or are described herein.

[0261] When the antigen-binding protein is secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis, and an antibiotic can be included to prevent the growth of accidental contaminants. Alternatively, or additionally, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0262] The antigen-binding protein prepared from the cells can be purified, for example, using ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the above. These methods are known in the art and are described, for example, in WO99 / 57134 or Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0263] One skilled in the art will also recognize that the protein can be modified to include tags for ease of purification or detection, such as a polyhistidine tag, such as a hexahistidine tag, or an influenza virus hemagglutinin (HA) tag, or a simian virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art such as affinity purification. For example, a protein containing a hexahistag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexahistag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively, or additionally, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0264] Binding of radioisotopes to antibodies In any embodiment of the invention, the antibodies described herein may be directly or indirectly linked to a therapeutic agent, preferably the therapeutic agent is a radioisotope.

[0265] As used herein, the term "radioisotope" is used interchangeably with "radionuclide".

[0266] Examples of suitable isotopes include actinium-225 ( 225 Ac), astatine-211 ( 211 At), bismuth-212 and bismuth-213 ( 212 Bi, 213 Bi), copper-64 and copper-67 ( 64 Cu, 67 Cu), iodine-123, -124, -125 or -131 ( 123 I, 124 I, 125 I, 131 I)( 123 I), lead-212 ( 212 Pb), lutetium-177 ( 177 Lu), radium-223 and radium-224 (223 Ra, 224 Ra), rhenium-186 and rhenium-188 ( 186 Re and 188 Re), samarium-153 ( 153 Sm), scandium-47 ( 47 Sc), strontium-90 ( 90 Sr), terbium-149 and terbium-161 ( 149 Tb and 161 Tb), and yttrium-90 ( 90 Y) are included.

[0267] In some embodiments, the antigen-binding protein is conjugated to a radioisotope capable of therapy and diagnosis, which can treat cancer in a subject and is useful for assessing the spread of cancer or the size of tumors expressing CAIX during treatment by binding to CAIX. Those skilled in the art will be familiar with which therapeutic radioisotopes can be used for imaging and which imaging techniques can be used.

[0268] It will be understood that the isotope can be conjugated directly to the antibody described herein (via a chelating agent or a family of bridging molecules or linkers) or indirectly via binding to a single or multiple amino acid residues in the antibody (e.g., halogenation of tyrosine residues).

[0269] In an alternative embodiment, a chelating agent or linker may be used to conjugate a radioisotope to an antibody. In one example, the antibody may be conjugated to a chelating moiety selected from the group consisting of: TMT (6,6’’-bis[N,N’’,N’’’-tetra(carboxymethyl)aminomethyl)-4’-(3-amino-4-methoxyphenyl)-2,2’:6’,2’’-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN’,N’’(N’’’-tetraacetic acid, also known as tetraxitane), TCMC (tetra primary amide of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), NOTA (1,4,7-triazacyclononane-triacetic acid), DiamSar (3,6,10,13,16,19-hexaazabicyclo[6,6,6]eicosane-1,8-diamine), DTPA (pentetic acid or diethylenetriaminepentaacetic acid), CHX-A’’-DTPA ([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8), 11-tetraacetic acid, Te2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), HBED, DFO (desferrioxamine), DFOsq (DFO-squaramide), and HOPO (3,4,3-(LI-1,2-HOPO), a chelating agent described in WO2022 / 133537 (incorporated herein by reference), or other chelating agents described herein.

[0270] Chelating agents containing radioactive metals and other halogenated radioisotopes may bind to the antibody via one or more amino acid residues or reactive moieties in the antibody, including but not limited to one or more lysine residues, tyrosine residues, or thiol moieties.

[0271] In another example, the modified antibody is conjugated using a bifunctional linker such as bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, or any amine or thiol modification chemistry known in the art.

[0272] One of ordinary skill in the art will be familiar with standard methods for conjugating chelating agents to antibodies and their derivatives or fragments. In addition, one of ordinary skill in the art will be familiar with approaches for selecting relevant chelating agents for pairing with radioactive metals, as described, for example, in Chem.Soc.Rev., 2014, 43, 260 (incorporated herein by reference).

[0273] Analysis of the activity of the antigen-binding protein Binding to CAIX From the disclosure herein, it will be apparent to one of ordinary skill in the art that the preferred antigen-binding proteins for use according to the present invention bind to CAIX. Methods for assessing binding to a protein are known in the art, for example, as described in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve immobilizing the antigen-binding protein and contacting it with a labeled antigen. After washing to remove non-specific binding proteins, the amount of label, and as a result the amount of bound antigen, is detected. Of course, the antigen-binding protein can be labeled and the antigen immobilized. Panning-type assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.

[0274] Therapeutic methods The antibodies for use according to the present invention are useful for the treatment of a number of conditions that require treatment by radioimmunotherapy. Typically, such conditions include cancer.

[0275] Exemplary cancers include cystic and solid tumors, bone and soft tissue tumors (including tumors in anal tissue, bile duct, bladder, blood cells, bowel, brain, breast, carcinoid, cervix, eye, esophagus, head and neck, kidney, larynx, leukemia, liver, lung, lymph nodes, lymphoma, melanoma, mesothelioma, myeloma, ovary, pancreas, penis, prostate, skin (e.g., squamous cell carcinoma), sarcoma, stomach, testis, thyroid, vagina, vulva). Soft tissue tumors include Benign schwannoma Monosomy, desmoid tumor, lipoblastoma, lipoma, uterine leiomyoma, clear cell sarcoma, dermatofibrosarcoma, Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, liposarcooma myxoid, alveolar rhabdomyosarcoma, and synovial sarcoma. Specific bone tumors include non-ossifying fibroma, unicameral bone cyst, enchondroma, aneurysmal bone cyst, osteoblastoma, chondroblastoma, chondromyxofibroma, osteofibroma, and adamantinoma, giant cell tumor of bone, fibrous dysplasia, Ewing's sarcoma, eosinophilic granuloma, osteosarcoma, chondroma, chondrosarcoma, malignant fibrous histiocytoma, and metastatic cancer. Leukemias include acute lymphoblastic, acute myeloblastic, chronic lymphocytic, and chronic myelogenous.

[0276] Other examples include breast tumors, colorectal tumors, adenocarcinoma, mesothelioma, bladder tumors, prostate tumors, germ cell tumors, liver tumors / bile duct cancer, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinoma, melanoma, atypical fibrotic tumors, spermatoma, non-testicular tumors, interstitial Leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilm's tumor.

[0277] In some embodiments, the cancer is metastatic cancer. The primary source of metastatic cancer can be any cancer type known in the art, including those described herein.

[0278] Preferably, the antigen-binding protein for use according to the present invention is useful for the treatment of cancers characterized by the presence of CAIX. For example, an antibody that binds to CAIX is useful for the treatment of cancers characterized by increased expression of CAIX, including renal cell carcinoma.

[0279] Antibody-binding domain containing a protein Single-domain antibody In some examples, the antigen-binding protein for use according to the present invention is a single-domain antibody or comprises one (which is used interchangeably with the term "domain antibody" or "dAb"). A single-domain antibody is a single polypeptide chain that comprises all or part of the variable region of the heavy chain of an antibody. In certain examples, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; for example, US6248516).

[0280] Diabody, triabody, tetrabody In some examples, the protein for use according to the present invention is a diabody, triabody, tetrabody, or higher-order protein complex (such as those described in WO98 / 044001 and / or WO94 / 007921) or comprises them.

[0281] For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain having the structure V L -X-V H or V H -X-V L wherein V L is the variable region of the light chain of an antibody, V H is the variable region of the heavy chain of an antibody, X comprises residues that are insufficient to allow V H and V L in a single polypeptide chain to associate (or form an Fv), or is absent, and V H of one polypeptide chain associates with V Lwhich binds to form an antigen-binding domain, i.e., an Fv molecule capable of specifically binding to one or more antigens. V L and V H may be the same in each polypeptide chain, or V L and V H may be different in each polypeptide chain such that they form a bispecific diabody (i.e., containing two Fvs with different specificities).

[0282] Single-chain Fv (scFv) Those skilled in the art will recognize that an scFv comprises a V H and V L region within a single polypeptide chain, and a polypeptide linker between V H and V L such that the scFv forms the desired structure for antigen binding (i.e., the V H and V L of the single polypeptide chain associate with each other to form an Fv). For example, the linker may contain more than 12 amino acid residues, and (Gly4Ser)3 is one of the more preferred linkers for scFv.

[0283] The invention also contemplates the use of disulfide-stabilized Fv (or diFv or dsFv) in which a single cysteine residue is introduced into the FR of V H and the FR of V L and cysteine residues linked by disulfide bonds to yield a stable Fv.

[0284] Alternatively, or additionally, the invention encompasses the use of a dimeric scFv, i.e., a protein comprising two scFv molecules linked by non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, the two scFvs are linked by a peptide linker of sufficient length such that both scFvs are formed and capable of binding to an antigen, as described, for example, in US2006 / 0263367.

[0285] Heavy chain antibody Heavy chain antibodies contain heavy chains but no light chains, so they are structurally different from many other forms of antibodies. Therefore, these antibodies are also referred to as "antibodies consisting only of heavy chains". Heavy chain antibodies are found, for example, in camels and cartilaginous fish (also called IgNAR).

[0286] The variable regions present in naturally occurring heavy chain antibodies are distinguished from the heavy chain variable regions (referred to as "V H domains") present in conventional four-chain antibodies and the light chain variable regions (referred to as "V L domains") present in conventional four-chain antibodies. In camelid antibodies, they are generally referred to as "V HH domains", and in IgNAR as V-NAR.

[0287] General descriptions of heavy chain antibodies derived from camelids and their variable regions, as well as methods for their production and / or isolation and / or use, can be found, inter alia, in the following references: WO94 / 04678, WO97 / 49805, and WO97 / 49805.

[0288] General descriptions of heavy chain antibodies derived from cartilaginous fish and their variable regions, as well as methods for their production and / or isolation and / or use, can be found, inter alia, in WO2005 / 118629.

[0289] Other antibodies and proteins containing antigen-binding domains The present invention also contemplates the use of other antibodies and proteins containing antigen-binding domains, such as: (i) The "key and hole" bispecific protein described in US5731168, (ii) Heteroconjugate proteins described, for example, in US4676980, (iii) Heteroconjugate proteins produced using chemical cross-linking agents, as described, for example, in US4676980, and (iv) Fab3 (described, for example, in EP1993 / 0302894).

[0290] Route of administration In some instances, the agents described herein can be administered by oral, parenteral, inhalation spray, via implantation in a depot in a dosage formulation containing an adsorbent, absorbent, topical, rectal, nasal, buccal, vaginal, intraventricular, or any other convenient dosage form containing a conventional non-toxic pharmaceutically acceptable carrier. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intracardiac, and intracranial injection or infusion techniques.

