Combination therapy

Abstract

The present invention relates to methods and compositions for combination radioimmunotherapy.

Description

Technical Field

[0001] The present invention relates to combination therapies of checkpoint inhibitors that bind to CAIX and radiolabeled agents, particularly agents for radioimmunotherapy, such as antibodies conjugated to radioisotopes, and kits and compositions for use in such combination therapies.

[0002] Related Applications

[0003] This application claims priority from Australian provisional applications AU 2022901582 and AU 2022902973, the entire contents of which are hereby incorporated by reference. Background Art

[0004] Radiotherapy is an important form of tumor therapy. Various radiotherapy methods have been developed to treat tumors. Among them, radioimmunotherapy (RAIT or RIT), also known as molecular targeted radiation (MTR) or targeted radionuclide therapy (TRT), is an emerging method for providing radiotherapy. It uses antibodies, antibody fragments or peptides that can bind to target cells so that radioisotopes are guided to specific tissues and cells, thereby enhancing the specificity of tumor treatment and reducing toxicity.

[0005] Radiation damage to healthy tissues and organs is a major problem associated with radiotherapy. Such damage is mainly attributed to reactive oxygen species generated by radiation, which oxidize functionally important biomolecules such as nucleic acids, carbohydrates, lipids and lipoproteins, and damage tissues and cells. It is associated with a variety of biological processes, such as antimicrobial defense, inflammation, carcinogenesis and aging. Myelosuppression and cytopenias, such as decreased white blood cell (WBC) and platelet counts, and hematopoietic toxicity, as reflected by weight loss, are the most significant consequences of radiation damage. Toxicity severely limits the radiation dose of RAIT and reduces the effectiveness of tumor treatment.

[0006] Many approaches have been developed to try to mitigate the hematopoietic toxicity of radiation. Stem cell transplantation (SCT) and bone marrow transplantation (BMT) are the most commonly used approaches. However, these approaches are invasive, expensive, and may result in longer hospital stays for individuals undergoing treatment.

[0007] Other approaches include using cytokines to stimulate the immune system and blood regulatory proteins such as HP5b to shut down hematopoiesis during radiation exposure. These approaches have had varying degrees of success in small-scale studies against hematopoietic toxicity, but still require patients to receive further medications and treatments and are therefore largely unused.

[0008] Immunotherapy has shown promise in treating cancer due to 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 by stimulating antigen presenting cells, T cells, or innate cells, by priming and enhancing the immune system, by modulating inhibitory pathways, for example, by enhancing adaptive or innate immune responses, or by reducing immunosuppression in the tumor environment.

[0009] An example of one class of immunotherapies is the checkpoint inhibitors. Checkpoint inhibitors currently approved by the U.S. Food and Drug Administration (FDA) target cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death receptor 1 (PD-1), programmed cell death ligand 1 (PD-L1), or LAG3. Such checkpoint inhibitors work by blocking T cell inhibitory signals. The first approved agent, ipilimumab, received FDA marketing authorization in 2011 for metastatic melanoma. Since ipilimumab, at least five additional checkpoint inhibitor drugs have been approved for at least 14 different indications.

[0010] Immune checkpoint inhibition (ICI) has dramatically transformed cancer treatment, but most patients still do not have a (long-term) response. Key determinants of ICI treatment success are high tumor mutational burden and pre-existing tumor-infiltrating lymphocytes (TILs).

[0011] There remains a need for new approaches to mitigate toxicities associated with radioimmunotherapy.

[0012] There is also a need for improved immunotherapies for treating cancer.

[0013] The reference to any prior art in this specification does not constitute an acknowledgement or implication that this prior art forms part of the common general knowledge in any jurisdiction, nor does it constitute an acknowledgement or implication that this prior art can be reasonably understood by a person skilled in the art, regarded as related to and / or combined with other prior art. Summary of the invention

[0014] The present invention provides combined immune checkpoint inhibition (ICI) and targeted radionuclide therapy (TRT). Surprisingly, the combination of ICI and TRT shows improved results relative to any type of therapy alone, allowing lower TRT doses, and in certain embodiments, allowing immune memory of the subject's tumor.

[0015] Accordingly, the present invention provides a method of treating, preventing or minimizing the progression of cancer in a subject, the method comprising:

[0016] - administering to the subject a radiolabeled agent that binds or specifically binds to carbonic anhydrase IX (CAIX) in combination with an immune checkpoint inhibitor therapy,

[0017] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0018] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising:

[0019] - administering to the subject a radiolabeled agent that binds or specifically binds to carbonic anhydrase IX (CAIX), and

[0020] - administering an immune checkpoint inhibitor to the subject,

[0021] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0022] In a further aspect, the present invention provides a method of treating, preventing, or minimizing progression of cancer in a subject who has received or is receiving checkpoint inhibitor therapy, the method comprising:

[0023] - administering to the subject a radiolabeled agent that binds or specifically binds to carbonic anhydrase IX (CAIX),

[0024] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0025] In any aspect or embodiment, the radiolabeled agent can be a small molecule, peptide or protein conjugated to a radionuclide, and the small molecule, peptide or protein is capable of binding to CAIX. Preferably, the agent is capable of specifically and / or selectively binding to CAIX.

[0026] In any embodiment, the agent for binding to CAIX is a small molecule, optionally selected from the group consisting of: SLC-0111, SLC-149, SLC-0121, SLC-101, PMI-05, sulfamide-nitroimidazole, JS-403, UB-TT220, HEHEHE-Z09781, -MIP-1486, MIP-1490, MIP-1504 (particularly 99m Tc-HEHEHE-Z09781, 99m Tc-MIP-1486, 99m Tc-MIP-1490 or 99mTc-MIP-1504 / 5) and PHC-102.

[0027] In any embodiment, the agent for binding to CAIX is a peptide, optionally selected from the group consisting of: 3B-301, 3B-302 or CAIX-P1.

[0028] In particularly preferred aspects and embodiments, the agent for binding to CAIX is a polypeptide, including an antibody or an antigen-binding fragment thereof.

[0029] In one aspect, the agent is a peptide or protein, such that the invention provides a method of treating, preventing or minimizing the progression of cancer in a subject, the method comprising:

[0030] - administering to the subject a radiolabeled peptide or radioconjugated protein that binds or specifically binds to carbonic anhydrase IX (CAIX) in combination with an immune checkpoint inhibitor therapy,

[0031] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0032] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising:

[0033] - administering to the subject a radiolabeled peptide or a radioconjugated protein that binds or specifically binds to carbonic anhydrase IX (CAIX), and administering to the subject an immune checkpoint inhibitor,

[0034] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0035] In another aspect, the agent is a radiolabeled antibody or antigen-binding fragment thereof, such that the invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising:

[0036] - administering to the subject an antibody or antigen-binding fragment thereof for binding to CAIX in combination with an immune checkpoint inhibitor therapy, wherein the antibody or antigen-binding fragment thereof is conjugated to a radionuclide,

[0037] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0038] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving a checkpoint inhibitor, the method comprising:

[0039] - administering to the subject an antigen binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX), wherein the antigen binding protein is conjugated to a radionuclide,

[0040] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0041] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving a checkpoint inhibitor, the method comprising:

[0042] - administering to the subject an antibody or an antigen-binding fragment thereof for binding to CAIX, wherein the antibody or the antigen-binding fragment thereof is conjugated to a radionuclide,

[0043] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0044] In any aspect of the invention, the radiolabeled agent (preferably a radiolabeled antigen binding protein, such as an antibody) and the check point inhibitor that bind or specifically bind to carbonic anhydrase IX (CAIX) can be administered simultaneously. Alternatively, they can be administered sequentially. For example, the check point inhibitor can be administered before the radiolabeled agent (preferably a radiolabeled antigen binding protein), or the radiolabeled agent (preferably a radiolabeled antigen binding protein) can be administered before the check point inhibitor. Alternatively, treatment with the check point inhibitor and / or the radiolabeled agent (preferably a radiolabeled antigen binding protein) can be staggered.

[0045] In any aspect or embodiment, the immune checkpoint inhibitor therapy targets any one or more of the following: 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, immune checkpoint inhibitor therapy involves the administration of inhibitors of PD-1 and CTLA-4. In any aspect or embodiment, immune checkpoint inhibitor therapy involves the administration of inhibitors of PD-L1 and CTLA-4.

[0046] In any aspect or embodiment described herein, the agent that binds or specifically binds to carbonic anhydrase IX (CAIX) can be an antibody or an antigen-binding fragment thereof that binds or specifically binds to CAIX. In some embodiments, the antibody is girentuximab or a variant or antigen-binding fragment thereof that retains the ability to bind to CAIX.

[0047] The radiolabeled agent (preferably a radiolabeled antibody or fragment thereof) may be conjugated to any radionuclide suitable for therapeutic use, including any of the following: 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、Zr( 89 Zr) and Yttrium-90 ( 90 Y). 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 .

[0048] As used herein, the terms radioconjugated and radiolabeled can be used interchangeably. The term will be understood to refer to an agent (such as a small molecule, peptide or polypeptide, such as an antibody or its antigen-binding fragment) conjugated to a radionuclide. It should be understood that the terms radionuclide and radioisotope can be used interchangeably herein.

[0049] On the other hand, the present invention provides a method of inducing an immune response, preferably a T cell immune response, to a cancer in a subject, the method comprising administering to the subject a combination of a radiolabeled agent for binding to CAIX and an immune checkpoint inhibitor, thereby inducing the T cell response to the cancer in the subject. Preferably, the radiolabeled agent is a radiolabeled peptide or a radiolabeled antibody or antigen-binding fragment thereof.

[0050] On the other hand, the present invention provides a method of inducing an adaptive immune response to a cancer in a subject, the method comprising administering to the subject a combination of a radiolabeled agent that binds or specifically binds to CAIX and an immune checkpoint inhibitor, thereby inducing a T cell response to the cancer in the subject. Preferably, the radiolabeled agent is a radiolabeled peptide or a radiolabeled antibody or antigen-binding fragment thereof.

[0051] In any aspect or embodiment of the invention, the methods described herein further comprise identifying a subject suffering from cancer. In one embodiment, the cancer may be precancerous or non-metastatic. In another embodiment, the cancer may be malignant or metastatic.

[0052] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising the steps of:

[0053] - Identify subjects with cancer who have received or are currently receiving a checkpoint inhibitor for treatment of cancer,

[0054] - assessing whether the subject responds to the checkpoint inhibitor,

[0055] Wherein if the subject does not respond to the checkpoint inhibitor:

[0056] - administering to said subject a radiolabeled agent (preferably a radiolabeled antigen binding protein) that binds or specifically binds to CAIX,

[0057] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0058] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising the steps of:

[0059] - Identify subjects who have cancer and are unresponsive to treatment comprising a checkpoint inhibitor,

[0060] - administering to the subject a radiolabeled agent comprising, consisting of, or consisting essentially of a radiolabeled antigen binding protein that binds or specifically binds to CAIX,

[0061] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0062] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising the steps of:

[0063] - identifying subjects suffering from cancer; and

[0064] - administering to the subject a radiolabeled agent (preferably a radiolabeled antigen binding protein) that binds or specifically binds to CAIX and a checkpoint inhibitor,

[0065] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0066] In another aspect, the present invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising the steps of:

[0067] - identifying subjects suffering from cancer; and

[0068] - administering to the subject a radiolabeled agent for binding to CAIX (preferably a radiolabeled antibody or antigen-binding fragment thereof) and a checkpoint inhibitor,

[0069] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0070] On the other hand, the present invention further provides a method for increasing the survival rate of a subject suffering from cancer, the method comprising administering to the subject a radiolabeled agent (preferably a radiolabeled antigen binding protein) that binds or specifically binds to CAIX and a checkpoint inhibitor, thereby increasing the survival rate of the subject suffering from cancer.

[0071] On the other hand, the present invention further provides a method for increasing the survival rate of a subject suffering from cancer, the method comprising administering to the subject a radiolabeled agent (preferably a radiolabeled antibody or antigen-binding fragment thereof) for binding to CAIX and a checkpoint inhibitor, thereby increasing the survival rate of the subject suffering from cancer.

[0072] On the other hand, the present invention further provides a method for minimizing, reducing or preventing the growth of a tumor in a subject suffering from cancer, the method comprising administering to the subject a combination of a radiolabeled agent (preferably a radiolabeled antigen binding protein) that binds or specifically binds to CAIX and an immune checkpoint inhibitor therapy, thereby minimizing, reducing or preventing the growth of the tumor in the subject suffering from cancer.

[0073] On the other hand, the present invention further provides a method for minimizing, reducing or preventing the growth of a tumor in a subject suffering from cancer, the method comprising administering to the subject a combination of a radiolabeled agent (preferably a radiolabeled antibody or antigen-binding fragment thereof) for binding to CAIX and an immune checkpoint inhibitor therapy, thereby minimizing, reducing or preventing the growth of the tumor in the subject suffering from cancer.

[0074] On the other hand, the present invention further provides a method for minimizing, reducing or preventing metastasis in a subject with cancer, the method comprising administering to the subject a combination of a radiolabeled agent (preferably a radiolabeled antigen binding protein) that binds or specifically binds to CAIX and an immune checkpoint inhibitor therapy, thereby minimizing, reducing or preventing metastasis in the subject with cancer.

[0075] In any aspect or embodiment, the present invention further provides a method of minimizing, reducing or preventing metastasis in a subject having cancer, the method comprising:

[0076] - Identification of subjects with primary tumors;

[0077] - removing the primary tumor from said subject; and

[0078] - administering to the subject a radiolabeled agent (such as a radiolabeled antigen binding protein) that binds or specifically binds to CAIX in combination with an immune checkpoint inhibitor therapy,

[0079] Metastasis in the subject suffering from cancer is thereby minimized, reduced or prevented.

[0080] On the other hand, the present invention further provides a method for minimizing, reducing or preventing the growth of a tumor in at least one site of a subject that is away from the site of another tumor (e.g., a primary tumor), the method comprising administering to the subject a combination of a radiolabeled agent (preferably a radiolabeled antigen binding protein) that binds or specifically binds to CAIX and an immune checkpoint inhibitor therapy, thereby minimizing, reducing or preventing the growth of a tumor in at least one site of the subject that is away from the site of the primary tumor.

[0081] In any aspect or embodiment of the invention, immune checkpoint inhibitor therapy may comprise administering one or more immune checkpoint inhibitors.

[0082] In any aspect or embodiment of the invention, the method may further comprise the step of conjugating the antigen binding protein that binds or specifically binds to CAIX to a radionuclide to provide a radiolabeled antigen binding protein that binds or specifically binds to CAIX.

[0083] On the other hand, the present invention further provides a use of an agent for preparing a drug for treating, preventing or minimizing the progression of a subject's cancer, the agent comprising, consisting of or consisting essentially of a small molecule, peptide or antigen binding protein and / or a checkpoint inhibitor that binds or specifically binds to CAIX. Preferably, the agent is conjugated to or capable of being conjugated to a radionuclide.

[0084] On the other hand, the present invention further provides a use of an agent (such as a small molecule, a peptide or an antigen binding protein, preferably an antigen binding protein) that binds or specifically binds to CAIX and a checkpoint inhibitor, wherein the agent is used to prepare a first drug, and the checkpoint inhibitor is used to prepare a second drug, wherein the first and second drugs are used for:

[0085] Treating, preventing or minimizing the progression of cancer in a subject,

[0086] minimizing, reducing or preventing the growth of a tumor in a subject,

[0087] Minimize, reduce or prevent metastasis in a subject, or

[0088] Increase the survival rate of subjects,

[0089] Preferably, wherein the agent is conjugated to a radionuclide.

[0090] Alternatively, the first and second medicaments are for use in any other method or use of the invention as described herein.

[0091] On the other hand, the present invention further provides an agent (such as a small molecule, a peptide or an antigen binding protein, preferably an antigen binding protein) that binds or specifically binds to CAIX, wherein the agent is conjugated to a radionuclide, or is capable of being conjugated to a radionuclide, for use in combination with immune checkpoint inhibitor therapy to treat, prevent or prevent the progression of cancer in a subject.

[0092] On the other hand, the present invention further provides the use of an agent (such as a small molecule, a peptide or an antigen binding protein, preferably an antigen binding protein) that binds or specifically binds to CAIX, wherein the agent is conjugated to a radionuclide and an immune checkpoint inhibitor for treating, preventing or preventing the progression of cancer in a subject.

[0093] On the other hand, the present invention further provides an agent (such as a small molecule, a peptide or an antigen binding protein, preferably an antigen binding protein) that binds or specifically binds to CAIX, wherein the agent is conjugated to a radionuclide for use in combination with immune checkpoint inhibitor therapy to treat, prevent or prevent the progression of cancer in a subject. Alternatively, the agent that binds or specifically binds to CAIX and the checkpoint inhibitor are used in any other method or use of the present invention as described herein, including a method for treating, preventing or minimizing the progression of cancer in a subject, minimizing the growth of a subject's tumor, reducing or preventing the growth of a subject's tumor, minimizing, reducing or preventing metastasis in a subject, or increasing the survival rate of a subject.

[0094] On the other hand, the present invention further provides the use of an agent (such as a small molecule, a peptide or an antigen binding protein, preferably an antigen binding protein) that binds or specifically binds to CAIX for preparing a medicament for:

[0095] Treat, prevent, or minimize the progression of cancer in a subject who has received or is currently receiving a checkpoint inhibitor,

[0096] Minimizing, reducing or preventing the growth of a tumor in a subject who has received or is currently receiving a checkpoint inhibitor,

[0097] Minimize, reduce, or prevent metastasis in subjects who have received or are currently receiving a checkpoint inhibitor, or

[0098] Increased survival in subjects who have received or are currently receiving checkpoint inhibitors,

[0099] Preferably, wherein the agent is conjugated to a radionuclide or is capable of being conjugated to a radionuclide.

[0100] In any aspect or embodiment of the invention, any drug described herein is suitable for intraperitoneal, intratumoral, topical, oral, intravenous administration to the respiratory tract, preferably by inhalation or intranasal, subcutaneous or intramuscular administration. Typically, any drug described herein is suitable for intravenous administration.

[0101] On the other hand, the present invention further provides an antigen binding protein that binds or specifically binds to CAIX for treating, preventing, or minimizing the progression of cancer in subjects who have received or are receiving checkpoint inhibitors, preferably wherein the antigen binding protein is conjugated to a radionuclide. Alternatively, the antigen binding protein that binds or specifically binds to CAIX is used in any other method or use of the present invention as described herein. In one aspect of the present invention, the antigen binding protein that preferably binds or specifically binds to CAIX is suitable for intraperitoneal, intratumoral, topical, oral administration to the respiratory tract, preferably by inhalation or intranasal, intravenous, subcutaneous or intramuscular administration, wherein the antigen binding protein is conjugated to a radionuclide. Preferably, the antigen binding protein that binds or specifically binds to CAIX is suitable for intravenous administration.

[0102] On the other hand, the present invention further provides a use of an antigen binding protein that binds or specifically binds to CAIX for treating, preventing, or minimizing the progression of cancer in subjects who have received or are receiving checkpoint inhibitors, preferably wherein the antigen binding protein is conjugated to a radionuclide. Alternatively, the use of the antigen binding protein that binds or specifically binds to CAIX is in any other method or use of the present invention as described herein.

[0103] In any of the uses described herein, the peptide or antigen binding protein that binds or specifically binds to CAIX may be conjugated to a radionuclide via a linker or chelator. The radionuclide may be any of the following: 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.

[0104] In any embodiment of the invention, the therapeutic effect of any radioconjugated agent (such as an antigen binding protein) and a check point inhibitor that binds or specifically binds to CAIX as described herein may be significant compared to the effect of a radioconjugated agent or a check point inhibitor alone (i.e., compared to a monotherapy with a radioconjugated agent that binds or specifically binds to CAIX or a monotherapy with a check point inhibitor). In one aspect, the effect may be additive or synergistic. Preferably, the effect is synergistic.

[0105] On the other hand, when a radioconjugated agent (preferably a radiolabeled antigen binding protein) that binds or specifically binds to CAIX and a checkpoint inhibitor are administered to a subject, the radioconjugated agent increases the effectiveness of the checkpoint inhibitor. Optionally, the radiolabeled agent (preferably an antigen binding protein) increases the effectiveness of the checkpoint inhibitor to such an extent that when the checkpoint inhibitor is administered alone (i.e., monotherapy), the total dose of the checkpoint inhibitor administered to the subject can be lower than the dose required to achieve the same effect. 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 one-half, or at least two-thirds of the dose required for monotherapy with the checkpoint inhibitor.

[0106] On the other hand, when the radioconjugated agent (preferably an antigen binding protein) and a checkpoint inhibitor described herein that bind or specifically bind to CAIX are administered to a subject, the checkpoint inhibitor can improve the effectiveness of the radioconjugated agent (preferably an antigen binding protein). Optionally, the checkpoint inhibitor improves the effectiveness of the agent (preferably an antigen binding protein) to such an extent that when the agent is administered alone, the total dose of the agent administered to the subject can be a dose lower than the dose required to achieve the same effect. Preferably, the improvement is such that the dose of the radioconjugated agent (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 radioconjugated agent (preferably an antigen binding protein). More preferably, the improvement is such that the dose of the radioconjugated agent (preferably an antigen binding protein) can be at least one-quarter, at least one-third, at least one-half or at least two-thirds of the dose required for monotherapy with the radioconjugated agent (antigen binding protein).

[0107] In certain embodiments, the effects of a radioconjugated agent (preferably an antigen binding protein) and a checkpoint inhibitor that binds or specifically binds to CAIX described herein on the survival rate of the subject may be significantly greater than the effects of the agent and the checkpoint inhibitor when administered alone. In further embodiments, the effects of any radioconjugated agent and a checkpoint inhibitor that binds or specifically binds to CAIX described herein on the tumor growth of the subject may be significantly greater than the effects of the agent and the checkpoint inhibitor when administered alone.

[0108] In any aspect of the invention, the radioactively conjugated agent (preferably an antigen binding protein) and / or a checkpoint inhibitor that is bound or specifically bound to CAIX is administered once. In another embodiment, the agent (preferably an antigen binding protein) and / or a checkpoint inhibitor is administered to the subject twice, three times, four times or more.

[0109] In any aspect of the invention, the radioactive conjugated agent (antigen binding protein) and / or check point inhibitor that binds or specifically binds to CAIX can be administered in the same composition or in a separate composition. On the other hand, the agent (antigen binding protein) and / or check point inhibitor can therefore be administered together or sequentially. Alternatively, administration can be staggered. The agent (antigen binding protein) and / or check point inhibitor can also be administered at the same frequency or at different frequencies.

[0110] Optionally, the radioconjugated agent (antigen binding protein) that binds or specifically binds to CAIX is administered to the subject prior to administration of the checkpoint inhibitor. In some embodiments, the radioconjugated agent that binds 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 radioconjugated agent (antigen binding protein) that binds or specifically binds to CAIX is administered at least 1 day prior to administration of the checkpoint inhibitor.

[0111] In any aspect of the invention, the antigen binding protein that binds or specifically binds to CAIX and the checkpoint inhibitor can be administered by any route of administration known in the art, including intraperitoneal, intratumoral, topical, oral administration to the respiratory tract, by inhalation or intranasal, intravenous, subcutaneous or intramuscular administration. Preferably, the antigen binding protein that binds or specifically binds to CAIX and is conjugated to a radionuclide and / or the checkpoint inhibitor is administered intravenously.

[0112] In any aspect of the invention, the amount of antigen binding protein that binds or specifically binds to CAIX conjugated to a radionuclide administered can be in the range of about 250 nanomoles / kg body weight / dose to 0.005 nanomoles / kg body weight / dose.

[0113] In any aspect of the invention, the amount of antigen binding protein that binds or specifically binds to CAIX and is conjugated to a radionuclide administered can 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.

[0114] In any aspect of the invention, the amount of the check point inhibitor administered can 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 can 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, or about 10 mg / kg body weight.

[0115] In any aspect of the invention, the cancer may be any cancer associated with or caused by CAIX expression. 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 cancer / cholangiocarcinoma, neuroendocrine cancer, pituitary tumor, small round cell tumor, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, non-seminoma, stromal stromal 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 a preferred embodiment, the cancer is melanoma, breast cancer or colon cancer. In some embodiments, the cancer is colorectal cancer or renal cancer, such as renal cortical adenocarcinoma or clear cell renal cell carcinoma (ccRCC).

[0116] In any aspect of the invention, the checkpoint inhibitor can be a PD-1, PD-L1 or CTLA-4 checkpoint inhibitor. On the one hand, 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, immune checkpoint inhibitor therapy involves the administration of inhibitors of PD-1 and CTLA-4. Examples of such inhibitors are further described herein.

[0117] Antigen binding protein that binds or specifically binds to CAIX

[0118] The present invention involves the administration of an antigen binding protein that binds or specifically binds to CAIX, wherein the antigen binding protein is conjugated to a radionuclide.

[0119] In any embodiment, the radiolabeled antigen binding protein is an antibody or antigen binding fragment thereof for binding to CAIX. Any suitable antibody or antigen binding fragment thereof for binding to CAIX can be used. In some embodiments, the radiolabeled antigen binding protein that binds or specifically binds to CAIX is a radiolabeled girentuximab or a radiolabeled functional variant or fragment thereof that retains the ability to bind to CAIX. 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 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 a radiolabeled humanized G250 (hG250).

[0120] In any embodiment, the antigen binding protein that binds or specifically binds to CAIX is as described in any of WO 2002 / 062972A2 (US2004 / 0219633 A1), WO 2004 / 002526 A1 (US 7,632,496 B2), WO 2006 / 002889A2 (US 7,691,375 B2), WO 2009 / 056342 A1 (US2014 / 0017252 A1), WO 2011 / 032973A1 (US2012 / 0207672 A1), WO 2014 / 128258 A1 (US10,620,208 B2), or US 11,629,199. An antibody for binding to CAIX described in these publications is referred to herein as TLX250, which is further described in the Examples herein. In any embodiment, the antibody or antigen-binding fragment for binding to CAIX is as described in WO 2021 / 000017 A1. The entire contents of each of these publications are incorporated herein by reference.

[0121] In any embodiment, the antigen binding protein comprises:

[0122] (a) three CDRs of an antigen binding domain having a heavy chain variable domain (VH) as shown in SEQ ID NO: 4, 20, 36, 52 or 68; and / or

[0123] (b) three CDRs of an antigen binding domain having a light chain variable domain (VL), wherein VL comprises the amino acid sequence as shown in SEQ ID NO: 84, 100, 116, 132, 148 or 164.

[0124] The skilled person will be familiar with various methods for determining any given heavy or light chain variable region, including methods routinely used in the art and methods described elsewhere herein. In addition, the skilled person will understand that the definition of the CDR boundaries of any given variable chain will depend on the specific naming convention used (such as IMGT, Chothia or Kabat numbering system).

[0125] In any embodiment, the antigen binding protein comprises:

[0126] (a) a heavy chain variable domain (VH) comprising three complementarity determining regions (CDRs) of the amino acid sequence shown in SEQ ID NO: 4, 20, 36, 52 or 68; and / or

[0127] (b) a light chain variable domain (VL), said VL comprising three complementarity determining regions (CDRs) of the amino acid sequence shown in SEQ ID NO: 84, 100, 116, 132, 148 or 164.