[0291] Methods for preparing the agents described herein into a form suitable for administration to a subject (e.g., a pharmaceutical composition) are well known in the art and include, for example, those described in Remington’s Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990), and U.S.Pharmacopeia:National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0292] The agents for use according to the invention are particularly useful for parenteral administration such as intravenous administration or administration into the body cavity or lumen of an organ or joint. In a particularly preferred embodiment, the antigen-binding protein for use according to the invention is preferably formulated for intravenous, intraperitoneal, subcutaneous, intramuscular, or intratumoral administration.

[0293] Compositions for administration generally include a pharmaceutically acceptable carrier, such as a solution of an antigen-binding protein dissolved in an aqueous carrier. For example, various aqueous carriers can be used, such as buffered saline. The composition can contain pharmaceutically acceptable adjuncts necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antigen-binding protein of the present invention in these formulations can vary widely and will be selected mainly based on fluid volume, viscosity, body weight, etc., according to the specific dosage form selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate can also be used. Liposomes may also be used as a carrier. The vehicle can contain small amounts of additives to enhance isotonicity and chemical stability, such as buffers and preservatives.

[0294] Immune checkpoint inhibitor Checkpoint inhibitor A "checkpoint inhibitor" inactivates proteins within the inhibitory checkpoint pathways of the immune response.

[0295] In the context of cancer, checkpoint inhibitors modulate the immune system by blocking proteins that prevent the immune system from attacking cancer cells. In particular, they control how immune evasion cancer cells and T cells interact, such that the T cells can recognize the tumor cells and mount an appropriate immune response against them. Non-limiting examples of checkpoint inhibitors that can be used according to the methods described herein include inhibitors that target PD-1 (programmed cell death protein 1), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), and PD-L1 (programmed death ligand 1). Those skilled in the art will understand that CTLA-4 and PD-1 are found on T cells, and PD-L1 is expressed on cancer cells and / or suppressive immune cells. Other non-limiting examples include PD-L2, TIM3, LAG3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD107), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF-beta.

[0296] Other immune checkpoints include indoleamine 2,3-dioxygenase (IDO) and CSF-R1. Inhibitors of these proteins are also contemplated as immune checkpoint inhibitors for use in the present invention.

[0297] "Programmed death-*1* (PD-1)" refers to an immune inhibitory receptor belonging to the CD28 family. PD-1 is expressed primarily on T cells activated in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found at GenBank accession number U64863.

[0298] When PD-1 binds to programmed cell death ligand 1 (PD-L1), the immune response is turned off to prevent T cells from damaging or killing cells. In the context of cancer, cancer cells are covered with PD-L1 protein, allowing them to camouflage themselves as healthy cells and thereby evade the immune response. Programmed death ligand-1 (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (HPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found at GenBank accession number Q9NZQ7.

[0299] Cytotoxic T-lymphocyte antigen-4 (CTLA-4) refers to an immune inhibitory receptor belonging to the CD28 family. CTLA-4 is expressed only on T cells in vivo and binds to two ligands, CD80 and CD86 (also called B7-1 and B7-2, respectively). As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, and analogs having at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found at GenBank accession number AAB59385.

[0300] Any checkpoint inhibitor described herein can be administered in the form of an antibody. An "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the Ab can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0301] The term "antibody" includes, for example, monoclonal and polyclonal Abs, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs, and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce immunogenicity in humans. Unless expressly stated otherwise and unless the context otherwise indicates, the term "antibody" also includes any antigen-binding fragment or antigen-binding portion of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain Abs.

[0302] "Isolated antibody" refers to an antibody that is substantially free of other Abs having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-1 is substantially free of Abs that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-1 may have cross-reactivity to other antigens such as PD-1 molecules from different species. Furthermore, an isolated antibody may be substantially free of other cellular materials and / or chemical substances. The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of an antibody molecule of a single molecular composition, i.e., an antibody molecule having an essentially identical primary sequence and exhibiting a single binding specificity and affinity for a particular epitope. mAbs are examples of isolated antibodies. mAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

[0303] "Human" antibody (huMAb) refers to an antibody having a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, when the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies described herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species such as a mouse have been transplanted into human framework sequences. The terms "human" Ab and "fully human Ab" are used synonymously.

[0304] The term "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human Ab have been replaced with the corresponding amino acids derived from a human immunoglobulin. In one embodiment of the humanized form of an Ab, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids derived from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions have not changed. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are tolerated (including variations as a result of codon degeneracy leading to synonymous codons) as long as they do not abrogate the ability of the Ab to bind to a particular antigen. A "humanized" Ab retains the same antigen specificity as the original Ab.

[0305] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, e.g., an antibody in which the variable region is derived from a mouse Ab and the constant region is derived from a human Ab.

[0306] The term "anti-antigen" Ab refers to an Ab that specifically binds to an antigen. For example, an anti-PD-1 Ab specifically binds to PD-1, and an anti-CTLA-4 Ab specifically binds to CTLA-4.

[0307] The "antigen-binding portion" of an Ab (also referred to as the "antigen-binding fragment") refers to one or more fragments of the Ab that retain the ability to specifically bind to the antigen bound by the whole Ab.

[0308] Examples of immune checkpoint and antibody inhibitors that target those checkpoints include anti-CTLA-4 (e.g., ipilimumab, tremelimumab, KAHR-102), anti-TIM3 (e.g., F38-2E2. ENUM005), anti-LAG3 (e.g., BMS-986016, IMP701. IMP321, C9B7W), anti-KIR (e.g., lirilumab, IPH2101, IPH4102), anti-PD-1 (e.g., nivolumab, pidilizumab, pembrolizumab, BMS-936559, atezolizumab, ramucirumab, MK-3475. AMP-224, AMP-514, STI-A1110, TSR-042), anti-PD-L1 (e.g., KY-1003 (EP2012 / 0194977), MCLA-145, atezolizumab. BMS-936559, MEDI-4736, MSB0010718C, AUR-012, STI-A1010, MPDL3280A, AMP-224, dapirolizumab pegol (CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42 (OSU-HDAC42, HDAC-42, AR42, AR 42, OSU-HDAC 42, OSU-HDAC-42, NSC D736012, HDAC-42, HDAC 42, HDAC42, NSC D736012, NSC-D736012), MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1, anti-B7-H4, anti-CD52 (such as alemtuzumab), anti-IL-10, anti-IL-35, anti-TGF-β (such as Fresolumimab), anti-CSF1R (e.g., FPA008), anti-NKG2A (e.g., monalizumab), anti-MICA (e.g., IPH43), and anti-CD39.

[0309] Anti-PD-1 antibody and anti-PD-L1 antibody Examples of suitable PD-1 inhibitors that can be used in accordance with the present invention include Keytruda (pembrolizumab), Opdivo (nivolumab), AGEN 2034, BGB-A317, BI-754091, CBT-501 (genolimzumab), MEDI0680, MGA012, PDR001, PF-06801591, REGN2810 (SAR439684), and TSR-042, or those disclosed in U.S. Patent No. 8,008,449. Other anti-PD-1 mAbs are described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, and PCT Publication No. 2012 / 145493 (incorporated herein by reference).

[0310] Nivolumab (also known as "Opdivo®" and previously called 5C4, BMS-936558, MDX-1106, or ONO4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor Ab that selectively prevents interaction with the PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of antitumor T cell function (U.S. Patent No. 8,008,449, incorporated herein by reference).

[0311] Pembrolizumab (also known as "Keytruda®", lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody for binding to the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587 (incorporated herein by reference). Pembrolizumab has been approved by the FDA for the treatment of recurrent or refractory melanoma.

[0312] Other suitable PD-1 inhibitors include Libtayo (cemiplimab), Blincyto (blinatumomab), dostarlimab, spartalizumab, cetrelimab, pidilizumab, and BI-754091.

[0313] An anti-PD-1 Ab suitable for use in the disclosed methods or uses of the present invention binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and / or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, the anti-PD-1 antibody comprises an antigen-binding portion or fragment that binds to the PD-1 receptor, inhibits ligand binding, and exhibits functional properties similar to those of the whole Ab in upregulating the immune system.

[0314] In certain embodiments, the anti-PD-1 antibody that can be used in accordance with the present invention can be replaced with another PD-1 or anti-PD-L1 antagonist. For example, an anti-PD-L1 antibody can prevent the interaction between PD-1 and PD-L1, and thereby exert an effect similar to that of the PD-1 signaling pathway. Thus, an anti-PD-L1 antibody can replace the use of an anti-PD-1 antibody in the methods disclosed herein. In any embodiment, suitable PD-L1 inhibitors include Imfinzi (durvalumab or MEDI4736), Tecentriq (atezolizumab or MPDL3280A), Bavencio (avelumab, MSB0010718C), MS-936559 (12A4 or MDX-1105), and CX-072.

[0315] Anti-CTLA-4 antibody An anti-CTLA-4 antibody that can be used in accordance with the present invention binds to human CTLA-4 so as to disrupt the interaction between CTLA-4 and the human B7 receptor. Since the interaction between CTLA-4 and B7 transduces a signal that leads to the inactivation of T cells carrying the CTLA-4 receptor, disruption of the interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging the immune response.

[0316] Suitable CTLA-4 inhibitors that may be used in accordance with the present invention include Yervoy (ipilimumab), tremelimumab and AGEN 1884, or those disclosed in U.S. Patent Nos. 6,984,720 and 7,605,238 (incorporated herein by reference). Ipilimumab is a fully human IgG1 monoclonal Ab that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation. Tremelimumab is a human IgG2 monoclonal anti-CTLA-4 antibody.

[0317] The immune checkpoint inhibitor can be administered in the form of a pharmaceutical composition, including those combined with any pharmaceutically acceptable excipient, carrier and / or diluent described herein. Typically, the immune checkpoint inhibitor is administered in a formulation as known in the art.

[0318] Dosage and administration time In any aspect or embodiment of the present invention, a therapeutically effective amount of an antigen-binding protein for binding to CAIX and an immune checkpoint inhibitor may be administered to a subject in need thereof.

[0319] The administrations can be simultaneous, separate, or sequential.

[0320] Administration refers to physically introducing a composition containing a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those skilled in the art, including those described herein. The pharmaceutical composition can be formulated from the active agents described herein according to any suitable route of administration. Typically, in addition to a therapeutic agent (e.g., an antibody for binding to CAIX and / or a checkpoint inhibitor), the pharmaceutical composition includes a pharmaceutically acceptable excipient, carrier, and / or diluent. Examples of suitable components for inclusion in the pharmaceutical composition are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington’s Pharmaceutical Sciences.

[0321] The terms "therapeutically effective amount" or "effective amount" generally refer to an amount of an antibody against CAIX and / or a checkpoint inhibitor of the present invention that (i) treats a particular disease, condition, or disorder, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. For example, undesirable effects such as side effects may occur along with the desired therapeutic effect. Thus, a practicing physician balances the potential benefits and potential risks in determining what the appropriate "effective amount" is. In some embodiments, an antigen-binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX) conjugated with a radionuclide and an immune checkpoint inhibitor is administered together in a "therapeutically effective amount" to a subject in need thereof. This therapeutically effective amount may include an amount of either or both of the antigen-binding protein or the immune checkpoint inhibitor that is less than a therapeutically effective amount by itself. In some embodiments, an antigen-binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX) conjugated with a radionuclide is administered in an amount less than a therapeutically effective amount that does not include an immune checkpoint inhibitor. In some embodiments, an immune checkpoint inhibitor is administered in an amount less than a therapeutically effective amount that does not include an antigen-binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX) conjugated with a radionuclide. In some embodiments, the "therapeutically effective amount" for a combination of an antigen-binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX) conjugated with a radionuclide and an immune checkpoint inhibitor may be a synergistic amount. A synergistic amount may be synergistic compared to monotherapy with either antigen-binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX) conjugated with a radionuclide or an immune checkpoint inhibitor.