[0128] In a preferred embodiment, the antigen binding protein comprises the amino acid sequences of VH and VL as shown below:

[0129] - SEQ ID NO: 4 and SEQ ID NO: 84

[0130] - SEQ ID NO:36 and SEQ ID NO:132

[0131] - SEQ ID NO:52 and SEQ ID NO:132

[0132] - SEQ ID NO: 52 and SEQ ID NO: 148;

[0133] or a variant thereof, wherein the VH and VL comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions, deletions or additions compared to the amino acid sequences as shown above; wherein the amino acid substitutions, deletions or additions are not in the CDRs, and the antigen binding protein retains the ability to bind to CAIX.

[0134] In any embodiment, the antigen binding protein comprises an antigen binding domain that specifically binds to carbonic anhydrase IX (CAIX) and comprises:

[0135] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a

[0136] in:

[0137] FR1, FR2, FR3 and FR4 are each framework regions;

[0138] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0139] FR1a, FR2a, FR3a and FR4a are each framework regions;

[0140] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0141] The sequence of any of the complementary determining regions has an amino acid sequence as described in Table 1 below. Preferably, the framework region has an amino acid sequence also as described in Table 1 below, including amino acid variations at specific residues, which can be determined by aligning various framework regions derived from each antibody. The CDR1, CDR2 and CDR3 may be sequences from the VH, and CDR1a, CDR2a and CDR3a may be sequences from the VL, or the CDR1, CDR2 and CDR3 may be sequences from the VL, and CDR1a, CDR2a and CDR3a may be sequences from the VH.

[0142] Reference herein to a protein or antibody that "binds to carbonic anhydrase IX (CAIX)" provides literal support for a protein or antibody that "binds specifically to" or "specifically binds to" CAIX.

[0143] In any embodiment, the antigen binding protein comprises:

[0144] (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:1, and 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 shown in SEQ ID NO:1. NO:2 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 shown in SEQ ID NO:2, and a CDR3 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:3;

[0145] (ii) a VH comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in any one of SEQ ID NOs: 4, 20, 36, 52, or 68;

[0146] (iii) a VL comprising 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 shown in SEQ ID NO:81, and 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 shown in SEQ ID NO:81. 82 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 shown in SEQ ID NO:82, and a CDR3 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:83;

[0147] (iv) a VL comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:84, 100, 116, 132, 148, or 164;

[0148] (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;

[0149] (vi) a VH comprising the sequence shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68;

[0150] (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;

[0151] (viii) a VL comprising the sequence set forth in any one of SEQ ID NOs: 84, 100, 116, 132, 148 or 164;

[0152] (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3; and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 81, a CDR2 comprising the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising the sequence set forth in SEQ ID NO: 83; or

[0153] (x) a VH comprising the sequence shown in any one of SEQ ID NOs: 4, 20, 36, 52 or 68, and a VL comprising the sequence shown in any one of SEQ ID NOs: 84, 100, 116, 132, 148 or 164.

[0154] In further embodiments, the antigen binding protein comprises:

[0155] (i) a VH comprising a framework region (FR) 1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in any one of SEQ ID NOs: 9, 25, 41, 57 or 73; and a FR2 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%, at least 99% identical to the sequence shown in any one of SEQ ID NOs: 9, 25, 41, 57 or 73. NO:10, 26, 42, 58 or 74, wherein the sequence 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 or consisting of a sequence as set forth in any one of SEQ ID NO:10, 26, 42, 58 or 74; FR3, wherein the FR3 comprises a sequence as set forth in SEQ ID NO:11, 27, 43, 59 or 75, wherein the sequence 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 or consisting of a sequence as set forth in any one of SEQ ID NO:11, 27, 43, 59 or 75; FR4, wherein the FR4 comprises a sequence as set forth in SEQ ID NO:12, 28, 44, 60 or 76 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 or consisting of a sequence as set forth in any of NO:12, 28, 44, 60 or 76; and

[0156] (ii) a VL comprising a framework region (FR) 1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in any one of SEQ ID NOs: 89, 105, 121, 137, 153, or 169; and a FR2 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%, at least 99% identical to the sequence shown in any one of SEQ ID NOs: NO:90, 106, 122, 138, 154 or 170, wherein the sequence 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 or consisting of a sequence as set forth in any one of SEQ ID NO:90, 106, 122, 138, 154 or 170; FR3, wherein the FR3 comprises a sequence as set forth in SEQ ID NO:90, 106, 122, 138, 154 or 170, wherein the sequence 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 or consisting of a sequence as set forth in SEQ ID NO: ID NO:91, 107, 123, 139, 155 or 171, wherein the sequence 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 or consisting of a sequence as set forth in any one of SEQ ID NO:91, 107, 123, 139, 155 or 171; FR4, wherein the FR4 comprises a sequence as set forth in SEQ ID NO:91, 107, 123, 139, 155 or 171, wherein the sequence 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 or consisting of a sequence as set forth in SEQ ID NO: NO:92, 108, 124, 140, 156 or 172, or consist 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 or consisting of a sequence shown in any of NO:92, 108, 124, 140, 156 or 172.

[0157] In further embodiments, the antigen binding protein comprises:

[0158] (i) a VH comprising a framework region (FR) 1 comprising or consisting of a sequence as shown in SEQ ID NO: 9, 25, 41, 57 or 73; a FR2 comprising or consisting of a sequence as shown in SEQ ID NO: 10, 26, 42, 58 or 74; a FR3 comprising or consisting of a sequence as shown in SEQ ID NO: 11, 27, 43, 59 or 75; a FR4 comprising or consisting of a sequence as shown in SEQ ID NO: 12, 28, 44, 60 or 76, and

[0159] (ii) a VL comprising a framework region (FR) 1 comprising or consisting of a sequence as shown in SEQ ID NO: 89, 105, 121, 137, 153 or 169; a FR2 comprising or consisting of a sequence as shown in any one of SEQ ID NO: 90, 106, 122, 138, 154 or 170; a FR3 comprising or consisting of a sequence as shown in SEQ ID NO: 91, 107, 123, 139, 155 or 171; and a FR4 comprising or consisting of a sequence as shown in SEQ ID NO: 92, 108, 124, 140, 156 or 172.

[0160] In any embodiment, the 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 NOs: 4, 20, 36, 52, or 68 and / or any one of SEQ ID NOs: 84, 100, 116, 132, 148, 164 (in order from N to C terminus or C to N terminus).

[0161] In any embodiment, the antigen binding protein comprises:

[0162] (a) a heavy chain variable domain (VH) comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO:4, 20, 36, 52 or 68; and / or

[0163] (b) a light chain variable domain (VL) comprising or consisting of a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO: 84, 100, 116, 132, 148 or 164. In some embodiments, the antigen binding protein is a modified IgG antibody comprising a heavy chain constant region having one or more amino acid substitutions compared to a wild-type IgG class antibody, wherein the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn) compared to a wild-type IgG class antibody, thereby shortening the serum half-life of the modified antibody.

[0164] 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, reducing the affinity of the IgG molecule to FcRn. Alternatively, the one or more amino acid substitutions may be in the heavy chain constant region 3 (CH3) of the IgG molecule, thereby reducing the affinity of the IgG molecule to FcRn. Still further, the amino acid substitutions may include at least one substitution in the CH2 region and at least one substitution in the CH3 region of the IgG molecule, thereby reducing the affinity of the IgG to FcRn.

[0165] In certain preferred embodiments, the one or more amino acid substitutions may be at one or more of residues His310, His433, His435, His436 or Ile253 of IgG. Preferably, the amino acid substitutions comprise substitutions in the heavy chain constant region at position His310 or His435. More preferably, the amino acid substitutions that reduce the affinity of the antibody for FcRn are at both His310 and His435.

[0166] In certain embodiments, the modified antibodies retain the ability to bind to one or more Fc-gamma receptors, and thus in certain embodiments, the modified antibodies retain the ability to stimulate effector responses, including ADCC.

[0167] In alternative embodiments, the one or more amino acid modifications that reduce affinity for FcRn receptors also reduce affinity for Fcγ receptors. Compared to wild-type IgG class antibodies, the modified antibodies may further comprise one or more amino acid substitutions, wherein the amino acid substitutions further reduce the affinity of the antibody for one or more Fcγ receptors.

[0168] In additional embodiments, the modified antibody further comprises one or more amino acid substitutions compared to a wild-type IgG antibody, wherein the amino acid substitutions increase the stability of the CH1-CH2 hinge region in the modified antibody compared to a wild-type IgG antibody.

[0169] In any embodiment, the antibody for binding to CAIX is conjugated to a therapeutic agent. The therapeutic agent can be conjugated to the antibody directly or indirectly, for example, by halogenation of amino acid residues. Preferably, the therapeutic agent is conjugated to the antibody indirectly through 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"'-tetrakis(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 chelator as described in WO 2022 / 133537 (incorporated herein by reference) or other chelators as described herein.

[0170] In another example, the antibody is conjugated to a bifunctional linker, such as bromoacetyl, thiol, succinimidyl ester, TFP ester, maleimide, or using any amine or thiol modification chemistry known in the art.

[0171] Preferably, the therapeutic agent is a radioactive isotope. 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), Samarium-153 ( 153 Sm), Scandium-47(47 Sc), Strontium-90 ( 90 Sr) and Yttrium-90 ( 90 Y).

[0172] In any embodiment, the heavy chain constant region of the antibody comprises amino acid substitutions at both His310 and His435. The antibody may also comprise amino acid substitutions at residues equivalent to Ser228 and Leu235 of the constant heavy chain region.

[0173] Preferably, the antibody comprises a heavy chain constant region comprising the sequence as shown in any one of SEQ ID NOs: 177 to 180, preferably as shown in SEQ ID NO: 178.

[0174] In still further embodiments, the antibody preferably comprises a heavy chain comprising the sequence shown in any one of SEQ ID NOs: 182 to 185, preferably as shown in SEQ ID NO: 183.

[0175] In any embodiment, the antibody comprises a light chain constant region comprising the amino acid sequence as shown in SEQ ID NO: 181. Preferably, the antibody comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO: 186.

[0176] In any embodiment, the antibody comprises the sequence shown in SEQ ID NO:183 and the sequence shown in SEQ ID NO:186.

[0177] The present invention also provides a use of a molecule comprising an immunoglobulin portion and a non-protein agent conjugated thereto,

[0178] wherein the immunoglobulin portion specifically binds to CAIX and 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 to C terminus or C to N terminus),

[0179] Optionally, wherein the immunoglobulin portion has reduced or eliminated affinity for the FcRn receptor compared to a wild-type immunoglobulin; and

[0180] The non-protein agent comprises a therapeutic moiety, such as a radioactive element.

[0181] Preferably, the immunoglobulin portion has an amino acid substitution at a residue equivalent to His310 and / or His435 in the constant heavy chain region. The immunoglobulin portion may also comprise an amino acid substitution at a residue equivalent to Ser228 and Leu235 in the constant heavy chain region. Preferably, the non-protein agent comprises a radioactive element.

[0182] The present invention also provides a use of a molecule comprising an immunoglobulin portion and a non-protein agent conjugated thereto, optionally wherein the affinity of the immunoglobulin portion for the FcRn receptor is reduced or eliminated compared to a wild-type immunoglobulin.

[0183] wherein the immunoglobulin portion specifically binds to CAIX and comprises at least one of:

[0184] (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence 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 shown in SEQ ID NO: 1, 17, 33, 49 or 65, a CDR2 comprising a sequence 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 shown in SEQ ID NO: 2, 18, 34, 50 or 66, and a CDR3 comprising a sequence 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 shown in SEQ ID NO: 3, 19, 35, 51 or 67;

[0185] (ii) a VH comprising a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 4, 20, 36, 52 or 68;

[0186] (iii) a VL comprising a CDR1 comprising a sequence 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 shown in SEQ ID NO:81, 97, 113, 129, 145 or 161, a CDR2 comprising a sequence 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 shown in SEQ ID NO:82, 98, 114, 130, 146 or 162, and a CDR3 comprising a sequence 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 shown in SEQ ID NO:83, 99, 115, 131, 147 or 163;

[0187] (iv) a VL comprising a sequence at least about 95% identical to the sequence shown in SEQ ID NO: 84, 100, 116, 132, 148 or 164;

[0188] (v) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1, 17, 33, 49 or 65, a CDR2 comprising the sequence shown in SEQ ID NO: 2, 18, 34, 50 or 66, and a CDR3 comprising the sequence shown in SEQ ID NO: 3, 19, 35, 51 or 67;

[0189] (vi) a VH comprising the sequence shown in SEQ ID NO: 4, 20, 36, 52 or 68;

[0190] (vii) 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;

[0191] (viii) a VL comprising the sequence shown in SEQ ID NO: 84, 100, 116, 132, 148 or 164;

[0192] (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 in 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

[0193] (x) VH comprising the sequence shown in SEQ ID NO:4, 20, 36, 52 or 68, and VL comprising the sequence shown in SEQ ID NO:84, 100, 116, 132, 148 or 164.

[0194] Preferably, the non-protein agent comprises a radioactive element. Preferably, the immunoglobulin portion has an amino acid substitution at a residue equivalent to His310 and / or His435 in the constant heavy chain region. The immunoglobulin portion may also comprise an amino acid substitution at a residue equivalent to Ser228 and Leu235 in the constant heavy chain region. Preferably, the non-protein agent comprises a radioactive element.

[0195] In any embodiment, the immunoglobulin comprises a heavy chain constant region comprising the sequence as shown in any one of SEQ ID NOs: 177 to 180, preferably as shown in SEQ ID NO: 178.

[0196] In still further embodiments, the immunoglobulin comprises a heavy chain comprising the sequence shown in any one of SEQ ID NOs: 182 to 185, preferably as shown in SEQ ID NO: 183.

[0197] In any embodiment, the immunoglobulin comprises a light chain constant region comprising the sequence shown in SEQ ID NO: 181. In one embodiment, the light chain comprises the sequence of SEQ ID NO: 186.

[0198] In a particularly preferred embodiment, the immunoglobulin comprises the amino acid sequence shown in SEQ ID NO:183 and 186.