[0322] For example, for the treatment of tumors, a therapeutically effective amount of the compounds or compositions described herein can inhibit tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to an untreated subject. Alternatively, the treatments described herein can cause complete regression of the tumor mass. In other embodiments of the invention, tumor regression is observed and can continue for at least about 10 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, or longer.

[0323] A therapeutically effective amount of a drug may also include a "prophylactic" or "prophylactically effective amount", which is administered to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or a subject suffering from cancer recurrence, and is any amount of an antibody and / or checkpoint inhibitor that binds to CAIX and inhibits the development or recurrence of cancer. In certain embodiments, a prophylactically effective amount completely prevents the development or recurrence of cancer. "Inhibiting" or "preventing" the occurrence or recurrence of cancer means either reducing the likelihood of the development or recurrence of cancer or completely preventing the occurrence or recurrence of cancer.

[0324] Suitable dosages of the antigen-binding proteins and immune checkpoint inhibitors for use according to the invention will vary depending on the particular active agent selected, the condition to be treated, and / or the subject to be treated. For example, it is within the ability of one of ordinary skill in the art to determine a suitable dosage by starting with a suboptimal dosage and progressively modifying the dosage to determine an optimal or useful dosage. Alternatively, data from cell culture assays or animal studies can be used to determine an appropriate dosage for treatment / prevention, and a suitable dosage is the ED of an active compound having little or no toxicity. 50within the range of circulating concentrations that include. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. A therapeutically / preventively effective dosage can first be estimated from cell culture assays. The IC 50 (i.e., the concentration or amount of the compound that achieves half-maximal inhibition of the symptoms) to achieve a range of circulating plasma concentrations, the dosage may be formulated in an animal model. Such information can be used to more accurately determine a useful dosage in humans. The levels in plasma can be measured, for example, by high performance liquid chromatography.

[0325] In some examples, the methods of the invention include administering a prophylactically or therapeutically effective amount of the proteins described herein.

[0326] For administration of a checkpoint inhibitor comprising a PD-1, PD-L1, or CTLA-4 inhibitor, the dosage can range from about 0.01 to about 20 mg / kg, about 0.1 to about 10 mg / kg, about 0.1 to about 5 mg / kg, about 1 to about 5 mg / kg, about 2 to about 5 mg / g, about 7.5 to about 12.5 mg / kg, or about 0.1 to about 30 mg / kg of the subject's body weight. For example, the dosage can be about 0.1, about 0.3, about 1, about 2, about 3, about 5, or about 10 mg / kg body weight, or about 0.3, about 1, about 2, about 3, or about 5 mg / kg body weight. The dosing schedule is typically designed to achieve an exposure that results in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of the Ab. Exemplary treatment regimens may require administration once every about 1 week, once every about 2 weeks, once every about 3 weeks, once every about 4 weeks, once every about 1 month, once every about 3 - 6 months, or more. In certain embodiments, the checkpoint inhibitor is administered to the subject once every about 2 weeks. In other embodiments, the antibody is administered once every about 3 weeks. The dosage and schedule can vary during the course of treatment. For example, dosing schedules for anti-PD-1 therapy can include administering the Ab (i) every about 2 weeks in a cycle of about 6 weeks, (ii) about 6 times every about 4 weeks, then every about 3 months, (iii) every about 3 weeks, (iv) about 3 - about 10 mg / kg once, followed by about 1 mg / kg every about 2 - 3 weeks. Considering that IgG4 Abs typically have a half-life of 2 - 3 weeks, dosing regimens for anti-PD-1 Abs for use according to the present invention include about 0.3 - 1, about 0 mg / kg body weight, 1 - 5 mg / kg body weight, or about 1 - about 3 mg / kg body weight by intravenous administration, and the Ab is administered every about 14 - 21 days in a cycle of up to about 6 weeks or about 12 weeks until complete response or confirmed progressive disease.

[0327] In some embodiments, treatment with a checkpoint inhibitor and / or an antibody for binding to CAIX disclosed herein is continued for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 18 months, at least about 24 months, at least about 3 years, at least about 5 years, or at least about 10 years. In some embodiments, treatment with a checkpoint inhibitor and / or an antibody for binding to CAIX disclosed herein is continued for a maximum duration of up to about 18 months, 16 months, 14 months, 12 months, 10 months, 8 months, or 6 months. The treatment period can be from any of these minimum periods to any of these maximum periods, for example, 1 to 18 months.

[0328] It will be understood that the specific dosage level for any particular patient depends on a variety of factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, and rate of excretion, drug combinations, i.e., other drugs used in treating the patient), as well as the severity of the particular disorder being treated.

[0329] As used herein, the terms "treatment" or "treating" include the application or administration to a subject of a compound described herein for the purpose of delaying, retarding, stabilizing, curing, healing, reducing, alleviating, modifying, repairing, lessening worsening, improving, or affecting a disease or condition, a symptom of a disease or condition, or a risk (or susceptibility thereto) of a disease or condition. The term "treating" includes any objective or subjective parameter in the treatment or improvement of an injury, condition or disease, including reduction, remission, reduction of the rate of worsening, reduction of the severity of a disease, stabilization, reduction of symptoms, or making an injury, condition or disease more tolerable to the subject, slowing the rate of degeneration or decline, not debilitating the ultimate point of degeneration, or improving the physical or mental well-being of the subject, and refers to any indication of success in the treatment or improvement of an injury, condition or disease.

[0330] As used herein, minimizing or preventing cancer progression means treating a subject to prevent or delay tumor recurrence or metastasis, or prevent growth of an existing tumor. Minimizing or preventing cancer progression includes preventing or delaying cancer recurrence or preventing growth of an existing tumor after treatment of cancer. Recurrences that are prevented include, for example, recurrence at the tumor bed after surgical resection. Alternatively, recurrence includes metastasis of cancer to another part of the body. As used herein, the terms "preventing recurrence" and "preventing relapse" are interchangeable.

[0331] The present invention also includes a method for preventing the onset of cancer in an individual. For example, an individual who requires cancer prevention may be considered at risk of developing cancer, but does not yet have a detectable cancer. An individual at risk of developing cancer may be an individual with a family history of cancer and / or an individual in whom genetic testing or other tests indicate a high or likely risk of developing cancer. An individual may have cancer stem cells but does not yet have a detectable tumor. It will be understood that the method for preventing the onset of cancer includes methods for delaying the onset of cancer in a subject.

[0332] The terms "subject" and "patient" will be understood to be interchangeable. Although the present invention is applicable to humans, the present invention is also useful for veterinary treatment purposes. The present invention is useful for livestock such as cows, sheep, horses, and poultry, companion animals such as cats and dogs, and zoo animals.

[0333] Kit A kit for use according to the present invention is also provided, comprising one or more of the following: (i) A drug for use according to the present invention, or an expression construct encoding the same.

[0334] In some embodiments, the kit may further comprise an immune checkpoint inhibitor optionally formulated in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and / or diluents.

[0335] In the case of a kit for detecting cancer, the kit can further comprise, for example, a detection means linked to an antigen-binding protein for use according to the present invention.

[0336] In the case of a kit for therapeutic / preventive use, the kit can further comprise a pharmaceutically acceptable carrier.

[0337] Optionally, the kit of the present invention is packaged with instructions for use in the methods described herein according to any of the examples.

[0338]

Table 1-1

[0339]

Table 1-2

[0340]

Table 1-3

[0341]

Table 1-4

[0342]

Table 1-5

[0343]

Table 1-6

[0344]

Table 1-7

[0345]

Table 1-8

[0346]

Table 1-9

[0347]

Table 1-10

[0348]

Table 1-11

Example

[0349] Example 1: Antibodies for binding to CAIX Summary The humanized antibody variable region gene was cloned into a vector encoding an unmodified human IgG1 heavy chain constant domain and a human kappa light chain constant domain. The chimeric antibody was further cloned into a vector encoding either a modified human IgG1 heavy chain constant domain containing mutations H310A and H435Q that abolish FcRn and protein A binding (Andersen et al, 2012) or mutations S228P (for hinge stabilization (Angal et al, 1993)) and L235E (for effector function removal (Reddy et al, 2000)) and the above-described FcRn-abolishing mutations in a modified human IgG4 heavy chain construct. The chimeric antibody and the humanized antibody were transiently expressed in HEK EBNA cells and tested for binding to human carbonic anhydrase IX (CAIX) using Biacore single-cycle kinetic analysis. The selected lead antibodies were purified by protein A or protein G and analyzed by analytical SEC, competitive ELISA, and Biacore multi-cycle kinetic analysis.

[0350] Three lead candidate humanized antibodies with similar binding to the chimeric antibody were identified. Subsequently, these were cloned into vectors encoding the human IgG1 heavy chain constant domain (H310A, H435Q) and the IgG4 heavy chain constant domain (S241P, L235E, H310A, H435Q) as described above. The chimeric antibody and the lead humanized antibody were transiently expressed in CHO cells and tested for binding to human carbonic anhydrase IX (CAIX) using Biacore single-cycle kinetic analysis. The antibody was purified by either protein A or protein G and analyzed by analytical SEC and Biacore multi-cycle kinetic analysis.

[0351] Method Design of the Variable Region Sequences of Composite Human Antibody (Trademark) The structural model of the V regions of the gilenzumab antibody was generated using Swiss PDB and analyzed to identify important "constrained" amino acids that are likely to be essential for the binding properties of the antibody. Most of the residues contained within the CDRs (using both Kabat and Chothia definitions), as well as a number of framework residues, were considered important. The VH and Vκ sequences of gilenzumab contain typical framework residues, as well as CDR1, 2, and 3 motifs comparable to those in many mouse antibodies.

[0352] From the above analysis, it was considered that the range of alternative residues outside the CDRs of the composite human sequences of gilenzumab that could be made was wide, but the menu of possible residues within the CDR sequences was very narrow. Preliminary analysis showed that corresponding sequence segments from several human antibodies could be combined to create CDRs that were similar or identical to those within the mouse sequences. For the regions outside and adjacent to the CDRs, a wide range of selected human sequence segments were identified as possible components of the novel humanized V regions.