[0199] In certain embodiments, the antibody is an antibody as described herein, including an antibody having any of the complementary determining regions, framework regions, variable light or variable heavy regions as described in Table 1 below. Preferably, the modified antibody further comprises amino acid substitutions at residues equivalent 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.

[0200] In any embodiment, the modified antibodies for use as described herein have reduced toxicity compared to unmodified antibodies. Reduced toxicity includes reducing many toxic effects that would otherwise be caused by long-term retention of radioisotopes in the circulation (including hematological toxicity, absorption into bone, and bone marrow irradiation).

[0201] In any embodiment, the toxicity of a radiolabeled antibody or radioimmunoconjugate described herein is assessed by determining the tumor:blood ratio of the antibody or immunoconjugate following administration to a subject.

[0202] In any embodiment of the invention, when the ratio is determined at least 8 hours after administration of the antibody, the tumor: blood ratio of the modified antibody of the invention is at least 2 times, at least 3 times, at least 4 times, at least 6 times, at least 8 times, or at least 10 times or more of the tumor: blood ratio of the unmodified antibody without the modification of the heavy chain constant region as described herein. Alternatively, the ratio is determined at least 24, 48, 72, or 120 hours after administration of the antibody to the individual. In certain embodiments, when the ratio is determined at least 120 hours after administration of the antibody, the tumor: blood ratio of the modified antibody of the invention is at least 50 times, at least 100 times, at least 200 times, or at least 300 times of the tumor: blood ratio of the unmodified antibody without the modification of the heavy chain constant region as described herein.

[0203] In any embodiment of the invention, the modified antibodies described herein with reduced or altered serum half-life have a serum clearance rate that is at least two times faster, at least three times faster, or more times faster than the serum clearance rate of the unmodified antibody, compared to the unmodified antibody.

[0204] In certain embodiments of the invention, the CAIX binding antibodies described herein are suitable for administration in a theranostic pair, wherein the theranostic pair comprises 1) an antibody conjugated to an imaging agent and 2) an antibody conjugated to an agent for therapy. For example, the antibody can be used first as a diagnostic agent when conjugated to a radioisotope suitable for use in radio-imaging, and secondly, as a therapeutic agent when conjugated to a radioisotope or cytotoxic agent suitable for use in therapy.

[0205] In another embodiment, the method of the present invention may further include the step of diagnosing, monitoring or prognosing a disease, disorder or infection in a subject, the method comprising:

[0206] (a) administering to a subject an antibody described herein that specifically binds to an antigen associated with a disease, disorder or infection;

[0207] (b) allowing antibodies to concentrate at the site where the antigen is found in the subject; and

[0208] (c) detecting the antibody;

[0209] Detection of the antibody above background or standard levels thereby indicates that the subject has the disease, disorder or infection.

[0210] In any aspect of the invention, the antigen binding domain further comprises at least one of the following:

[0211] (i) a VH comprising a framework region (FR) 1 comprising a sequence 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 shown in SEQ ID NO:9, 25, 41, 57 or 76, a FR2 comprising a sequence 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 shown in SEQ ID NO:10, 26, 42, 58 or 74, a FR3 comprising a sequence 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 shown in SEQ ID NO:11, 27, 43, 59 or 75, and a FR4 comprising a sequence 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 shown in SEQ ID NO:11, 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 shown in NO:12, 28, 44, 60 or 76;

[0212] (ii) a VL comprising a FR1 comprising a sequence 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 shown in SEQ ID NO:89, 105, 121, 137, 153 or 169, a FR2 comprising a sequence 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 shown in SEQ ID NO:90, 106, 122, 138, 154 or 170, a FR3 comprising a sequence 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 shown in SEQ ID NO:91, 107, 123, 139, 155 or 171, and a FR4 comprising a sequence 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 shown in SEQ ID NO: 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 shown in NO:92, 108, 124, 140, 156 or 172;

[0213] (iii) a VH comprising a FR1 comprising the sequence shown in SEQ ID NO: 9, 25, 41, 57 or 76, a FR2 comprising the sequence shown in SEQ ID NO: 10, 26, 42, 58 or 74, a FR3 comprising the sequence shown in SEQ ID NO: 11, 27, 43, 59 or 75, and a FR4 comprising the sequence shown in SEQ ID NO: 12, 28, 44, 60 or 76;

[0214] (iv) a VL comprising a FR1 comprising the sequence set forth in SEQ ID NO: 89, 105, 121, 137, 153 or 169, a FR2 comprising the sequence set forth in SEQ ID NO: 90, 106, 122, 138, 154 or 170, a FR3 comprising the sequence set forth in SEQ ID NO: 91, 107, 123, 139, 155 or 171, and a FR4 comprising the sequence set forth in SEQ ID NO: 92, 108, 124, 140, 156 or 172; or

[0215] (v) a VH comprising a FR1 comprising the sequence shown in SEQ ID NO:9, 25, 41, 57 or 76, a FR2 comprising the sequence shown in SEQ ID NO:10, 26, 42, 58 or 74, a FR3 comprising the sequence shown in SEQ ID NO:11, 27, 43, 59 or 75, and a FR4 comprising the sequence shown in SEQ ID NO:12, 28, 44, 60 or 76; and a VL comprising a FR1 comprising the sequence shown in SEQ ID NO:89, 105, 121, 137, 153 or 169, a FR2 comprising the sequence shown in SEQ ID NO:90, 106, 122, 138, 154 or 170, and a FR3 comprising the sequence shown in SEQ ID NO:11, 27, 43, 59 or 75 The sequence shown in NO:91, 107, 123, 139, 155 or 171, and FR4, wherein the FR4 comprises the sequence shown in SEQ ID NO:92, 108, 124, 140, 156 or 172.

[0216] In one embodiment, the VH comprises a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 36 or 52, or comprises the sequence shown therein, and the VL comprises a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 116, 132 or 148, or comprises the sequence shown therein. Preferably, the VH comprises a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 36 or 52, or comprises the sequence shown therein, and the VL comprises a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 132 or 148, or comprises the sequence shown therein. More preferably, the VH comprises a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 36, or comprises the sequence shown therein, and the VL comprises a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 148, or comprises the sequence shown therein. In one embodiment, the VH comprises a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 52, or comprises the sequence shown therein, and the VL comprises a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 132 or 148, preferably a sequence that is at least about 95%, or 96%, or 97%, or 98% or 99% identical to the sequence shown in SEQ ID NO: 148, or comprises the sequence shown therein.

[0217] In any of the embodiments herein, the antigen binding protein may also be referred to as an antigen binding domain of an antibody.

[0218] Preferably, the antigen binding protein as 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, such as a monoclonal antibody.

[0219] As described herein, the antigen binding protein may be in the form of:

[0220] (i) single-chain Fv fragment (scFv);

[0221] (ii) dimeric scFv (di-scFv);

[0222] (iii) one of (i) or (ii) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3; or

[0223] (iv) one of (i) or (ii) linked to a protein that binds to immune effector cells.

[0224] Further, as described herein, the antigen binding protein may be in the form of:

[0225] (i) Bifunctional antibodies;

[0226] (ii) trifunctional antibodies;

[0227] (iii) tetrafunctional antibodies;

[0228] (iv) Fab;

[0229] (v) F(ab')2;

[0230] (vi) Fv;

[0231] (vii) one of (i) to (vi) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3; or

[0232] (viii) one of (i) to (vi) linked to a protein that binds to immune effector cells.

[0233] In any aspect or embodiment, the antibody is a naked antibody. In particular, the antibody is in a non-conjugated form and is not suitable for forming a conjugate.

[0234] In any embodiment, the antigen binding protein for use according to the present invention can be a fusion protein comprising an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab (single domain antibody), a bi-scFv, scFv, Fab, Fab', F(ab')2, an Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody as described herein.

[0235] In any embodiment, the antigen binding protein for use according to the present invention can be a conjugate in the form of an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, a bi-scFv, a scFv, a Fab, a Fab', a F(ab')2, a Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody or fusion protein conjugated to a label or a cytotoxic agent as described herein.

[0236] In a preferred embodiment, the antigen binding protein is an IgG immunoglobulin, which comprises one or more amino acid substitutions in the antibody constant domain CH2-CH3 region, which modify the binding of the antibody to the neonatal Fc receptor (FcRn) relative to the wild-type antibody Fc region. The one or more amino acid modifications change the affinity of the antibody constant domain, Fc region or its FcRn binding fragment to FcRn, and thereby change the serum half-life of the antigen binding protein.

[0237] Preferably, the substitution changes the binding affinity and / or serum half-life of the modified antibody to FcRn relative to the unmodified wild-type antibody. The present invention further provides a modified antibody having reduced binding affinity and / or shortened serum half-life to FcRn compared to the unmodified antibody, wherein any one or more amino acid residues at positions Ile253 or His310 from the CH2 domain and / or residue His435 from the CH3 domain are substituted with another amino acid different from that present in the unmodified antibody or unmodified IgG.

[0238] In one example, the 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 His310 and His435 residues.

[0239] In further embodiments, the antibody may comprise an amino acid substitution at residue Lys322. Preferably, the substitution is K322A. In particularly preferred embodiments, the antibody comprises the substitutions K322A, H310A and H435Q.

[0240] The amino acid substitution may include a substitution from a histidine residue to: alanine, glutamine, glutamic acid or aspartic acid. Preferably, the amino acid substitution at His310 is a substitution for alanine. Preferably, the amino acid substitution at His435 is a substitution for glutamine. Preferably, the amino acid substitution at Ile253 is alanine.

[0241] In another embodiment, the antigen binding protein is an antibody or antigen binding fragment thereof comprising one or more amino acid substitutions, wherein the one or more amino acid substitutions modify the binding of the antibody to an activated Fcγ receptor. The one or more amino acid modifications change the affinity of the antibody constant domain, Fc region or Fcγ receptor binding fragment to any one or more Fcγ receptors. Preferably, the amino acid modification is at a residue equivalent to Leu235. More preferably, the amino acid modification is from Leu235 to glutamic acid.

[0242] In one embodiment, the amino acid modification is a hinge stabilizing mutation at Ser 228. Preferably, the amino acid modification at Ser 228 is a substitution for proline.

[0243] In one embodiment of the present invention, the antibody comprises mutations at Ser228, Leu235, His310 and His435. Preferably, the amino acid modifications are Ser228Pro, Leu235Glu, His310Ala and His435Gln.

[0244] The amino acid modifications are preferably made in antibodies having an IgG1 isotype, or on an IgG4 isotype.

[0245] In a preferred embodiment, the antibody comprises a heavy chain constant region as shown in any one of SEQ ID NOs: 177-180.

[0246] An antigen binding protein, immunoglobulin variable domain, antibody, dab, bi-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, linear antibody, single-chain antibody molecule or multispecific antibody, fusion protein or conjugate as described herein can be obtained by expressing a nucleic acid encoding it.

[0247] In one example, such nucleic acid is included in an expression construct, wherein the nucleic acid is operably linked to a promoter. Such expression constructs may be in a vector, such as a plasmid. In examples for single polypeptide chain antigen-binding proteins, the expression construct may include a promoter connected to a nucleic acid encoding the polypeptide chain. In examples for multiple polypeptide chains forming antigen-binding proteins, the expression construct includes a nucleic acid encoding a polypeptide comprising, for example, a VH operably linked to a promoter and a nucleic acid encoding a polypeptide comprising, for example, a VL operably linked to a promoter.

[0248] In another example, the expression construct is a bicistronic expression construct, for example, comprising the following operably linked components in 5' to 3' order:

[0249] (i) Promoter

[0250] (ii) a nucleic acid encoding a first polypeptide;

[0251] (iii) an internal ribosome entry site; and

[0252] (iv) a nucleic acid encoding a second polypeptide,

[0253] wherein the first polypeptide comprises VH and the second polypeptide comprises VL, or vice versa.

[0254] The present invention also contemplates the use of separate expression constructs, one of which encodes a first polypeptide comprising VH, and the other of which encodes a second polypeptide comprising VL. For example, the present invention also provides a composition comprising:

[0255] (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and

[0256] (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

[0257] Antigen binding proteins or antibodies or antigen binding fragments thereof as described herein may comprise a human constant region, such as an IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region or a mixture thereof. Where an antibody or protein comprises a VH and a VL, the VH may be linked to a heavy chain constant region, and the VL may be linked to a light chain constant region.

[0258] In one example, a protein or antibody as described herein or a composition of a protein or antibody as described herein comprises a heavy chain constant region comprising a stabilized heavy chain constant region comprising a mixture of sequences that are fully or partially with or without a C-terminal lysine residue.

[0259] In one example, an antibody for use according to the invention comprises a VH disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as discussed above), and the VL is linked or fused to a kappa light chain constant region.

[0260] The functional properties of the antigen binding proteins for use in accordance with the present invention will be used to modify the antibodies of the present invention mutatis mutandis.

[0261] The antigen binding proteins as described herein may be purified, substantially purified, isolated and / or recombinant.

[0262] 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 the recurrence of the condition, and this is considered to prevent the condition.

[0263] Other exemplary cancers include renal cancer.It will be appreciated that antibodies of the invention having affinity for CAIX will be useful for this purpose.

[0264] The invention also includes the step of performing an in vivo method of diagnosing, monitoring or prognosing a disease, condition or infection in a subject prior to or concurrently with administering a treatment as described herein.

[0265] As used herein, the term "comprise" and variations of the term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, components, integers or steps, unless the context requires otherwise.

[0266] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0267] Figure 1 Figure 3: Survival-corrected area under the tumor growth curve for the following treatment groups of Renca hCAIX tumor-bearing mice following treatment as described in Example 3: (1) control, targeted radionuclide therapy (TRT) administered with (2) 12 MBq, (3) 18 MBq, and (4) 24 MBq— 177 Lu-hG250, (5) administration of immune checkpoint inhibitor (ICI) anti-PD-1Ab (200 μg), and (6) anti-PD-1Ab (200 μg) with 18 MBq 177 Combination of Lu-hG250. Analysis: Kruskal-Wallis test with Dunn's multiple comparison test *p<0.05, ***p<0.001. The experimental groups were excluded from the analysis. For the control group and TRT monotherapy group, N=10 mice per group, and for the ICI group and combination group, N=7.

[0268] Figure 2 : Graph of body weight changes of Renca-hCAIX tumor-bearing mice in each treatment group as described in Example 2. N=10 mice per group for the control group and TRT monotherapy group, and N=7 for the ICI group and combination group.

[0269] Figure 3 : Graph showing the reasons for euthanasia of each Renca-hCAIX tumor bearing mouse of the treatment groups as described in Example 2. N=10 mice per group were started for the control and TRT monotherapy groups, and N=7 mice were started for the ICI and combination groups.

[0270] Figure 4 : (A). Survival curves of Renca-hCAIX tumor-bearing mice treated as described in Example 2. (B). Number of animals with complete tumor regression, and the number of animals that rejected subsequent Renca cell or Renca-hCAIX cell transplants in that animal with complete tumor regression. N=10 mice per group for the control and TRT monotherapy groups, and N=7 for the ICI and combination groups.

[0271] Figure 5 : Graph of tumor volume of treatment groups as described in Example 2. N=10 mice per group for the control group and TRT monotherapy group, and N=7 for the ICI group and combination group.

[0272] Figure 6 : Graph of tumor volume of individual Renca-hCAIX tumor-bearing mice. Individual mice are shown in gray and the average is shown in black. (A) Control; (B) 12MBq 177Lu-hG250; (C) 18MBq 177Lu-hG250; (D) 24MBq 177Lu-hG250; (E) Immune checkpoint inhibitors aPD1 and aCTLA4; (E) ICI+TRT, 18MBq 177Lu-hG250+aPD1+aCTLA4 - as described in Example 2. For the control group and TRT monotherapy group, N=10 mice per group, and for the ICI group and combination group, N=7.

[0273] Figure 7 : Experimental design of low-dose combination TRT+ICI therapy (as described in Example 3).

[0274] Figure 8 : Plots of tumor volumes of treatment groups as described in Example 3. In (A), individual mice are shown in gray and the mean of the group is shown in black. In (B), the mean of tumor volumes is plotted. (C) The area under the tumor growth curve corrected for survival of mice bearing Renca hCAIX tumors after the treatments described in the legend is plotted. N=10 mice per group.

[0275] Fig. 9 : (A) Survival curves of Renca-hCAIX tumor-bearing mice treated as described in Example 3. (B) The reasons for euthanasia of mice in each treatment group are shown. (C) The number of animals with complete tumor regression and the number of animals that rejected subsequent Renca cell or Renca-hCAIX cell transplants in those animals with complete tumor regression are shown. N = 10 mice per group.

[0276] Fig.10: Experimental design of low-dose combination TRT+ICI therapy in the CT26-huCAIX model.

[0277] Fig.11 : (A) Plot of tumor volume of treatment groups in mice bearing CT26-hCAIX tumors. (A) Individual mice are shown in gray and the group average is shown in black. (B) The area under the tumor growth curve corrected for survival of mice bearing CT26-hCAIX tumors after the treatments described in the legend is plotted. N=10 mice per group.

[0278] Fig.12 : (A) Survival curves of treated groups of mice bearing CT26-hCAIX tumors. (B) Shows the percentage of animals with complete tumor regression, and the number of animals that rejected subsequent CT26 cell or CT26hCAIX cell transplants in those animals with complete tumor regression. N = 10 mice per group were used to initiate the experiment. N = 10 mice per group.

[0279] Fig.13 :177-Lu-hG250 treatment induces expression of several genes involved in anti-tumor immunity, and this may promote checkpoint inhibition. Tumor gene expression profiling of mice bearing Renca-hCAIX tumors was performed using the nanostring platform. The TRT group was treated with 177-Lu-hG250, 12MBq of radiation. Tumors were harvested 7 days later for gene expression analysis. The graph is derived from a list of genes that passed a filter with at least 1.5-fold differential expression and p-value <0.05. N=3 mice per group. Expression values ​​represent the mean minus the normalized log2 value.

[0280] Fig.14 : Schematic diagram of the experimental design used to determine tumor gene expression profiling after low-dose TRT+ICI therapy (as described in Example 5). DETAILED DESCRIPTION

[0281] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the various features mentioned or evident in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0282] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings.

[0283] Reference will now be made in detail to certain embodiments of the present invention. Although the present invention will be described in conjunction with the embodiments, it will be appreciated that it is not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to encompass all alternatives, modifications and equivalents which may be included within the scope of the present invention as defined in the claims.

[0284] The present invention relates to a combination of immune checkpoint inhibitor (ICI) therapy and targeted radionuclide therapy (TRT). Surprisingly, the inventors have found that the combination of these individual treatment methods produces improved treatment outcomes relative to either individual immune checkpoint inhibitor therapy or targeted radionuclide therapy.

[0285] The present invention relates in part to the identification of a novel method for reducing the toxicity of radioimmunoconjugates for use in radioimmunotherapy. Specifically, the method of the present invention enables the use of relatively lower doses of radioimmunoconjugates to achieve comparable, and in some cases superior, therapeutic effects.

[0286] Additionally, the present invention also relates in part to improved immune checkpoint inhibitor therapies.

[0287] As previously outlined above, radiation damage to healthy tissues and cells is a major problem associated with radioimmunotherapy. Toxicity severely limits the radiation dose of RAIT and reduces the effectiveness of tumor treatment. However, the inventors have recognized that lower doses of radionuclides can be used to achieve at least comparable (and preferably improved) results.

[0288] Overview

[0289] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter are to be considered to encompass both one and more (i.e., one or more) of these steps, compositions of matter, groups of steps, or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the context clearly indicates otherwise. For example, reference to "a" includes a single as well as two or more; reference to "a" includes a single as well as two or more; reference to "the" includes a single as well as two or more, etc.

[0290] Those skilled in the art will appreciate that the present invention is susceptible to variations and modifications, except those specifically described. It should be understood that the present invention includes all such changes and modifications. The present invention also includes all steps, features, compositions and compounds mentioned or indicated individually or collectively in this specification, as well as any and all combinations of any two or more of the steps or features.

[0291] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the present invention.The present invention is in no way limited to the methods and materials described.

[0292] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0293] The present invention is not to be limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention.

[0294] Unless expressly stated otherwise, any example or embodiment of the invention herein should be considered applicable mutatis mutandis to any other example or embodiment of the invention.

[0295] Unless specifically defined otherwise, all technical and scientific terms used herein should be interpreted as having 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).

[0296] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in 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), TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991), DM Glover and BD Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (eds.), Current Protocols in Molecular Biology: A Laboratory Manual. Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0297] The description and definition of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further illustrated by a discussion of the Kabat sequences of proteins of immunological significance, 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.

[0298] The term "and / or" (eg, "X and / or Y") should be understood to mean "X and Y" or "X or Y," and should be considered to provide clear support for both or either meanings.

[0299] As used herein, the term "derived from" shall indicate that the specified integer may be obtained from a particular source, although not necessarily directly from said source.

[0300] CAIX

[0301] As used herein, carbonic anhydrase is also known as: CA-IX, CA9, CAIX, carbonic anhydrase IX, carbonic anhydrase 9, carbonic anhydrase IX, carbonic anhydrase, G250, membrane antigen MN, P54 / 58N, pMW1, RCC-associated antigen G250, RCC-associated protein G250, and renal cell carcinoma-associated antigen G250.

[0302] Cancer cells predominantly express the plasma-membrane-associated CA isoforms CAIX and CAXII, as well as intracellular CAs such as CAI and CAII. Among cancer-associated CAs, CAIX has received the most attention, as the expression of this isoform in healthy tissues is restricted to epithelial cells of the stomach and intestine, but is strongly upregulated in renal cancer.

[0303] The expression of CAIX is controlled by hypoxia-inducible factor 1 (HIF-1), which is mainly localized in chronically hypoxic tumor areas. However, CAIX can also be found in mildly hypoxic or even normoxic areas, as its expression can be activated by components of the mitogen-activated protein kinase (MAPK) pathway.

[0304] Agents for binding to CAIX

[0305] The agent for binding to CAIX and for use according to the methods of the invention can be any compound that specifically recognizes or binds to CAIX, mediates its activity by binding to CAIX or a fragment or splice variant thereof; binds irreversibly at the entrance to the active site, and / or inhibits CAIX by coordinating with the zinc ion at the CAIX active site.

[0306] Preferably, the agent used to bind to CAIX specifically interacts with the CAIX polypeptide. Specific interaction (e.g., recognition or binding) means that the agent, such as an antibody, has a greater affinity for CAIX than other polypeptides. In one embodiment, the agent interacts with (i.e., binds to or recognizes) the CAIX polypeptide or modulates the activity of the CAIX polypeptide and / or mediates antibody-dependent cellular cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). Therefore, according to one embodiment, the agent is a CAIX inhibitor. The CAIX inhibitor may act at the protein level or at the nucleic acid level. Examples of CAIX inhibitors that act at the protein level, for example, include, but are not limited to, peptides and anti-CAIX antibodies and functional fragments of those antibodies or small organic molecules, preferably with a molecular weight of less than 500 g / mol.

[0307] Examples of anti-CAIX antibodies or antibodies for binding to CAIX are described in EP 637 336, WO 93 / 18152, WO 95 / 34650, WO 00 / 24913, WO 02 / 063010, WO 04 / 025302, WO 05 / 037083, WO 2011 / 139375, US 11,629,199, Murri-Plesko et al., Eur J Pharmacol 201 1, 657: 173-183.

[0308] Examples of small organic molecules for binding to CAIX include, but are not limited to, sulfonamides, heteroaromatic sulfonamides, sulfamates, coumarins and thiocoumarins, and BAY-79-4620. Examples of inhibitors that act at the nucleic acid level are siRNA molecules, ribozymes, and / or antisense molecules.

[0309] As used herein, the term "specifically binds" or "binds specifically" should be taken 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 period of time, and / or with greater affinity than with alternative antigens or cells. For example, the affinity of an antigen binding protein that binds to CAIX is substantially greater (e.g., 1.5-fold, or 2-fold, or 5-fold, or 10-fold, or 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 150-fold or 200-fold) than its affinity for binding to other antigens.

[0310] Methods for assessing binding to proteins (e.g., CAIX) are known in the art, for example, as described in Scopes (Protein purification: principles and practice, 3rd edition, Springer Verlag, 1994). Such methods typically involve fixing an agent (e.g., an antibody) and contacting it with a labeled target (in the case of an antibody, an antigen). After washing to remove non-specifically bound proteins, the amount of the label is detected, and thus the bound antigen is detected. Of course, the antigen binding site can be labeled and the antigen fixed. A panning type assay can also be used. Alternatively or additionally, a surface plasmon resonance assay can be used.

[0311] Other standard methods for assessing binding to targets such as CAIX are also known in the art.

[0312] In any embodiment, the agent used to bind to CAIX is the small molecule SLC-0111 (CAS 178606-66-1), SLC-149 (as described in EP 3317255 B1, which is incorporated herein by reference), SLC-0121 or SLC-101.

[0313] In any embodiment, the agent used to bind to CAIX is the small molecule / contrast dye PMI-05 (as described in US2019 / 0192699A1, which is incorporated herein by reference).

[0314] In any embodiment, the agent used to bind to CAIX is a small molecule sulfonamide-nitroimidazole (as described in Rami et al., (2013), J. Med. Chem, 56:8512-8520, which is incorporated herein by reference).

[0315] In any embodiment, the agent used to bind to CAIX is the small molecule JS-403 (as described in WO 2010 / 089752 A1, which is incorporated herein by reference).

[0316] In any embodiment, the agent for binding to CAIX is the small molecule UB-TT220 (as described in WO 2022 / 015955A1, which is incorporated herein by reference).

[0317] In any embodiment, the agent used to bind to CAIX is a small molecule 99m Tc-HEHEHE-Z09781 (Kim et al. (2017) Advanced Science, 4:1600471; Gebauer and Skerra (2009) Current Opin in Chem Biol, 13(3):245-55; Schardt et al. (2017) Mol Pharmaceutics, 14(4):1047-56; Tolmachev et al. (2008) Bioconjugate Chem, 19(8):1579-87; Liu et al. (2022) Analytical and Bioanalytical Chemistry, 414:1095-1104; Grindel et al. (2022) ACS Chem Biol, 414:1095-1104). Biol, 17(6):1543-55, which is incorporated herein by reference); 99m Tc-MIP-1486, 99m Tc-MIP-1490 (4-(2-bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide, wherein X = ethyl) or 99m Tc-MIP-1504 (4-(2-bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino-X)benzenesulfonamide, where X = n-butoxy) (Hillier et al., (2012) Journal of Nuclear Medicine, 53(s1):217, incorporated herein by reference)

[0318] In any embodiment, the agent for binding to CAIX is the small molecule PHC-102 (as described in WO 2015 / 114171 A1; WO 2018154517 A1; US2014 / 0357650 A1; WO 2015 / 114171 A1, which are incorporated herein by reference)

[0319] In any embodiment, the agent for combining with CAIX is a peptide. As used herein, a peptide will be understood as a chain comprising more than 1 amino acid residue. Typically, a peptide can 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. The peptide can have a length of at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 amino acids. Typically, a peptide is no longer than about 40 amino acids, such as no longer than about 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids.