[0353] CD4+ T Cell Epitope Avoidance Based on structural analysis, a large preliminary set of array segments that can be used to generate a humanized variant of girentuximab was identified. These segments were selected and analyzed using iTope™ technology (Perry et al, 2008) for in silico analysis of peptide binding to human MHC class II alleles and using TCED™ of known antibody sequence-related T cell epitopes (Bryson et al, 2010). Array segments identified as significant non-human germline binding factors to human MHC class II or scoring significant hits to TCED™ were discarded. This reduced the set of segments, and these combinations were re-analyzed as above to confirm that the junctions between segments did not contain potential T cell epitopes. The selected array segments were assembled into complete V region sequences lacking significant T cell epitopes. Then, five heavy chain (VH0 (chimeric), VH1, VH3, VH4, and VH5) and six light chain (Vκ0 (chimeric), Vκ1-Vκ5) sequences were selected for gene synthesis and expression in mammalian cells (see Table 1).

[0354] Construction of chimeric antibodies and humanized variants Chimeric VH0 and Vκ0 girentuximab sequences and their humanized variants were synthesized with restriction enzyme sites flanking both sides for cloning into the Abzena pANT expression vector system for unmodified human IgG1 heavy chain (allotype G1m 1,17, equivalent to the girentuximab allotype) (pANT68) and kappa light chain (pANT13.2). The VH region was cloned between the Mlu I and Hind III restriction sites, and the Vκ region was cloned between the BssH II and BamH I restriction sites. All constructs were confirmed by sequencing.

[0355] The FcRn - eliminating mutations, H310A and H435Q, were incorporated into the expression vectors of either human IgG1 heavy chain (allotype equivalent to girentuximab) (pANT69) or human IgG4 (S228P, L235E) heavy chain (pANT70) by site - directed mutagenesis. VH0 was cloned into these two vectors in addition to pANT36.2 encoding IgG4 (S228P, L235E).

[0356] Antibody expression in HEK293 EBNA cells Chimeric girentuximab (VH0 / Vκ0), two control antibodies (VH0 / Vκ1, VH1 / Vκ0), and combinations of VH and Vκ chains (a total of 20 pairings) were transiently expressed as IgG1 in HEK EBNA cells (LGC Standards, Teddington, UK) using the PEI transfection method. The cells were incubated for 7 days after transfection. Additionally, HEK EBNA cells were transfected with chimeric girentuximab VH0 and Vκ0 in the IgG1 (H310A, H435Q) heavy - chain vector, IgG4 (S228P, L235E) heavy - chain vector, and IgG4 (S228P, L235E H310A, H435Q) heavy - chain vector. The supernatant antibody titers were determined by ELISA.

[0357] Kinetic analysis of chimeric and humanized variant binding to carbonic anhydrase IX To evaluate the binding of all Gilenuximab Composite Human Antibody (trademark) variants, single-cycle kinetic analysis was performed on supernatants from transiently transfected HEK EBNA cell cultures. Kinetic experiments were performed on a Biacore T200 (serial number 1909913) running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (GE Healthcare, Uppsala, Sweden). All single-cycle kinetic experiments were run at 25 °C using HBS-P+ running buffer (pH 7.4) (GE Healthcare, Little Chalfont, UK). For direct comparison, all antibodies were captured on a Protein G chip (GE Healthcare, Uppsala, Sweden).

[0358] The antibody was diluted in running buffer to a final concentration of 0.5 μg / ml based on the concentration evaluated by ELISA titer. At the start of each cycle, the antibody was loaded onto Fc2, Fc3, and Fc4 of a Protein G chip (GE Healthcare, Little Chalfont, UK). IgG was captured at a flow rate of 10 μl / min to obtain an immobilization level (RL) of approximately 79 RU, which is the theoretical value, and an Rmax of approximately 50 RU. Subsequently, the surface was stabilized. Single-cycle kinetic data were obtained using carbonic anhydrase IX (CAIX, Stratech, Newmarket, UK) as the analyte at a flow rate of 30 μl / min, minimizing potential mass transport limitations. Multiple repetitions were performed using a reference chimeric antibody (VH0 / Vκ0 IgG1) to confirm the stability of the surface and the analyte over the kinetic cycle. The signal from the reference channel Fc1 (without antibody) was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to the reference surface. A four-point two-fold dilution range of 3.125 nM - 25 nM CAIX was used without regeneration between each concentration. The association phase of four injections of increasing concentrations of CAIX was monitored for 200 seconds each time, and a single dissociation phase was measured for 300 seconds after the last injection of CAIX. Regeneration of the Protein G surface was performed using an injection of 10 mM glycine-HCL (pH 1.5), followed by an injection of 10 mM glycine-HCL (pH 1.5) containing 0.5% P20.

[0359] The variants were analyzed using a reference chimera (VH0 / Vκ0 IgG1) used in the calculation of relative KD. The difference in surface stability was corrected by subtracting the signal from each antibody blank run. Single-cycle kinetic data showed that the humanized variants exhibited lower affinity for CAIX. Therefore, with the exception of those containing VH5 and / or V K 5, it was demonstrated that all of these variants bound to CAIX within two-fold of the reference chimeric antibody.

[0360] Purification of Chimeric and Lead Humanized Antibodies Six IgG1 lead antibodies VH3 / Vκ2, VH3 / Vκ3, VH3 / Vκ4, VH4 / Vκ2, VH4 / Vκ3 and VH4 / Vκ4 were purified based on human and single-cycle kinetic data. Chimeric IgG1 antibodies, lead humanized IgG1 antibodies, and chimeric IgG4(S228P, L235E) antibodies were purified from cell culture supernatants using a Protein A Sepharose column (GE Healthcare, Little Chalfont, UK). Chimeric IgG1(H310A, H435Q) and IgG4(S228P, L325E, H310A, H435Q) antibodies were purified from cell culture supernatants using a HiTrap™ Protein G HP column (GE Healthcare, Little Chalfont, UK). This is because the double mutation of H310A H435Q has been shown to adversely affect the binding to some Protein A resins. All antibodies were buffer-exchanged into 1×PBS (pH 7.2) and quantified by OD280nm using the extinction coefficient (Ec(0.1%)) based on the predicted amino acid sequence. Antibodies were analyzed by reducing the SDS-PAGE of 2 μg of the loaded antibody, and bands corresponding to the typical antibody profile were observed.

[0361] Multi-cycle kinetic analysis of chimeric and lead antibodies To establish the exact affinity for carbonic anhydrase IX, multi-cycle kinetic analysis was performed on the purified chimeric antibody and six lead IgG1 antibodies using a Biacore T200 (serial number 1909913) instrument running Biacore T200 Evaluation Software V3.0.1 (Uppsala, Sweden). The purified antibodies were diluted to a concentration of 1 μg / ml in HBS-P+. At the start of each cycle, each antibody was captured on the protein G surface, and approximately 79 RU of RL was obtained. After capture, the surface was stabilized. Kinetic data were obtained using a flow rate of 30 μl / min to minimize potential mass transfer effects. Carbonic anhydrase IX (CAIX) was used for kinetic analysis. To check the stability of both the surface and the analyte over the kinetic cycle, multiple repetitions of the blank (CAIX) and repetitions of a single concentration of analyte were programmed into the kinetic run. For kinetic analysis, a two-fold dilution range was selected from 50 nM to 0.078 nM of CAIX. The association phase of CAIX was monitored for 280 seconds, and the dissociation phase was monitored for 300 seconds. Regeneration of the protein G surface was performed using an injection of 10 mM glycine-HCL (pH 1.5), followed by an injection of 10 mM glycine-HCL (pH 1.5) containing 0.5% P20. The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to the reference surface, and the global Rmax parameter was used with a 1:1 binding model. The relative KD was calculated by dividing the KD of the humanized variant by the KD of the chimeric antibody on the same chip. All lead humanized IgG1 variants and chimeric antibodies on different IgG backbones showed relative KD within two-fold of the chimeric IgG1 antibody.

[0362] Carbonic anhydrase IX competitive ELISA Lead-purified IgG1 variants and chimeric antibodies expressed with different IgG constant domains (IgG1, IgG1(H310A, H435Q), IgG4(S228P, L235E) and IgG4(S228P, L235E, H310A, H435Q)) were tested for binding to CAIX using competition with biotinylated (biotinylation kit from Innova Biosciences, Cambridge, UK) chimeric Girentuximab IgG1. An irrelevant human IgG1 antibody was tested as a negative control for binding to CAIX.

[0363] CAIX was diluted to 0.5 μg / ml in 1×PBS and 100 μl / well was coated overnight at 4 °C on a 96-well ELISA plate. The next day, the plate was washed three times with 1×PBS / 0.05% Tween (PBST) and blocked with 200 μl of 2% BSA / PBS for 1 h at room temperature. In a 96-well dilution plate, an equal volume of a fixed concentration of biotinylated Girentuximab chimeric IgG1 antibody (final concentration 0.3 μg / ml) was added to a three-fold titration series of test antibodies (starting from 20 μg / ml diluted in blocking buffer (final concentration 10 μg / ml)).

[0364] After washing the plate three times with PBS-T, 100 μl of the chimeric / test antibody mixture was added to the ELISA plate.

[0365] After incubation for 1 h at room temperature, the plate was washed three times with PBS-T and 100 μl of streptavidin-peroxidase conjugate secondary antibody (Sigma, Dorset, UK) diluted 1:1000 in PBS-T was applied for 1 h at room temperature to detect the bound biotinylated Girentuximab chimeric IgG1 antibody. For color development, the plate was washed three times with PBS-T, followed by addition of 100 μl of TMB substrate and incubation for approximately 5 min at room temperature. The reaction was stopped with 50 μl of 3.0 M hydrochloric acid and the absorbance was immediately read using a Dynex plate reader at 450 nm.

[0366] IC 50Values were calculated for each variant, and the relative IC50 values were calculated by dividing the IC50 of the humanized variant by the IC50 of the gemtuzumab chimeric IgG1 antibody assayed on the same plate. All lead humanized variants and chimeric antibodies on different scaffolds showed IC50 values within 2-fold of the gemtuzumab chimeric IgG1 antibody.

[0367] Analysis of all data generated (including iTope™ analysis, percentage human, multi-cycle kinetic data, and competitive ELISA data) led to the selection of three antibodies VH3 / V K 4, VH4 / V K 3 and VH4 / V K 4 (sequence a provided in Table 1) as leads for further expression and analysis as IgG1, IgG1(H310A, H435Q), and IgG4(S228P, L235E, H310A, H435Q).

[0368] Expression of lead humanized gemtuzumab antibodies in CHO cells The heavy chain variable domains were cloned into the Fc null vectors pANT69 (IgG1(H310A, H435Q)) and pANT70 (IgG4(S228P, L235E, H310A, H435Q)) as outlined above.

[0369] FreeStyle™ CHO-S cells (ThermoFisher, Loughborough, UK) were transiently transfected with the corresponding plasmids and then, using the MaxCyte STX® electroporation system (MaxCyte Inc., Gaithersburg, USA.), chimeric (VH0 / Vκ0), VH3 / V K 4, VH4 / V K 3 and VH4 / V K4 was expressed. After cell harvest, the cells were diluted to 3×106 cells / ml in CD Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM L-glutamine (ThermoFisher, Loughborough, UK) and 1× hypoxanthine-thymidine (ThermoFisher, Loughborough, UK). Twenty-four hours after transfection, the culture temperature was lowered to 32 °C and 1 mM sodium butyrate (Sigma, Dorset, UK) was added. The cultures were fed on day 1 with 30% CD Efficient Feed B (ThermoFisher, Loughborough, UK) and 3.3% FunctionMAX™ TiterEnhancer (ThermoFisher, Loughborough, UK), and again on day 7 with 15% CD Efficient Feed B (ThermoFisher, Loughborough, UK) and 1.65% FunctionMAX™ TiterEnhancer (ThermoFisher, Loughborough, UK) (percentages based on the starting culture volume). IgG supernatant titers were monitored by IgG ELISA and the transfected cells were cultured for up to 14 days before the supernatant was harvested.