[0320] In any embodiment, the agent used to bind to CAIX is peptide 3B-301 (also known as Debio 0228; Queen et al., (2018) Int J of Biol Macromol, 106:840-850; Eldehna et al., (2019) Bioorganic Chem, 90:103102; Lavecchia et al., (2011) Carbohydrate Res, 346(3):442-48; Krymov et al., (2022) Eur J of Medicinal Chem, 228:113997; Supuran (2008) BJI Int, 101(s4):39-40; Koyuncu et al., (2019) J of Enzyme Inhibition and Medicinal Chem, 101(s4):39-40). Enzyme Inhibition and Medicinal Chem), 34(1):703-11; Kumar et al., (2017) European Journal of Medicinal Chemistry, 136:52-62, the references of which are incorporated herein by reference) or 3B-302.

[0321] In any embodiment, the agent for binding to CAIX is the peptide CAIX-P1 having the amino acid sequence YNTNHVPLSPKY (as described in Askoxylakis et al., (2010), PLoS One, 5(12):e15962), optionally wherein the peptide is labeled with 125I or 131I to enable its detection (although it will be appreciated that any suitable radiolabel or other detectable moiety may be used). As used herein, the term 'theranostic' refers to compounds / materials that are capable of diagnosis as well as therapy. The term "theranostic agent" relates to any agent that is suitable for detecting, diagnosing and / or treating a disease or condition in a patient. The purpose of a therapeutic diagnostic compound / material is to overcome undesirable differences in biodistribution and selectivity that may exist between different diagnostic and therapeutic agents. Using a therapeutic diagnostic pairing, a therapeutic diagnostic compound containing an imaging radionuclide is first administered to a patient to identify a disease or locate an affected area in the body. Once identified / localized, the disease can be treated by administering a theranostic compound containing the therapeutic radionuclide in a target-specific manner, since the biodistribution of the imaging and therapeutic radionuclides is identical.

[0322] Antibodies for binding to CAIX

[0323] 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. The fragment of the anti-CAIX antibody may have substantially the same CAIX binding and / or inhibitory activity as the full-length anti-CAIX antibody, and / or be an epitope-binding fragment of the anti-CAIX antibody.

[0324] 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.

[0325] The antibody and / or its antibody fragment can be selected from the group consisting of: polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2, Fab', scFv, dsFv and chimeric, humanized and fully human variants thereof. The antibody can be multivalent, or multivalent and multispecific. The antibody can include the human constant region of IgG1, IgG2a, IgG3 or IgG4.

[0326] According to a further preferred embodiment, the anti-CAIX antibody or epitope-binding fragment thereof for use according to the present invention binds to the amino acid sequence LSTAFARV and / or ALGPGREYRAL.

[0327] In any embodiment, the agent for binding to CAIX is in the form of the antibody BAY-794620 or an antigen-binding fragment thereof (as described in WO 2003 / 100029 A2; WO 2003 / 033674 A2; Theiner et al., (2021) Tierarztl Prax Ausg G Grosstiere Nutztiere, 49(6):392-402; Kimani et al., (2011) Photochemistry and Photobiology, 88(1):175-87; NCT01065623 (v24, September 30, 2014); NCT01028755 (v30, January 19, 2015), which are incorporated herein by reference).

[0328] In any embodiment, the agent for binding to CAIX is in the form of the antibody SLC-0131 or an antigen-binding fragment thereof.

[0329] In any embodiment, the agent for binding to CAIX is in the form of an antibody or antigen-binding fragment thereof, as described in US 1 1,629,199, which is incorporated herein by reference.

[0330] According to another particularly preferred embodiment, the agent for binding to CAIX is an anti-G250 antibody and / or an antigen-binding fragment thereof. Anti-G250 antibodies are described, for example, in EP-B-0 637 336. The antibody or fragment thereof may be a chimeric or humanized G250 antibody. In some embodiments, the antigen binding protein that binds or specifically binds to CAIX is as described in any of WO 2002 / 062972A2 (US2004 / 0219633 A1), WO 2004 / 002526 A1 (US 7,632,496 B2), WO 2006 / 002889A2 (US 7,691,375 B2), WO 2009 / 056342 A1 (US2014 / 0017252 A1), WO 2011 / 032973A1 (US2012 / 0207672 A1) and WO 2014 / 128258 A1 (US10,620,208 B2), or WO 2021 / 000017A1, the entire contents of each of these publications are incorporated herein by reference.

[0331] Antibodies for use in the present invention may be produced by any suitable method known in the art, including but not limited to the methods described in PCT / EP02 / 01282 and PCT / EP02 / 01283, which are incorporated herein by reference.

[0332] A particularly preferred antibody is cG250, preferably girentuximab (INN). Another particularly preferred embodiment is the monoclonal antibody G250 produced by the hybridoma cell line DSMACC 2526. Antibody cG250 is an IgG1 kappa light chain chimeric version of the original murine monoclonal antibody mG250.

[0333] Variants of the original chimeric G250 (cG250) antibody, including WX-G250 and WX-G250RIT (131 Iodine) (Janssen Global Services LLC) are known.

[0334] In particularly preferred embodiments, the antibody is 89 Zr-gilenulomab (i.e. 89 Zr-cG250), 123 I-, 124 I-or 131 I- Girentuximab or 177 Lu-girentuximab.

[0335] In the context of the present invention, antibodies for use according to the invention are 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 the diagnostic radioisotope to the site of the tumor to facilitate diagnosis (including tumor distribution, tumor size, tumor density), while the same antigen binding domain of the antibody directs the radioisotope to the tumor for therapy.

[0336] The term "Fc region", sometimes referred to as "Fc" or "Fc domain" as used herein, refers to the portion of an IgG molecule associated with a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region consists of the C-termini of half of the two heavy chains of the IgG molecule connected by a disulfide bond. The Fc region does not have antigen binding activity but contains a carbohydrate portion and binding sites for complementarity and Fc receptors, including FcRn receptors. The Fc region contains a complete second constant domain CH2 (residues 231-340 of human IgG1 according to the EU index numbering system, also defined as residues 244-360 in the Kabat system) and a third constant domain CH3 (residues 341-447EU index / 361-478Kabat) (see, for example, SEQ ID NO: 1 of WO 2015175874 or for the sequence of CH2 and SEQ ID NO: 2 see Figure 1 C; for the sequence of CH3 see Figure 1 D, which is incorporated herein by reference; see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs for a comparison of numbering conventions for various residues in the Fc region of immunoglobulins).

[0337] As used herein, the "EU index" or "EU numbering scheme" refers to the EU numbering for 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, the "Kabat system" refers to the Kabat sequences for proteins of immunological significance, National Institutes of Health, Bethesda, Maryland, 1987 and 1991. A skilled artisan will be able to readily determine whether a given amino acid sequence is numbered according to the EU or Kabat system.

[0338] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or source of derivation, is not associated with naturally associated components with which it is naturally associated; and is substantially free of other proteins from the same source. Proteins can be rendered substantially free of naturally associated components or substantially purified by isolation using protein purification techniques known in the art. "Substantially purified" means that the protein is substantially free of contaminants, for example, 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.

[0339] The term "recombinant" should be understood to mean the product of artificial gene recombination. Therefore, in the context of a recombinant protein comprising an antibody antigen-binding domain, the term does not encompass naturally occurring antibodies in a subject, which are the product of natural recombination that occurs during B cell maturation. However, if such an antibody is separated, it is considered to be a separated protein comprising an antibody antigen-binding domain. Similarly, if a nucleic acid encoding a protein is separated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen-binding domain. Recombinant protein also encompasses proteins expressed by artificial recombinant means when it is in a cell, tissue, or subject, such as proteins expressed therein.

[0340] The term "protein" should be considered to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently linked to each other (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using suitable chemicals or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Vander Waals forces, and hydrophobic interactions.

[0341] The term "polypeptide" or "polypeptide chain" will be understood from the preceding paragraphs to mean a series of consecutive amino acids linked by peptide bonds.

[0342] As used herein, the term "antigen binding site" can be used interchangeably with "antigen binding domain" and should be taken to mean a region of an antibody that is capable of specific binding to an antigen, i.e., a VH or VL or an Fv comprising both VH and VL. An antigen binding domain need not be in the context of an intact antibody, e.g., it can be in isolated form (e.g., a domain antibody) or in another form, e.g., as described herein, such as an scFv.

[0343] For the purposes of this disclosure, the term "antibody" includes proteins that can specifically bind to one or more closely related antigens through an antigen-binding domain included in an Fv. The term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synthetic humanized antibodies, half antibodies, bispecific antibodies). Antibodies generally include constant domains, which can be arranged into constant regions or constant fragments or crystallizable fragments (Fc). An exemplary form of an antibody includes a four-chain structure as its basic unit. A full-length antibody includes two covalently linked heavy chains (about 50 to 70 kD) and two light chains (about 23 kDa each). A light chain generally includes a variable region (if present) and a constant domain, and is a kappa light chain or a lambda light chain in mammals. A heavy chain generally includes a variable region and one or two constant domains connected to another constant domain by a hinge region. The heavy chain of mammals is one of the following types: α, δ, ε, γ or μ. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains and heavy and light chains are held together by interchain disulfide bonds and by non-covalent interactions. In different types of antibodies, the number of interchain disulfide bonds can be different. Each chain has an N-terminal variable region (VH or VL, each of which is about 110 amino acids in length) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL with a length of about 110 amino acids) is aligned and disulfide bonded with the first constant domain of the heavy chain (CH1 with a length of 330 to 440 amino acids). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain may include 2 or more additional CH domains (such as CH2, CH3, etc.), and may include a hinge region between the CH1 and CH2 constant domains. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), species (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (e.g., human) antibody. In one example, the antibody heavy chain has lost the C-terminal lysine residue. In one example, the antibody is humanized, synthetically humanized, chimeric, CDR-grafted, or deimmunized.

[0344] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in substantially complete form, rather than an antigen-binding fragment of an antibody. Specifically, a complete antibody includes an antibody with heavy and light chains having a ratio Fc region. The constant domain may be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0345] As used herein, "variable region" refers to the part of the light chain and / or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen, and includes the amino acid sequence of the complementary determining region (CDR); i.e., CDR1, CDR2 and CDR3, and framework region (FR). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) and three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0346] As used herein, the term "complementarity determining region" (synonymous CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the variable region of an antibody, the presence of which is a major factor in specific antigen binding. Each variable region domain (VH or VL) generally has three CDRs identified as CDR1, CDR2, and CDR3. The CDRs of VH are also referred to herein as CDR H1, CDR H2, and CDR H3, respectively, wherein CDR H1 corresponds to CDR 1 of VH, CDR H2 corresponds to CDR 2 of VH, and CDR H3 corresponds to CDR 3 of VH. Similarly, the CDRs of VL are referred to herein as CDR L1, CDR L2, and CDR L3, respectively, wherein CDR L1 corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL, and CDR L3 corresponds to CDR 3 of VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to the Kabat sequences of proteins of immunological significance, National Institutes of Health, Bethesda, Maryland, 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 the canonical numbering system or the numbering systems in the following references: Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989; and / or the numbering systems of Al-Lazikani et al., J. Mol. Biol. 273:927-948, 1997; Honnegher and Plükthun, 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 five C-terminal amino acids listed herein, or any one or more of these amino acids are substituted by another naturally occurring amino acid. In this regard, Padlan et al., Journal of the Federation of American Societies for Experimental Biology (FASEB J.), 9: 133-139, 1995 confirmed that the five C-terminal amino acids of the heavy chain CDR2 are generally not involved in antigen binding.

[0347] "Framework regions" (FRs) are those variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, wherein FR H1 corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FRH3 corresponds to FR 3 of VH, and FR H4 corresponds to CDR 4 of VH. Similarly, the FRs of VL are referred to herein as FRL1, FR L2, FR L3, and FR L4, respectively, wherein FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL, and FR L4 corresponds to CDR 4 of VL.

[0348] As used herein, the term "Fv" should be deemed to mean any protein, whether it is composed of multiple polypeptides or a single polypeptide, in which VL and VH associate and form a complex with an antigen binding domain, that is, capable of specific binding to an antigen. The VH and VL that form the antigen binding domain may be in a single polypeptide chain, or may be in different polypeptide chains. In addition, 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. The term should be understood to cover fragments directly derived from antibodies and proteins corresponding to such fragments produced using recombinant means. In some instances, VH is not connected to the heavy chain constant domain (CH) 1, and / or VL is not connected to the light chain constant domain (CL). Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies or higher-order complexes, or any of the aforementioned substances connected to their constant regions or domains, such as CH2 or CH3 domains, such as mini antibodies. "Fab fragments" consist of monovalent antigen-binding fragments of immunoglobulins and can be produced by digesting whole antibodies with papain to produce fragments consisting of a complete light chain and a portion of a heavy chain, or can be produced using recombinant means. "Fab' fragments" of antibodies can be obtained by treating whole antibodies with pepsin and then reducing to produce molecules consisting of a complete light chain and a portion of a heavy chain containing VH and a single constant domain. Two Fab' fragments are obtained by treating each antibody in this way. Fab' fragments can also be produced by recombinant means. "F(ab')2 fragments" of antibodies consist of dimers of two Fab' fragments held together by two disulfide bonds and are obtained by treating complete antibody molecules with pepsin without subsequent reduction. "Fab2" fragments are recombinant fragments comprising two Fab fragments connected using, for example, leucine zippers or CH3 domains. "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 flexible polypeptide linker.

[0349] As used herein, when referring to the interaction of an antigen binding protein or its antigen binding domain with an antigen, the term "binding" means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes a specific protein structure rather than a general protein and binds to it. If an antibody binds to an epitope "A", then in a reaction containing a labeled "A" and an antibody, the presence of a molecule containing an epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0350] As used herein, the term "specifically binds" or "binds specifically" should be taken to mean that an antigen binding protein for use in accordance with the present invention reacts or associates with a particular antigen or cell expressing the antigen more frequently, more rapidly, for a longer period of time, and / or with greater affinity than with alternative antigens or cells. For example, the affinity of an antigen binding protein that binds to CAIX is substantially greater (e.g., 1.5-fold, or 2-fold, or 5-fold, or 10-fold, or 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 150-fold or 200-fold) than its affinity for binding to other antigens.

[0351] As used herein, the term "epitope" (synonymous with "antigenic determinant") should be understood to mean the region of a cell surface protein (such as CAIX) to which an antigen binding protein comprising an antigen binding domain of an antibody binds.

[0352] As used herein, the term "condition" refers to a disruption or interference with normal function, and is not limited to any particular condition, and would include diseases or disorders.

[0353] As used herein, the terms "preventing," "prevent," or "prevention" include administering an active species, such as an antigen binding protein or a checkpoint inhibitor as described herein, to stop or hinder the development of at least one symptom of a condition. The term also encompasses treatment of subjects in remission to prevent or stop relapse.

[0354] As used herein, the terms "treating," "treat," or "treatment" include the administration of an active species, 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.

[0355] As used herein, the term "subject" should be taken to mean any animal, such as a mammal, including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0356] Modified Antibodies

[0357] The present invention relates in part to the use of antibodies comprising modifications to an IgG antibody, said modifications comprising one or more amino acid substitutions to regions of the antibody which reduce or eliminate the affinity of the antibody for FcRn, thereby shortening the serum half-life of the antibody.

[0358] It should be understood that according to the present invention, any antibody for which a shortened serum half-life is desired can be modified according to the methods described herein.

[0359] In certain embodiments, antibodies suitable for modification according to the present invention to reduce affinity for FcRn are antibodies having one or more of the sequences shown in Table 1.

[0360] The present invention also provides the use of antigen binding proteins with at least 80% identity to the sequences disclosed herein. In one example, the antigen binding proteins for use according to the present invention comprise sequences disclosed herein 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.

[0361] Alternatively or additionally, according to any embodiment, the antigen binding protein comprises a V H or V L In some embodiments, the present invention relates to CDRs of the present invention that are 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 CDRs (e.g., three CDRs).

[0362] The present invention also contemplates mutant forms of the antigen-binding proteins used according to the invention, which mutant forms comprise one or more conservative amino acid substitutions compared to the sequences shown herein. In some instances, the antigen-binding proteins comprise 10 or less, such as 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2 or 1 conservative amino acid substitutions. "Conservative amino acid substitutions" are substitutions in which amino acid residues are substituted with amino acid residues having similar side chains and / or hydrophilicity and / or hydrophilicity.

[0363] Families of amino acid residues having similar side chains have been defined in the art, including 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indices are described, for example, in Kyte and Doolittle, Journal of Molecular Biology, 157: 105-132, 1982, and hydropathic indices are described, for example, in U.S. Pat. No. 4,554,101.

[0364] In some embodiments, the antigen binding protein comprises non-conservative amino acid changes. For example, particular attention is paid to replacing a charged amino acid with another charged amino acid and a neutral or positively charged amino acid. In some instances, the antigen binding protein comprises 10 or less, such as 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2 or 1 non-conservative amino acid substitutions.

[0365] In one example, the mutation occurs in the FR of the antigen binding domain of the antigen binding protein. In another example, the mutation occurs in the CDR of the antigen binding protein.

[0366] Exemplary methods for generating mutant forms of antigen binding proteins include:

[0367] Mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525:309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al., BMC Biotechnology, 7:18, 2007; and WO 1999 / 058661):

[0368] Introducing a nucleic acid encoding a polypeptide into mutant cells, such as XL-1 Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);

[0369] DNA shuffling, e.g., as disclosed in Stemmer, Nature 370:389-91, 1994; and

[0370] - Site-directed mutagenesis, e.g., as described in Dieffenbach (eds.) and Dveksler (eds.) (PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1995).

[0371] Exemplary methods for determining the biological activity of antigen binding proteins for use in accordance with the invention will be apparent to the skilled artisan and / or described herein, such as antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein.

[0372] Constant region

[0373] The present invention also provides the use of antigen binding proteins and / or antibodies described herein comprising the constant region of an antibody. This includes antigen binding fragments of antibodies fused to Fc.

[0374] The sequences of the constant regions that can be used to produce proteins of the present invention can be obtained from many different sources. In some instances, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgG1, IgG2, IgG3 and IgG4. In one example, the constant region is a human isotype IgG4 or a stable IgG4 constant region.

[0375] Preferred modifications

[0376] The present invention also contemplates modifications to antibodies or antigen binding proteins for use according to the present invention which comprise an Fc region or a constant region.

[0377] Neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG class antibodies in vivo. The function of FcRn is to salvage IgG from the lysosomal degradation pathway, so that the clearance rate is reduced and the half-life is extended. It is a heterodimeric protein composed of two polypeptides: 50kDa I class major histocompatibility complex-like protein (a-FcRn) and 15kDa p2-microglobulin (β2ηι). FcRn is combined with the CH2-CH3 part of the Fc region of IgG class antibodies with high affinity. The interaction between IgG class antibodies and FcRn is pH dependent, and occurs with a stoichiometry of 1:2, i.e., an IgG antibody molecule can interact with two FcRn molecules through its two heavy chain Fc region polypeptides (see, e.g., Huber, AH et al., Journal of Molecular Biology 230 (1993) 1077-1083).

[0378] Therefore, the in vitro FcRn binding properties / characteristics of IgG are indicative of its in vivo pharmacokinetic properties in blood circulation.In the interaction between FcRn and the Fc region of antibodies of the IgG class, different amino acid residues of the heavy chain CH2- and CH3-domains are involved.

[0379] Different mutations that affect the half-life of FcRn binding and blood circulation are known. Fc region residues that are critical to mouse Fc region-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF et al., Journal of Immunology (J.Immunol) 169 (2002) 5171-5180). Residues Ile253, His310, His433, Asn434, and His435 (numbered according to the EU index numbering system) are involved in the interaction (Medesan, C et al., European Journal of Immunology (Eur.J.Immunol.) 26 (1996) 2533-2536; Firan, M. et al., International Immunology (Int.Immunol.) 13 (2001) 993-1002; Kim, JK et al., European Journal of Immunology 24 (1994) 542-548). (Using the Kabat system, the relevant residues are Ile266, His329, His464, Asn465 and His466.) Residues Ile253, His310 and His435 were found to be critical for the interaction of the human Fc region with murine FcRn (Kim, JK et al., Eur. J. Immunol. 29 (1999) 2819-2885).

[0380] More specifically, the antibody may comprise one or more amino acid substitutions that shorten the half-life of the protein. For example, the antibody comprises an Fc region comprising one or more amino acid substitutions that reduce the affinity of the Fc region to the neonatal Fc region (FcRn).

[0381] The present invention also provides the use of an antibody having a constant region substantially identical to a naturally occurring IgG class antibody constant region, wherein at least one amino acid residue selected from the group consisting of residues His310, His435 and Ile253 is different from an amino acid residue in a naturally occurring IgG class antibody, thereby altering the FcRn binding affinity and / or serum half-life of the antibody relative to the naturally occurring antibody. In a preferred embodiment, the naturally occurring IgG class antibody comprises a heavy chain constant region of a human IgG1, IgG2, IgG2M3, IgG3 or IgG4 molecule.

[0382] Also in a preferred embodiment, amino acid residue 310 or residue 435 of the heavy chain constant region of an antibody having a constant region substantially identical to a naturally occurring IgG class antibody is any amino acid that is not histidine and 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.

[0383] Preferably, the residue at position 310 is selected from alanine, 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 IgG class antibody has an alanine residue at position 310 and a glutamine residue at position 435.

[0384] In preferred embodiments of the invention, the binding affinity and / or serum half-life of the modified antibody to FcRn is reduced by at least about 30%, 50%, 80%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold. In preferred embodiments of the invention, the binding affinity and / or serum half-life of the modified antibody to FcRn is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99%.

[0385] Additionally, antibodies for use in accordance with the present invention may contain one or more mutations that alter the affinity of the antibody for any one or more Fc gamma receptors.

[0386] In some embodiments, the Fc region of the constant region retains the ability to induce effector function. In one example, the Fc region of the constant region contains one or more amino acid substitutions that modulate effector function, including increasing effector function compared to wild-type IgG.

[0387] In one example, the ability of the Fc region of the constant region to induce effector functions is reduced, for example, compared to native or wild-type human IgG1 or IgG3 Fc regions. In one example, the 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 proteins containing Fc regions are known in the art and / or described herein.

[0388] In one example, the amino acid replacement that changes the ability of antibody inducing effector function is the amino acid replacement from the residue Ile253 of heavy chain constant region. In one example, replacement is to be selected from any amino acid of alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine or glycine replacement, wherein replacement reduces the ability of antibody inducing effector function. In a preferred embodiment, replacement from the Ile at residue 253 is to arginine, proline or aspartic acid, more preferably alanine replacement.

[0389] In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4 constant region), such as a human IgG4 Fc region. The sequence of a suitable IgG4 Fc region will be apparent to the skilled person and / or available from public databases (e.g., available from the National Center for Biotechnology Information).

[0390] In one example, constant region is a stable IgG4 constant region. The term "stable IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce the exchange of Fab arms or experience the exchange of Fab arms or the tendency to form half antibodies or the tendency to form half antibodies."Fab arm exchange" refers to a type of protein modification for human IgG4, in which the IgG4 heavy chain and the attached light chain (half molecule) are exchanged by the heavy-light chain pair from another IgG4 molecule. Therefore, IgG4 molecules can obtain two different Fab arms (producing bispecific molecules) that recognize two different antigens. Fab arm exchange occurs naturally in vivo, and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. When the IgG4 antibody dissociates to form two molecules, each molecule contains a heavy chain and a light chain, a "half antibody" is formed.

[0391] In one example, according to Kabat's system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC United States Department of Health and Human Services, 1987 and / or 1991), a stable IgG4 constant region comprises a proline at position 241 of the hinge region. According to the EU numbering system, this position corresponds to position 228 of the hinge region. In human IgG4, this residue is usually serine. After serine replaces proline, the IgG4 hinge region comprises the sequence CPPC. In this regard, the technician will appreciate that the "hinge region" is a proline-rich portion of the antibody heavy chain constant region connecting the Fc and Fab regions, which imparts mobility to the two Fab arms of the antibody. The hinge region includes cysteine ​​residues involved in the inter-heavy chain disulfide bond. According to Kabat's numbering system, it is generally defined as extending from Glu226 of human IgG1 to Pro243 (or from Glu216 to Pro230 using the EU index). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues forming inter-heavy chain disulfide bonds (SS) in the same position (see, eg, WO 2010 / 080538).

[0392] Another example of a stable IgG4 antibody is an antibody in which the arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) is substituted with lysine, threonine, methionine or leucine (e.g., as described in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively comprise a residue selected from the group consisting of: alanine, valine, glycine, isoleucine and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e., a CPPC sequence) (as described above).

[0393] In another example, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region". For example, the Fc region is an IgG1 Fc region comprising substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region comprising one or more of the following changes E233P, L234V, L235A, and deletion of G236 and / or one or more of the following changes A327G, A330S, and P331S (Armour et al., Eur. J. Immunol. 29:2613-2624, 1999; Shields et al., J. Biol. Chem. 276(9):6591-604, 2001). Other 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.

[0394] In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one C from an IgG4 antibody. H 2 domains and at least one C from an IgG1 antibody H 3 domains, wherein the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S and 427F.

[0395] Antibody production

[0396] Preferably, the antigen binding protein described herein according to any example is recombinant.

[0397] 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 a host cell (such as an E. coli cell, a yeast cell, an insect cell, or a mammalian cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a myeloma cell) that does not otherwise produce the protein. Exemplary cells for expressing proteins 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., (eds.), Contemporary Molecular Biology Experimental Guide, Green Publishing Associates and Wiley International Scientific Publishing Company (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). Various cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see for example US4816567 or US5530101.

[0398] After isolation, the nucleic acid is inserted into, operably linked to, a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or expression in a cell-free system or cell.

[0399] As used herein, the term "promoter" is understood in its broadest context and includes transcriptional regulatory sequences of genomic genes, including TATA boxes or initiation elements, which are necessary for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers and silencers) that alter nucleic acid expression, for example, in response to development and / or external stimuli, or in a tissue-specific manner. In this context, the term "promoter" is also used to describe recombinant, synthetic or fusion nucleic acids, or derivatives that confer, activate or enhance expression of nucleic acids operably connected thereto. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter spatial expression and / or temporal expression of the nucleic acid.

[0400] As used herein, the term "operably linked" means that a promoter is positioned relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0401] Many vectors are available for expression in cells. Vector components typically include, but are not limited to, one or more of the following: 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. The skilled artisan will know suitable sequences 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, alpha factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0402] Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-alpha promoter (EF1), small nuclear RNA promoters (U1a and U1b), alpha-myosin heavy chain promoter, simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, beta-actin promoter; hybrid regulatory elements comprising CMV enhancer / beta-actin promoter or immunoglobulin promoter or active fragments thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0403] Typical promoters suitable for expression in yeast cells, e.g., a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, ADH1 promoter, GAL1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter or TEF1 promoter.

[0404] For separating nucleic acid or the expression construct comprising nucleic acid is introduced into the cell for the means of expression is known to those skilled in the art. The technology for given cells depends on known successful technology. For the means of recombinant DNA introduced into the cell include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposome, such as by using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), DNA uptake, electroporation and microparticle bombardment mediated by PEG, such as by using tungsten or gold particles (Agracetus Inc., WI, USA) etc. of DNA coating.

[0405] Depending on the cell type used, host cells used to produce proteins can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing mammalian cells. Culture media for culturing other cell types discussed herein are known in the art.

[0406] Protein separation

[0407] Methods for isolating proteins are known in the art and / or described herein.

[0408] When the antigen-binding protein is secreted into the culture medium, the supernatant from such expression system can first be concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the preceding steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of exogenous contaminants. Alternatively or additionally, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0409] Antigen binding proteins prepared from cells can be purified using, for example, 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 thereof. These methods are known in the art and are described in, for example, WO 99 / 57134 or Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0410] The skilled artisan will also appreciate that the protein can be modified to include a tag that facilitates purification or detection, such as a polyhistidine tag, such as a hexa-histidine 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 hexa-his tag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively or in addition, a ligand or antibody that binds to the tag is used in an affinity purification method.