[0370] Purification of chimeric and lead humanized antibodies expressed as different IgGs Chimeric (VH0 / Vκ0), VH3 / V K 4, VH4 / V K 3 and VH4 / V K 4 IgG1 antibodies and VH4 / Vκ4 IgG4 (S228P, L235E) antibodies were purified from cell culture supernatants using a protein A sepharose column. Chimeric (VH0 / Vκ0), VH3 / V K 4, VH4 / V K 3 and VH4 / V K4 was purified using a Protein G column. The purified antibody was processed as outlined above. The antibody was analyzed by reducing SDS-PAGE of 2 μg of the loaded antibody, and bands corresponding to a typical IgG profile were observed.

[0371] Multi-cycle kinetic analysis of chimeric antibodies and lead humanized antibodies expressed as different IgGs To establish the exact affinity for carbonic anhydrase IX, a Biacore T200 (serial number 1909913) instrument running Biacore T200 Evaluation Software V 3.0.1 (Uppsala, Sweden) was used to perform multi-cycle kinetic analysis on chimeric (VH0 / Vκ0), VH3 / V K 4, VH4 / V K 3 and VH4 / V K 4 antibodies. As outlined in Section 2.7, multi-cycle kinetics were performed. As a reference for Fc2 of each run, VH0 / V K 0 IgG1 was run. The relative KD was calculated by dividing the KD of the humanized variant by the KD of the chimeric antibody having the same IgG constant domain. In all three IgG backbones, the lead variants all bound within 2-fold of VH0 / V K 0 having the same IgG backbone.

[0372] Conclusions and Discussion The V-region genes from the Girentuximab antibody were cloned into IgG1 (the same allotype as Girentuximab) heavy-chain and kappa light-chain vectors together with four humanized VH regions and five humanized Vκ regions (designed using Composite Human Antibody™ technology). The chimeric (VH0 / V K 0) was further cloned into VH vector IgG1 (H310A, H435Q) and IgG4 (S228P, L235E, H310A, H435Q). Here, the mutations H310A and H435Q abolish FcRn binding.

[0373] Antibodies transiently transfected into HEK293 EBNA cells were tested for binding to CAIX by single - cycle Biacore analysis. Six IgG1 lead antibodies (VH3 / Vκ2, VH3 / Vκ3, VH3 / Vκ4, VH4 / Vκ2, VH4 / Vκ3, and VH4 / Vκ4) were identified based on expression levels and single - cycle kinetic data. Purified leads tested by Biacore multi - cycle analysis and competitive ELISA were shown to bind within 2 - fold of an IgG1 chimeric antibody.

[0374] Based on iTope™ analysis, human percentage, multi - cycle kinetic data, and competitive ELISA data, the six leads were narrowed down to three; VH3 / V K 4 (SEQ ID NO: 36 / SEQ ID NO: 132), VH4 / V K 3 (SEQ ID NO: 52 / SEQ ID NO: 132) and VH4 / V K 4 (SEQ ID NO: 52 / SEQ ID NO: 148). These three lead variants were recloned and the antibodies were transiently expressed in CHO - S cells on three different backbones (IgG1, IgG1(H310A, H435Q), and IgG4(S228P, L235E, H310A, H435Q)). All antibodies were purified and then tested by Biacore multi - cycle kinetic analysis. In all three IgG backbones, the lead humanized variants all showed binding within 2 - fold of VH0 / V K 0 having the same IgG backbone.

[0375] Example 2 - Combination of Targeted Radionuclide Therapy and Immune Checkpoint Inhibition Immune checkpoint inhibition (ICI) has substantially changed cancer treatment, but (long - term) responses have not yet been seen in the majority of patients. Key determinants for successful ICI treatment are high tumor mutation burden and existing tumor - infiltrating lymphocytes (TIL). Ionizing radiation can increase TIL and induce inflammatory signaling that can cause mutations recognized by T cells. A mouse model was used to evaluate the therapeutic effects of targeted radionuclide therapy (TRT) and combination therapy of TRT + ICI.

[0376] Preparation of Radioactive Labeled Girentuximab Antibody Conjugate

[0377]

Table 2

[0378] Conjugate Preparation Protocol 1: 334 μL of 1.25 M NaHCO3 (pH 8.2) was added dropwise per 20 mg of humanized girentuximab (GmAb) antibody. Next, 1.25 mg of NHS-DOTA per 20 mg of GmAb was added while gently stirring the mixture until the NHS-DOTA was completely dissolved. The reaction mixture was incubated at 37 °C for 1 hour and then cooled to room temperature. To ensure removal of free NHS-DOTA in the solution, the GmAb-DOTA reaction solution was exchanged to 0.25 M ammonium acetate pH 6.0 up to a maximum of 15 volumes using a 50 kDa MWCO membrane or sufficient PD10. The GmAb-DOTA conjugate concentration was determined by UV-vis and corrected to 5 mg / mL using 0.25 M ammonium acetate at pH 6.0.

[0379] Conjugate Preparation Protocol 2: Alternatively, the conjugate may be prepared according to the following "Protocol 2" steps. 1. Using a 50 kDa MWCO membrane, buffer exchange the GmAb into reaction buffer: 100 mM NaPO4 + 20 mM EDTA (pH 7.5). There is an approximately 10-fold volume change and an approximate concentration of 5 mg / mL. Confirm that the pH has changed to pH 7.5. 2. Prepare a 5 mg / ml NHS-DOTA stock solution by dissolving NHS-DOTA in cold phosphate buffered saline at pH 7.5 within 30 minutes before use (i.e., use immediately). Add 12.1 molar equivalents of the NHS-DOTA stock solution to the buffer-exchanged GmAb solution. [Alternatively, 1.25 mg of solid NHS-DOTA per 20 mg of GmAb is mixed while gently stirring until the solid NHS-DOTA is completely dissolved.] 3. Incubate the bioconjugation reaction at 22 °C (21 - 24 °C) for 2 hours to complete the DOTA-conjugation reaction. 4. Use a 50 kDa MWCO membrane. To ensure the removal of free NHS-DOTA in the solution, use a diafiltration volume more than 15-fold to buffer-exchange the GmAb-DOTA reaction solution into 0.25 M ammonium acetate (pH 6.0). 5. Check the final pH and protein concentration of GmAb-DOTA and concentrate it to 5 mg / ml using 0.25 M ammonium acetate (pH 6.0).

[0380] According to either Protocol 1 or 2, the generated GmAb-DOTA is exposed to a source of 177-Lu for a time sufficient for the radioisotope to chelate with the DOTA moiety. The source of 177-Lu may be any suitable source as known in the art.

[0381] Method: CT26 (mouse colorectal cancer cell line) and Renca (mouse renal epithelial) cells transfected with human CAIX were subcutaneously injected into the flanks of Balb / C mice. When the tumor volume reached 50 - 100 mm 3 the mice were administered with lutetium-177 labeled CAIX-specific antibody 177 Lu-hG250 (intravenously), and subsequently an ex vivo biodistribution study was performed 24 hours post-injection (p.i.). The antibody was prepared as described in Examples 1 and 2.

[0382] The anti-PD-L1 and anti-CTLA4 antibodies used in this study were InVivoMab anti-mouse CTLA-4 (CD152) (BioXCell, BE0032) and InVivoMab anti-mouse PD-1 (CD279) (BioXCell, BE0146).

[0383] Furthermore, in Renca-CAIX tumor-bearing mice, (1) 12, 18, or 24 MBq 177Lu-hG250 monotherapy, (2) ICI consisting of anti-PD-1 Ab (200 μg) + anti-CTLA-4 Ab (200 μg), (3) 18 MBq 177 Tumor growth inhibition and survival rate were determined at the time of injection of (4) vehicle, combination of Lu-hG250 and ICI, and (4) vehicle. The body weight of the mice was monitored (Figure 2). The mice were euthanized according to ethical principles (Figure 3). The tumor growth curve was quantified using the area under the curve (AUC) corrected for the lifespan of the animals (Figures 1, 5, and 6). Mice with complete regression were rechallenged with tumor cells.

[0384] Results, 177 Tumor uptake of Lu-hG250 was 17 ± 2% and 32 ± 9% ID / g in CT26-CAIX and Renca-CAIX, respectively. Immunohistochemistry (IHC) confirmed CAIX expression in both tumors. Furthermore, Renca-CAIX tumors showed high expression of PD-L1 and very few CD8 + TILs. CT26-CAIX also had significant but heterogeneous PD-L1 expression, and a large number of CD8 + TILs infiltrated the tumor.

[0385] Compared with untreated animals (AUC = 449 ± 204 mm 3 / day), 18 and 24 MBq 177 Lu-hG250 monotherapy and TRT + ICI combination therapy significantly inhibited Renca-CAIX tumor growth (AUC = 202 ± 201 mm 3 / day, p < 0.001, AUC = 95 ± 78 mm 3 / day, p < 0.0001, and AUC = 66 ± 64 mm 3 / day, p < 0.0001). Furthermore, these treatments significantly prolonged survival compared to untreated animals (p < 0.01, p < 0.001, and p < 0.005, respectively) (Figure 4). Complete tumor regression was observed in 50%, 40%, and 57% of the mice, respectively. When these animals were rechallenged with tumor cells, Renca-CAIX was 100% rejected in all groups. In the TRT+ICI combination therapy group, the parental Renca tumors were also 100% rejected, while in the 18 and 24 MBq TRT monotherapy groups, 40% and 75% were rejected, respectively.

[0386] Conclusion: 177 TRT with Lu-hG250 and its combination with ICI were therapeutically effective. The combination of TRT and ICI enabled equivalent antitumor efficacy with lower doses of TRT compared to TRT alone. The tumor rechallenge experiments suggest the induction of tumor-specific T cell responses after TRT.

[0387] Example 3 - Combination of Targeted Radionuclide Therapy and Immune Checkpoint Inhibition A study similar to that described in Example 2 was conducted using lower doses of targeted radiotherapy (TRT), either alone or in combination with ICI therapy. Briefly, Renca cells transfected with human CAIX were subcutaneously injected into the flanks of Balb / C mice. When the tumor volume reached 50 - 100 mm 3 the mice were administered lutetium-177-labeled CAIX-specific antibody 177 Lu-hG250 (intravenously), followed by administration of anti-PD-1 and anti-CTLA4 antibodies.

[0388] The experimental design is summarized in Figure 7. Briefly, the mice were divided into the following treatment groups: 1. Control (vehicle + vehicle) 2. ICI monotherapy (vehicle + PD-1Ab + anti-CTLA-4Ab), 3. Low-dose TRT monotherapy (4 MBq 177 Lu-hG250 + vehicle), 4. TRT+ICI low-dose therapy (4 MBq 177 Lu-hG250 + PD-1Ab + anti-CTLA-4Ab) 5. Single - dose therapy in TRT (12 MBq 177 Lu - hG250 + vehicle), 6. Medium - dose therapy in TRT + ICI (12 MBq 177 Lu - hG250 + PD - 1Ab + anti - CTLA - 4Ab)

[0389] The inhibition of tumor growth and survival was determined, and the results are shown in Figures 8 and 9. Briefly, the results are consistent with the observations made in Example 2, showing that the combination of TRT and ICI, which includes lower - dose radiation compared to Example 2, shows a significant decrease in tumor growth compared to the control or single - therapy (either TRT or ICI alone).

[0390]

Table 3

[0391] Mice with complete regression were rechallenged with tumor cells having either the "parental" Renca cell line on one side and the Renca - CAIX that the mice received in the primary experiment on the other side.

[0392] The following table shows that a high percentage of animals treated with the combined TRT + ICI therapy had complete tumor regression, and almost all of these animals rejected tumor rechallenge. These results indicate that the animals induced a tumor - specific memory T - cell response.

[0393]

Table 4

[0394] Example 4 - Combination of Targeted Radionuclide Therapy and Immune Checkpoint Inhibition (CT26 Model) A study similar to that described in Example 3 was conducted using the CT26 - huCAIX model. CT26 cells transfected with human CAIX were subcutaneously injected into the flanks of Balb / C mice. When the tumor volume reached 50 - 100 mm 3 the mice were given a lutetium - 177 - labeled CAIX - specific antibody 177Lu-hG250 (intravenously), followed by administration of an anti-PD-1 antibody.

[0395] The experimental design is summarized in Figure 10. Briefly, mice were divided into the following treatment groups: 1. Control (vehicle + vehicle) 2. ICI monotherapy (vehicle + anti-PD-1 antibody) 3. Low-dose TRT monotherapy (4 MBq 177 Lu-hG250 + vehicle) 4. TRT + low-dose ICI therapy (4 MBq 177 Lu-hG250 + anti-PD-1 Ab)

[0396] Tumor growth and survival inhibition were determined, and the results are shown in Figures 11 and 12. Briefly, the results are consistent with the observations made in Example 2, indicating that the combination of TRT and ICI containing a lower dose of radiation, compared to the control or monotherapy (either TRT or ICI alone), shows a significant decrease in tumor growth compared to the control or monotherapy (either TRT or ICI alone).

[0397] Example 5 - Evaluation of Changes in Gene Expression after 177-Lu-hG250 Treatment To better understand the mechanism by which 177-Lu-hG250 treatment modulates the tumor microenvironment and increases the efficacy of ICI treatment, the inventors performed gene expression profiling of Renca-hCAIX tumor-bearing mice using the IO360 PanCancer panel from the Nanostring platform.

[0398] The TRT group was treated with 177-Lu-hG250 at 12 MBq of radiation. Tumors were harvested after 7 days for gene expression analysis.

[0399] The results are shown in Example 13, indicating that 177-Lu-hG250 treatment promotes anti-tumor immunity, including genes related to interferon (IFN) signaling, antigen presentation, cytotoxicity, and DNA damage repair, and induces the expression of several genes involved in processes related to the response to checkpoint inhibition.

[0400] The graph is based on a list of genes passing a filter of at least 1.5-fold differential expression and p-value < 0.05. N = 3 mice / group. The expression values represent mean-subtracted normalized log2 values.

[0401] Example 6 - Evaluation of Cell Profiling and Changes in Gene Expression after 177-Lu-hG250 Treatment To better understand the mechanism by which 177-Lu-hG250 treatment, which increases the efficacy of ICI treatment, modulates the tumor microenvironment, further investigations are conducted. In this series of experiments, compared with Example 4, a lower dose of 177-Lu-hG250 treatment was administered (e.g., 4 MBq 177-Lu-hG250 compared to 12 MBq 177-Lu-hG250 in Example 4).

[0402] The experimental protocol is outlined in Figure 14. Briefly, Renca-hCAIX-expressing mice are treated according to the following protocol: Part 1: Animal groups · Control (no treatment) · 4 MBq 177Lu-hG250 · Anti-PD-1 + anti-CTLA-4 antibodies · 4 MBq 177Lu-hG250 + anti-PD-1 + anti-CTLA-4 antibodies

[0403] Tumors are harvested on either day 0, ~day 5, or ~day 8 after the start of treatment. Flow cytometry is performed to profile immune cells in the tumor microenvironment, including markers for effector T cells, T cell activation / exhaustion, regulatory T cells, myeloid cells, and tumor antigen-specific T cells.

[0404] The results show that low-dose targeted radiation / low-dose targeted radiation in combination with checkpoint inhibition changes the type / ratio of immune cells in a manner that promotes anti-tumor immunity. For example, an increase in the activated state of effector T cells and a decrease in suppressive immune cell types are observed.

[0405] Part 2: Animal groups: · Control (no treatment) ·4MBq 177Lu-hG250 ·Anti-PD-1 + Anti-CTLA-4 antibody ·4MBq 177Lu-hG250 + Anti-PD-1 + Anti-CTLA-4 antibody

[0406] Tumors are harvested at any one of D0, ~D5, ~D8 after the start of treatment, and gene expression profiling is performed using Nanostring to evaluate changes in the expression of over 700 immunological genes. Furthermore, immunohistochemistry of T cell and bone marrow markers is performed to evaluate the effect of the combination of 177Lu-hG250 and Anti-PD-1 + Anti-CTLA-4 antibody on the tumor microenvironment.

[0407] The results indicate that when low TOSE target radiation / low-dose target radiation is combined with checkpoint inhibition, similar to the observations in Example 4, the gene expression profile of cells in the tumor microenvironment that promotes anti-tumor immunity changes. For example, the results will show an increase in the expression of genes involved in IFN signaling, T cell chemoattraction, cytotoxicity, and antigen presentation.

[0408] Embodiment 1. A method for treating, preventing, or minimizing the progression of cancer in a subject, comprising: administering to the subject a therapeutically effective amount of an antigen-binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX), wherein the antigen-binding protein is conjugated to a radionuclide in combination with an immune checkpoint inhibitor therapy, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0409] 2. The antigen-binding protein specifically binds to carbonic anhydrase IX (CAIX) and comprises an antigen-binding domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-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, The method according to Embodiment 1, wherein any one of the sequences of the complementarity-determining regions has the amino acid sequence set forth in Table 1.

[0410] 3. The antigen-binding domain is (i) a VH comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 1, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 3, and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 81, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 83, or (iii) A complementarity-determining region (CDR) 1 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO: 1, a CDR2 that contains, or consists of, a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 2, and a CDR3 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO: 3, and a VH, and (iv) A complementarity-determining region (CDR) 1 that comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 81, a CDR 2 that comprises or consists of a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 82, and a CDR 3 that comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 83; and a VH comprising the same; the method according to Embodiment 2.

[0411] 4. The antigen-binding domain is (i) A variable heavy chain (VH) comprising a framework region (FR) 1 that comprises or consists of a sequence set forth in any of SEQ ID NOs: 9, 25, 41, 57, or 73, an FR 2 that comprises or consists of a sequence set forth in any of SEQ ID NOs: 10, 26, 42, 58, or 74, an FR 3 that comprises or consists of a sequence set forth in any of SEQ ID NOs: 11, 27, 43, 59, or 75, and an FR 4 that comprises or consists of a sequence set forth in any of SEQ ID NOs: 12, 28, 44, 60, or 76; and (ii) VL comprising a framework region (FR) 1 comprising or consisting of the sequence set forth in any of SEQ ID NOs: 89, 105, 121, 137, 153, or 169, an FR2 comprising or consisting of the sequence set forth in any of SEQ ID NOs: 90, 106, 122, 138, 154, or 170, an FR3 comprising or consisting of the sequence set forth in any of SEQ ID NOs: 91, 107, 123, 139, 155, or 171, and an FR4 comprising or consisting of the sequence set forth in any of SEQ ID NOs: 92, 108, 124, 140, 156, or 172. or (iii) A framework region (FR) 1 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any of SEQ ID NOs: 9, 25, 41, 57, or 73; an FR2 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any of SEQ ID NOs: 10, 26, 42, 58, or 74; an FR3 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any of SEQ ID NOs: 11, 27, 43, 59, or 75; an FR4 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in any of SEQ ID NOs: 12, 28, 44, 60, or 76; and a VH that includes...(iv) a framework region (FR) 1 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in any of SEQ ID NOs: 89, 105, 121, 137, 153, or 169; an FR2 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in any of SEQ ID NOs: 90, 106, 122, 138, 154, or 170; an FR3 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in any of SEQ ID NOs: 91, 107, 123, 139, 155, or 171; and an FR4 that contains, or consists of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,The method according to embodiment 2 or 3, comprising VL, comprising FR4, comprising at least 99% identical sequences or consisting thereof.

[0412] 5. The antigen-binding protein is (a) a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 4, 20, 36, 52, or 68, and / or (b) a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164 or (c) a heavy chain variable domain (VH) comprising 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 sequences to the sequence set forth in SEQ ID NO: 4, 20, 36, 52, or 68, and / or (d) a light chain variable domain (VL) comprising 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 sequences to the sequence set forth in SEQ ID NO: 84, 100, 116, 132, 148, or 164, the method according to any one of embodiments 1 to 4.

[0413] 6. The method according to any one of embodiments 1 to 5, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

[0414] 7. The method according to embodiment 1, wherein the antigen-binding protein is the antibody Girentuximab.

[0415] 8. An antigen-binding protein is a modified antibody of class IgG that includes a heavy-chain constant region having one or more amino acid substitutions compared to a wild-type antibody of class IgG, and the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn), thereby reducing the serum half-life of the modified monoclonal antibody compared to the wild-type antibody of class IgG. The method according to any one of Embodiments 1 to 6.

[0416] 9. The method according to Embodiment 8, wherein the one or more amino acid substitutions are selected from substitutions in the heavy-chain constant region at one or more of positions His310, His433, His435, His436, and Ile253.

[0417] 10. The method according to Embodiment 9, wherein the one or more amino acid substitutions include a substitution in the heavy-chain constant region at position His310 or His435, and preferably, the amino acid substitution is at both His310 and His435.

[0418] 11. The method according to any one of Embodiments 8 to 10, wherein the modified antibody further includes (a) one or more amino acid substitutions that reduce the affinity of the antibody for one or more Fc gamma receptors compared to the wild-type antibody of class IgG, and / or (b) one or more amino acid substitutions that increase the stability of the CH1-CH2 hinge region in the modified antibody compared to the wild-type antibody of class IgG.

[0419] 12. The method according to Embodiment 11, wherein the one or more amino acid substitutions that reduce the affinity of the antibody for the Fc gamma receptor include an amino acid substitution at position Leu235.

[0420] 13. The method according to Embodiment 11 or 12, wherein the one or more amino acid substitutions that increase the stability of the CH1-CH2 hinge region in the antibody include a substitution of Ser228.

[0421] 14. The method according to any one of embodiments 8 to 13, wherein the modified antibody is conjugated to a radionuclide via a linker or chelator moiety.

[0422] 15. The radionuclide is actinium-225 ( 225 Ac), astatine-211 ( 211 At), bismuth-212 and bismuth-213 ( 212 Bi, 213 Bi), copper-64 and copper-67 ( 64 Cu, 67 Cu), iodine-123, -124, -125 or -131 ( 123 I, 124 I, 125 I, 131 I)( 123 I), lead-212 ( 212 Pb), lutetium-177 ( 177 Lu), radium-223 and radium-224 ( 223 Ra, 224 Ra), samarium-153 ( 153 Sm), scandium-47 ( 47 Sc), strontium-90 ( 90 Sr), and yttrium-90 ( 90 Y), the method according to any one of embodiments 1 to 14.

[0423] 16. The antigen-binding protein is a modified antibody having a reduced FcRn binding affinity compared to the unmodified form of the antibody or a wild-type antibody of class IgG, -One or more amino acid residues at positions His310, His433, His435, His436, and Ile253 are different from the residues present in the unmodified antibody or a wild-type antibody of class IgG, and the method according to any one of embodiments 1 to 15, comprising a heavy chain constant region.

[0424] 17. The method according to any one of embodiments 1 to 16, wherein the antigen-binding protein comprises a heavy chain comprising the sequence set forth in any one of SEQ ID NOs: 182 to 185, and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 181 or SEQ ID NO: 186.

[0425] 18. The method according to embodiment 16, wherein the antibody comprises the amino acid sequences set forth in SEQ ID NOs: 183 and 186.

[0426] 19. The antigen-binding protein is a molecule comprising an immunoglobulin moiety and a non-protein agent conjugated thereto, the immunoglobulin moiety specifically binds to CAIX, comprises an antigen-binding protein consisting essentially of or consisting of the amino acid sequence of any one of SEQ ID NOs: 4, 20, 36, 52, or 68 and any one of SEQ ID NOs: 84, 100, 116, 132, 148, 164 (in order from N-terminus to C-terminus or C-terminus to N-terminus), and the immunoglobulin moiety has a reduced or abolished affinity for the FcRn receptor as compared to a wild-type immunoglobulin, the non-protein agent comprises a radioactive element, the method according to embodiment 1.

[0427] 20. The radioactive nuclide is actinium-225 ( 225 Ac), astatine-211 ( 211 At), and iodine-123 ( 123 I), the method according to any one of the preceding embodiments.

[0428] 21. The antigen-binding protein is - a heavy chain and a light chain, each chain comprising a variable region and a constant region, - the constant region of the heavy chain comprises one or more amino acid substitutions as compared to a wild-type antibody of class IgG, and the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn), thereby reducing the serum half-life of the modified monoclonal antibody as compared to a wild-type antibody of class IgG, the heavy chain and the light chain, The method according to any one of the preceding embodiments, which is a therapeutic IgG molecule comprising a radioisotope conjugated to one or more amino acid residues of a heavy chain or a light chain.

[0429] 22. The method according to any one of the preceding embodiments, wherein the antigen-binding protein is an antibody, preferably a naked antibody.

[0430] 23. The radionuclide is 177 Lu, and the method according to any one of the preceding embodiments.

[0431] 24. The radioactivity of the antigen-binding protein for binding to CAIX conjugated with a radionuclide is about 18 MBq or less, and the method according to any one of the preceding embodiments.

[0432] 25. The radionuclide is conjugated to an antibody for binding to CAIX via a linker or a chelating agent, and the method according to any one of the preceding embodiments.

[0433] 26. The chelating agent is selected from TMT (6,6’’-bis[N,N’’,N’’’-tetra(carboxymethyl)aminomethyl)-4’-(3-amino-4-methoxyphenyl)-2,2’:6’,2’’-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN’,N’’(N’’’-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A’’-DTPA, TETE, Te2A, HBED, DFO, DFOsq, and HOPO, and the method according to embodiment 25.

[0434] 27. The method according to any one of the preceding embodiments 1 to 68, further comprising conjugating the antigen-binding protein with a radionuclide before the step of administration.

[0435] 28. The antigen-binding protein is a modified antibody for binding to CAIX, and the method further comprises generating a modified antibody for binding to CAIX comprising a non-peptide moiety prior to the step of conjugating, the method according to embodiment 27.

[0436] 29. The method according to embodiment 28, wherein the non-peptide moiety is a linker or a chelating agent.

[0437] 30. The method according to embodiment 29, wherein the chelating agent is selected from the group consisting of TMT (6,6’’-bis[N,N’’,N’’’-tetra(carboxymethyl)aminomethyl)-4’-(3-amino-4-methoxyphenyl)-2,2’:6’,2’’-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN’,N’’(N’’’-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, DiamSar, DTPA, CHX-A’’-DTPA, TETE, Te2A, HBED, DFO, DFOsq, and HOPO.

[0438] 31. The method according to any one of the preceding embodiments, wherein the immune checkpoint inhibitor therapy comprises targeting a protein selected from PD-1, PD-L1, and CTLA-4, or combinations thereof.

[0439] 32. The method according to embodiment 31, wherein the immune checkpoint inhibitor therapy comprises targeting PD-1 and CTLA4.

[0440] 33. The method according to embodiment 32, wherein the immune checkpoint inhibitor therapy comprises administering to the subject an antibody for binding to PD-1 and an antibody for binding to CTLA4.

[0441] 34. The method according to any one of the preceding embodiments, the method further comprising administering to the subject one or more immune checkpoint inhibitors.

[0442] 35. The method according to embodiment 34, wherein the administration of the antigen-binding protein and the immune checkpoint inhibitor is separate, sequential, or continuous.

[0443] 36. A method of inducing a T cell response against cancer in a subject, comprising administering to the subject an effective amount of an antibody or an antigen-binding fragment thereof to CAIX in combination with an immune checkpoint inhibitor, thereby inducing a T cell response against cancer in the subject.

[0444] 37. A method of inducing an adaptive immune response against cancer in a subject, comprising administering to the subject an effective amount of an antibody or an antigen-binding fragment thereof to CAIX in combination with an immune checkpoint inhibitor, thereby inducing a T cell response against cancer in the subject.

[0445] 38. The method according to any one of the preceding embodiments, wherein the cancer is renal cancer.

[0446] 39. Use of an antigen-binding protein for binding to CAIX in the manufacture of a medicament for use in combination with an immune checkpoint inhibitor therapy for the treatment of cancer, preferably renal cancer.

[0447] 40. Use of an immune checkpoint inhibitor in the manufacture of a medicament for use in combination with an antigen-binding protein for binding to CAIX conjugated to a radionuclide for the treatment of cancer, preferably renal cancer.

[0448] 41. An antigen-binding protein that binds to CAIX for use in combination with an immune checkpoint inhibitor therapy for the treatment of cancer, preferably renal cancer.

[0449] 42. An immune checkpoint inhibitor for use in combination with an antigen-binding protein for binding to CAIX conjugated to a radionuclide for the treatment of cancer, preferably renal cancer.

[0450] A pharmaceutical composition comprising an antigen-binding protein for binding to CAIX, wherein the pharmaceutical composition is for use in the treatment of cancer, preferably renal cancer, in combination with an immune checkpoint inhibitor.

[0451] A pharmaceutical composition comprising an immune checkpoint inhibitor for use in the treatment of cancer, preferably renal cancer, in combination with an antigen-binding protein for binding to CAIX conjugated to a radionuclide.

[0452] 45. A kit for use according to, or when used according to, any method described herein, · Optionally, an antigen-binding protein for binding to CAIX conjugated to a radionuclide, and · Instructions for use in combination with an immune checkpoint inhibitor.

[0453] 46. The kit according to embodiment 87, further comprising one or more immune checkpoint inhibitors in a separate part.

[0454] 47. A kit for use according to, or when used according to, any method described herein, · One or more immune checkpoint inhibitors, and · Instructions for use in combination with an antigen-binding protein for binding to CAIX conjugated to a radionuclide.

[0455] 48. The kit according to embodiment 47, further comprising, in a separate part, an antigen-binding protein for binding to CAIX optionally conjugated to a radionuclide.

[0456] The kit according to any one of embodiments 45 to 48, wherein the antigen-binding protein and / or immune checkpoint inhibitor for binding to CAIX is provided in the form of a pharmaceutical composition optionally comprising one or more pharmaceutically acceptable excipients, carriers, and / or diluents.

Claims

1. A pharmaceutical composition for use in treating, preventing, or minimizing the progression of cancer in a subject, A pharmaceutical composition comprising a radiolabeled agent that binds to or specifically binds to carbonic anhydrase IX (CAIX), and an immune checkpoint inhibitor.

2. A pharmaceutical composition for use in inducing a T-cell response to cancer in a target, A pharmaceutical composition comprising a radiolabeled agent that, in combination with an immune checkpoint inhibitor, binds to or specifically binds to CAIX.

3. A pharmaceutical composition for use in inducing an adaptive immune response to cancer in a target, A pharmaceutical composition comprising a radiolabeled agent that, in combination with an immune checkpoint inhibitor, binds to or specifically binds to CAIX.

4. A pharmaceutical composition for use in treating, preventing, or minimizing the progression of cancer in subjects who have been or are being treated with immune checkpoint inhibitors, A pharmaceutical composition comprising a radiolabeled agent that binds to or specifically binds to carbonic anhydrase IX (CAIX).

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the drug is selected from small molecules, peptides, or proteins that can bind to or specifically bind to CAIX.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the radiolabeled drug is a radiolabeled peptide.

7. The pharmaceutical composition according to any one of claims 1 to 4, wherein the radiolabeled drug is an antibody or antigen-binding fragment thereof that is capable of binding to or specifically binding to CAIX, and the antibody or antigen-binding fragment thereof is conjugated to a radionuclide.

8. The pharmaceutical composition according to claim 7, wherein the radiolabeled antibody or its antigen-binding fragment is a radiolabeled gylenetuximab antibody or its antigen-binding fragment.

9. The radiolabeled antibody or antigen-binding protein includes an antigen-binding domain that binds to or is specific to carbonic anhydrase IX (CAIX), and the antigen-binding domain is Includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, During the ceremony, FR1, FR2, FR3, and FR4 are each framework domains. CDR1, CDR2, and CDR3 are complementarity-determining regions, FR1a, FR2a, FR3a, and FR4a are each framework domains. CDR1a, CDR2a, and CDR3a are complementarity-determining regions, Preferably, the complementarity-determining region has one of the amino acid sequences listed in Table 1, the pharmaceutical composition according to any one of claims 1 to 4.

10. The antigen-binding domain, (i) Complementarity Determination Region (CDR) 1 containing at least approximately 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% of the sequence is identical to the sequence set in Sequence ID No.

2. VH, comprising: CDR2 containing a sequence identical to %, 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%; and CDR3 containing a sequence identical to at least approximately 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% of the sequence described in Sequence ID No.

3. (ii) A VH containing 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% of the sequence described in any of sequence numbers 4, 20, 36, 52, or 68, (iii) CDR1 containing 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% of the sequence described in SEQ ID NO: 82, and at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, VL, comprising CDR2 containing 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% of the same sequence as described in Sequence ID No. 83, and CDR3 containing at least approximately 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% of the same sequence as described in Sequence ID No.

83. (iv) A VL containing 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% of the sequence described in SEQ ID NOs. (v) VH comprising CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 2, and CDR3 containing the sequence described in SEQ ID NO: 3 (vi) VH containing the sequence described in any of sequence numbers 4, 20, 36, 52, or 68, (vii) A VL comprising CDR1 containing the sequence described in SEQ ID NO: 81, CDR2 containing the sequence described in SEQ ID NO: 82, and CDR3 containing the sequence described in SEQ ID NO:

83. (viiii) VL containing the sequence described in any of sequence numbers 84, 100, 116, 132, 148, or 164, (ix) VH, which includes CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 2, and CDR3 containing the sequence described in SEQ ID NO: 3, and VL, which includes CDR1 containing the sequence set of SEQ ID NO: 81, CDR2 containing the sequence described in SEQ ID NO: 82, and CDR3 containing the sequence described in SEQ ID NO: 83, or (x) A pharmaceutical composition according to claim 9, comprising VH containing the sequence described in any of sequence numbers 4, 20, 36, 52, or 68, and VL containing the sequence described in any of sequence numbers 84, 100, 116, 132, 148, or 164, preferably VH and VL, wherein VH contains the sequence described in sequence number 4 and VL contains the sequence described in sequence number 100, or VH contains the sequence described in sequence number 52 and VL contains the sequence described in sequence number 148.

11. The antigen-binding domain, (i) VH, and a framework region (FR) 1 containing or consisting of the sequence described in any of sequence numbers 9, 25, 41, 57, or 73; FR2 containing or consisting of the sequence described in any of sequence numbers 10, 26, 42, 58, or 74; FR3 containing or consisting of the sequence described in any of sequence numbers 11, 27, 43, 59, or 75; and FR4 containing or consisting of the sequence described in any of sequence numbers 12, 28, 44, 60, or 76. (ii) A VL containing or comprising a framework region (FR) 1 containing or comprising the sequence described in any of sequence numbers 89, 105, 121, 137, 153, or 169; FR2 containing or comprising the sequence described in any of sequence numbers 90, 106, 122, 138, 154, or 170; FR3 containing or comprising the sequence described in any of sequence numbers 91, 107, 123, 139, 155, or 171; or FR4 containing or comprising the sequence described in any of sequence numbers 92, 108, 124, 140, 156, or 172; or (i) A framework containing or consisting of at least approximately 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% of the sequence described in any of sequence numbers 9, 25, 41, 57, or 73. Region (FR) 1, containing or comprising 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% of the same sequence as the sequence described in any of sequence numbers 10, 26, 42, 58, or 74.

2. FR3, sequence number 11, 27, 43, 59, or 75, containing or consisting of at least approximately 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% of the sequence described in any of sequence numbers 11, 27, 43, 59, or 75. VH, and FR4 containing 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%, and at least 99% of the sequence described in any of numbers 12, 28, 44, 60, or 76, or consisting of FR4, and (ii) Contains 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% of the sequence described in any of sequence numbers 89, 105, 121, 137, 153, or 169, or Framework region (FR) 1 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% of the sequence described in any of sequence numbers 90, 106, 122, 138, 154, or 170. 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 FR3, sequence numbers 92, 108, 124, 140, 156, or 1722, which contain or consist of 99% identical sequences, are at least approximately 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%, and at least 98% identical to any of the sequences described in FR3, sequence numbers 92, 108, 124, 140, 156, or 1722.The pharmaceutical composition according to claim 9, comprising VL, which includes FR4, which contains at least 99% identical sequences or consists thereof.

12. The aforementioned antigen-binding protein (a) Heavy chain variable domains (VHs) containing 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%, and / or (b) The pharmaceutical composition according to claim 9, comprising 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%, and at least 99% of the sequence described in any of SEQ ID NOs: 84, 100, 116, 132, 148, or 164, or comprising a light chain variable domain (VL) consisting thereof.

13. The aforementioned antigen-binding protein (a) A heavy chain variable domain (VH) comprising the amino acid sequence described in any of SEQ ID NOs: 4, 20, 36, 52, or 68, and / or (b) comprising a light chain variable domain (VL) containing the amino acid sequence described in any of SEQ ID NOs: 84, 100, 116, 132, 148, or 164, Preferably, the antigen-binding protein comprises VH and VL amino acid sequences selected from SEQ ID NO: 4 and SEQ ID NO: 84, or SEQ ID NO: 36 and SEQ ID NO: 132, or SEQ ID NO: 52 and SEQ ID NO: 132, or SEQ ID NO: 52 and SEQ ID NO: 148, and more preferably, the antigen-binding protein comprises VH and VL amino acid sequences described in SEQ ID NO: 52 / SEQ ID NO: 148, the pharmaceutical composition according to claim 9.

14. The pharmaceutical composition according to claim 9, wherein the antigen-binding protein is a modified class IgG antibody comprising a heavy chain constant region having one or more amino acid substitutions compared to a wild-type class IgG antibody, and the one or more amino acid substitutions reduce the affinity of the antibody to the neonatal Fc receptor (FcRn), thereby reducing the serum half-life of the modified monoclonal antibody compared to a wild-type class IgG antibody.

15. The antigen-binding protein is a modified antibody having a reduced FcRn binding affinity compared to the unmodified form of the antibody or the wild-type antibody of class IgG. - The pharmaceutical composition according to claim 9, comprising a heavy chain constant region in which one or more amino acid residues at His310, His433, His435, His436, and Ile253 are different from those present in the unmodified antibody or the wild-type antibody of class IgG.

16. The radiolabeled agent is conjugated to a radionuclide selected from the group consisting of actinium-225 ( ２２５ Ac), astatine-211 ( ２１１ At), bismuth-212 and bismuth-213 ( ２１２ Bi, ２１３ Bi), copper-64 and copper-67 ( ６４ Cu, ６７ Cu), iodine-123, -124, -125 or -131 ( １２３ I, １２４ I, １２５ I, １３１ I)( １２３ I), lead-212 ( ２１２ Pb), lutetium-177 ( １７７ Lu), radium-223 and radium-224 ( ２２３ Ra, ２２４ Ra), rhenium-186 and rhenium-188 ( １８６ Re and １８８ Re), samarium-153 ( １５３ Sm), and scandium-47 ( ４７ Sc), strontium-90 ( ９０ Sr), terbium-149 and terbium-161 ( １４９ Tb and １６１ Tb), and yttrium-90 ( ９０ Y), the pharmaceutical composition according to any one of claims 1 to 4.

17. The aforementioned radioactive nuclide is lutetium-177 ( １７７ Lu) or Actinium-225 ( ２２５ The pharmaceutical composition according to claim 16, which is Ac.

18. The aforementioned radioactive nuclide is １７７ The pharmaceutical composition according to claim 16, wherein the component is Lu.

19. The pharmaceutical composition according to any one of claims 1 to 4, wherein the radioactivity of the radiolabeled drug is about 18 MBq or less, and optionally, the radioactivity is about 12 MBq or less.

20. The pharmaceutical composition according to any one of claims 1 to 4, wherein a radionuclide is conjugated to the drug via a linker or chelating agent.

21. The pharmaceutical composition according to claim 20, wherein the chelating agent is selected from TMT (6,6''-bis[N,N'',N'''-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2''-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N''(N'''-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A''-DTPA, TETE, Te2A, HBED, DFO, DFOsq, DFO-NCS, HOPO, or a chelating agent listed in WO2022 / 133537.

22. The pharmaceutical composition according to any one of claims 1 to 4, wherein the drug is conjugated to the radionuclide before administration.

23. A pharmaceutical composition according to any one of claims 1 to 4, wherein the immune checkpoint inhibitor therapy comprises inhibiting a protein selected from PD-1, PD-L1, and CTLA-4, or a combination thereof.

24. The pharmaceutical composition according to claim 23, wherein the immune checkpoint inhibitor therapy comprises inhibiting PD-1 and / or CTLA4.

25. The pharmaceutical composition according to claim 24, wherein the immune checkpoint inhibitor comprises an antibody for binding to PD-1 and / or an antibody for binding to CTLA4.

26. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject is administered the immune checkpoint inhibitor after administration of the radiolabeled agent for binding to CAIX.

27. ​​The pharmaceutical composition according to claim 26, wherein the subject is administered the immune checkpoint inhibitor at least 24 hours after administration of the radiolabeling agent for binding to CAIX.

28. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is renal cancer.

29. Use of a radiolabeled antigen-binding protein that binds to or specifically binds to CAIX in the manufacture of a pharmaceutical product for use in combination with immune checkpoint inhibitor therapy for the treatment of cancer, preferably renal cancer.

30. Use of immune checkpoint inhibitors in the manufacture of pharmaceuticals for use in combination with radiolabeled antigen-binding proteins that bind to or specifically bind to CAIX in the treatment of cancer, preferably renal cancer.

31. A radiolabeled antigen-binding protein that binds to or specifically binds to CAIX, for use in combination with immune checkpoint inhibitor therapy in the treatment of cancer, preferably renal cancer.

32. An immune checkpoint inhibitor for use in the treatment of cancer, preferably renal cancer, in combination with a radiolabeled antigen-binding protein that binds to or specifically binds to CAIX.

33. A pharmaceutical composition comprising a radiolabeled antigen-binding protein that binds to or specifically binds to CAIX, wherein the pharmaceutical composition is intended for use in combination with an immune checkpoint inhibitor in the treatment of cancer, preferably renal cancer.

34. A pharmaceutical composition comprising an immune checkpoint inhibitor for use in the treatment of cancer, preferably kidney cancer, in combination with a radiolabeled antigen-binding protein for binding to CAIX.

35. A kit for use as defined in any one of claims 1 to 4, comprising a separate part - A drug that is selectively conjugated with a radionuclide and specifically binds to CAIX, A kit including instructions for using it in combination with an immune checkpoint inhibitor.

36. The kit according to claim 35, wherein the drug is an antigen-binding protein for binding to CAIX, and preferably the antigen-binding protein is a gylenetuximab antibody or an antigen-binding fragment thereof.

37. The kit according to claim 35, further comprising one or more immune checkpoint inhibitors in a separate part.

38. A kit for use as defined in any one of claims 1 to 4, • One or more immune checkpoint inhibitors, A kit including instructions for using it in combination with an antigen-binding protein to bind to a CAIX conjugated with a radionuclide.

39. The kit according to claim 38, further comprising in a separate part an agent (preferably an antigen-binding protein) for optionally binding to CAIX conjugated with a radionuclide.

40. The kit according to claim 35, wherein the agent and / or the immune checkpoint inhibitor for binding to CAIX is provided in the form of a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and / or diluents, optionally selected.

41. The kit according to claim 38, wherein the agent and / or the immune checkpoint inhibitor for binding to CAIX is provided in the form of a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and / or diluents, optionally selected.

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