[0411] Radioisotope attachment to antibodies

[0412] In any embodiment of the invention, the antibodies described herein may be linked directly or indirectly to a therapeutic agent, preferably, the therapeutic agent is a radioactive isotope.

[0413] As used herein, the terms "radioisotope" and "radionuclide" are used interchangeably.

[0414] 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).

[0415] In some embodiments, the antigen binding proteins are conjugated to radioisotopes that enable both therapy and diagnosis, which can be used to treat cancer in a subject and can also assess the spread of cancer by binding to CAIX or the size of a treated CAIX-expressing tumor. The skilled artisan will be familiar with which therapeutic radioisotopes can also be used for imaging and which imaging techniques can be used.

[0416] It will be appreciated that the isotope may be conjugated to the antibodies described herein directly (via a chelator or prosthetic group or linker) or indirectly via binding to single or multiple amino acid residues in the antibody (eg, halogenation of tyrosine residues).

[0417] In alternative embodiments, chelators or linkers may be used to conjugate the radioisotope to the antibody. In one example, the antibody can be conjugated to a chelating moiety selected from the group consisting of TMT (6,6"-bis(N,N",N"'-tetrakis(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N" (N"'-tetraacetic acid, also known as tetraxetan), TCMC (tetraprimary amine of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tri(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 (deferoxamine), DFOsq (DFO-squaramide) and HOPO (3,4,3-(LI-1,2-HOPO)), such as WO 2022 / 133537 (incorporated herein by reference), or other chelating agents as described herein.

[0418] Chelators with radioactive metals and other halogenated radioisotopes may be bound 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.

[0419] In another example, the modified antibody is conjugated to a bifunctional linker, such as bromoacetyl, thiol, succinimidyl ester, TFP ester, maleimide, or using any amine or thiol modification chemistry known in the art.

[0420] The skilled person will be familiar with standard methods for conjugating chelators to antibodies and derivatives or fragments thereof. In addition, the skilled person will be familiar with methods for selecting relevant chelators for pairing with radioactive metals, such as described in Chem. Soc. Rev., 2014, 43, 260, which is incorporated herein by reference.

[0421] Determining the activity of antigen binding proteins

[0422] Combination with CAIX

[0423] From the disclosure herein, it will be clear to the skilled person that the preferred antigen-binding proteins for use according to the present invention bind to CAIX. Methods for evaluating binding to proteins are known in the art, for example, as described in Scopes (Protein Purification: Principles and Practice, 3rd edition, Springer, 1994). Such methods generally involve fixing the antigen-binding protein and contacting it with a labeled antigen. After washing to remove non-specifically bound proteins, the amount of labeling is detected, and thus the bound antigen is detected. Of course, the antigen-binding protein can be labeled and the antigen fixed. A panning type assay can also be used. Alternatively or additionally, a surface plasmon resonance assay can be used.

[0424] Treatment

[0425] Antibodies for use according to the present invention can be used to treat a number of conditions that require treatment by radioimmunotherapy. Typically, such conditions include cancer.

[0426] Exemplary cancers include cystic and solid tumors, bone and soft tissue tumors, including anal tissue, bile duct, bladder, blood cells, intestine, 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 in tumors. Soft tissue tumors include benign schwannoma monomers, desmoid tumors, lipoblastomas, lipomas, uterine leiomyoma, clear cell sarcoma, cutaneous fibrosarcoma, Ewing sarcoma, extraosseous myxochondrosarcoma, liposarcoma myxo, alveolar rhabdomyosarcoma and synovial sarcoma. Specific bone tumors include nonossifying fibroma, unilocular bone cyst, enchondroma, aneurysmal bone cyst, osteoblastoma, chondroblastoma, chondromyxofibroma, ossifying fibroma and ameloblastoma, giant cell tumor, fibrous dysplasia, eosinophilic granuloma of Ewing's sarcoma, osteosarcoma, enchondroma, chondrosarcoma, malignant fibrous histiocytoma, and metastatic carcinoma. Leukemias include acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia.

[0427] Other examples include breast tumors, colorectal tumors, adenocarcinomas, mesotheliomas, bladder tumors, prostate tumors, germ cell tumors, hepato / cholangiocarcinoma, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, non-seminoma, stromal stromal 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 Wilms' tumor.

[0428] In some embodiments, the cancer is a metastatic cancer. The primary source of the metastatic cancer can be any cancer type known in the art, including those described herein.

[0429] Preferably, the antigen binding proteins for use according to the present invention can be used to treat cancers characterized by the presence of CAIX. For example, antibodies that bind to CAIX can be used to treat cancers characterized by increased expression of CAIX, including renal cell carcinoma.

[0430] Proteins containing antibody binding domains

[0431] Single domain antibodies

[0432] In some examples, the antigen binding protein for use according to the present invention is or comprises a single domain antibody (which is used interchangeably with the term "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain that comprises all or a portion of the heavy chain variable region of an antibody. In certain examples, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US6248516).

[0433] Bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies

[0434] In some examples, the protein for use according to the present invention is or comprises a bifunctional antibody, a trifunctional antibody, a tetrafunctional antibody or a higher order protein complex, such as those described in WO98 / 044001 and / or WO94 / 007921.

[0435] For example, a bifunctional antibody is a protein comprising two associated polypeptide chains, each of which comprises structure V L -XV H or V H -XV L , where V L is the variable region of the antibody light chain, V H is the variable region of the antibody heavy chain, X is insufficient to allow V H and V L The linker of the residues associated (or forming Fv) or not present, and the V of one polypeptide chain H V L Combine to form an antigen binding domain, that is, to form an Fv molecule that can specifically bind to one or more antigens. L and V H Can be the same or V in each polypeptide chain L and V H The Fv's may be different so as to form a bispecific bifunctional antibody (ie, comprising two Fv's with different specificities).

[0436] Single-chain Fv (scFv)

[0437] The skilled artisan will appreciate that a scFv comprises a V H and V L Area and V H With V L A polypeptide linker between the scFv and the polypeptide linker that enables the scFv to form a desired structure for antigen binding (i.e., V of a single polypeptide chain H and V LFor example, the linker comprises more than 12 amino acid residues, among which (Gly4Ser)3 is one of the more favorable linkers for scFv.

[0438] The present invention also contemplates the use of disulfide-stabilized Fvs (or diFvs or dsFvs) in which a single cysteine ​​residue is introduced into the V H FR and V L The cysteine ​​residues are linked by disulfide bonds to produce a stable Fv.

[0439] Alternatively or in addition, the present invention encompasses the use of dimeric scFvs, i.e. proteins comprising two scFv molecules linked by non-covalent or covalent bonds, 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 to allow the two scFvs to form and bind to an antigen, e.g., as described in US20060263367.

[0440] Heavy chain antibody

[0441] Heavy chain antibodies are structurally different from many other forms of antibodies because they contain heavy chains but no light chains. Therefore, these antibodies are also referred to as "heavy chain only antibodies." For example, heavy chain antibodies are found in camels and cartilaginous fish (also known as IgNARs).

[0442] The variable region of naturally occurring heavy chain antibodies is usually referred to as “V HH domain” and is referred to as V-NAR in IgNAR to distinguish it from the heavy chain variable region (referred to as “V H domain”) and the light chain variable region present in conventional 4-chain antibodies (referred to as “V L domains”).

[0443] A general description of heavy chain antibodies from camelids and their variable regions and methods for their production and / or isolation and / or use is found in, inter alia, the following references WO 94 / 04678, WO 97 / 49805 and WO 97 / 49805.

[0444] A general description of heavy chain antibodies from cartilaginous fish and variable regions thereof as well as methods for their production and / or isolation and / or use is found inter alia in WO 2005 / 118629.

[0445] Other antibodies and proteins comprising their antigen binding domains

[0446] The present invention also contemplates the use of other antibodies and proteins comprising the antigen binding domains thereof, such as:

[0447] (i) "key and hole" bispecific proteins as described in US5731168;

[0448] (ii) heteroconjugate proteins, for example, as described in US4676980;

[0449] (iii) heteroconjugated proteins generated using chemical cross-linkers, for example, as described in US4676980; and

[0450] (iv) Fab3 (eg as described in EP19930302894).

[0451] Route of administration

[0452] In some embodiments, the agents described herein may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, buccally, vaginally, intraventricularly, by implanted reservoir in dosage formulations containing conventional nontoxic pharmaceutically acceptable carriers, or by any other convenient dosage form. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion techniques.

[0453] Methods for preparing the agents described herein into a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Co., Easton, Pa., 1984).

[0454] The medicament for use according to the present invention is particularly suitable for parenteral administration, such as intravenous administration or intraluminal administration of a body cavity or organ or joint. In a particularly preferred embodiment, the antigen-binding proteins for use according to the present invention are preferably formulated for intravenous, intraperitoneal, subcutaneous, intramuscular or intratumoral administration.

[0455] The composition for administration will generally comprise a solution of the antigen-binding protein dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers can be used, such as buffered saline, etc. The composition can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as 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 over a wide range, and will be selected mainly based on fluid volume, viscosity, body weight, etc. according to the selected specific mode of administration 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 can also be used as carriers. The vehicle can contain a small amount of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0456] Immune checkpoint inhibitors

[0457] Checkpoint inhibitors

[0458] "Checkpoint inhibitors" inactivate proteins in the inhibitory checkpoint pathways of the immune response.

[0459] In the context of cancer, checkpoint inhibitors regulate the immune system by blocking proteins that prevent the immune system from attacking cancer cells. Specifically, it controls how cancer cells and T cells that escape detection interact so that T cells can recognize tumor cells and produce an appropriate immune response to them. Non-limiting examples of checkpoint inhibitors that can be used according to the methods described herein include inhibitors targeting PD-1 (programmed cell death protein 1), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), and PD-L1 (programmed death ligand 1). Technicians will understand that CTLA-4 and PD-1 are found on T cells, and PD-L1 is expressed on cancer cells and / or inhibitory 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-β.

[0460] 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.

[0461] "Programmed death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein 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 under GenBank accession number U64863.

[0462] When PD-1 binds to programmed cell death ligand 1 (PD-L1), the immune response is shut down, preventing T cells from destroying or killing cells. In the context of cancer, cancer cells can be covered with PD-L1 protein to disguise themselves as healthy cells, thereby avoiding immune responses. Programmed death ligand-1 (PD-L1) is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion after binding to PD-1. The term "PD-L1" as used herein 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 under GenBank accession number Q9NZQ7.

[0463] Cytotoxic T lymphocyte antigen-4 (CTLA-4) refers to an immunosuppressive 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 referred to as B7-1 and B7-2, respectively). The term "CTLA-4" as used herein 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 under GenBank accession number AAB59385.

[0464] Any of the checkpoint inhibitors described herein can be administered in the form of an antibody. "Antibodies" (Ab) should include, but are not limited to, glycoprotein immunoglobulins that specifically bind to an antigen and include at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region includes three constant domains CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes a constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions referred to as complementary determining regions (CDRs), which are interspersed with more conservative regions referred to as framework regions (FRs). Each VH and VL includes three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the Ab may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0465] 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 their immunogenicity in humans. In the absence of explicit instructions, and unless the context indicates otherwise, the term "antibody" also includes an antigen-binding fragment or antigen-binding portion of any of the above-mentioned immunoglobulins, and includes monovalent and divalent fragments or portions, as well as single-chain Abs.

[0466] "Isolated antibody" refers to an Ab that is substantially free of other Abs with different antigenic specificities (e.g., an isolated Ab that specifically binds to PD-1 is substantially free of Abs that specifically bind to antigens other than PD-1). However, the isolated Ab that specifically binds to PD-1 may have cross-reactivity to other antigens such as PD-1 molecules from different species. In addition, the isolated Ab may be substantially free of other cellular substances and / or chemicals. The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of Ab molecules of a single molecular composition, i.e., an Ab molecule that has substantially the same primary sequence and exhibits a single binding specificity and affinity for a specific epitope. mAb is an example of an isolated Ab. mAbs can be produced by hybridomas, recombination, transgenics, or other techniques known to those skilled in the art.

[0467] "Human" antibody (huMAb) refers to an Ab with a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the Ab contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human Ab described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include Abs in which CDR sequences derived from germlines of another mammalian species such as mice have been transplanted onto human framework region sequences. The terms "human" Ab and "completely human Ab" are used synonymously.

[0468] "Humanized antibody" refers to an Ab in which some, most or all of the amino acids outside the CDR domain of a non-human Ab are replaced with corresponding amino acids derived from human immunoglobulin. In one embodiment of a humanized form of Ab, some, most or all of the amino acids outside the CDR domain have been replaced with amino acids from human immunoglobulin, while some, most or all of the amino acids in one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible (including variations of synonymous codons resulting from codon degeneracy), as long as they do not eliminate the ability of Ab to bind to a specific antigen. "Humanized" Ab retains an antigenic specificity similar to that of the original Ab.

[0469] A "chimeric antibody" refers to an Ab in which the variable region is derived from one species and the constant region is derived from another species, such as an Ab in which the variable region is derived from a mouse Ab and the constant region is derived from a human Ab.

[0470] An "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.

[0471] An "antigen-binding portion" of an Ab (also referred to as an "antigen-binding fragment") refers to one or more fragments of an Ab that retain the ability to specifically bind to the antigen bound by the entire Ab.

[0472] Examples of immune checkpoints and antibody inhibitors targeting these 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), umab), BMS-936559, atezolizumab, lambrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, TSR-042), anti-PD-L1 (e.g., KY-1003 (EP20120194977), MCLA-145, atezolizumab, BMS-936559, MEDI-4736, MSB0010718C, AUR-012, STI-A1010, MPDL3280A, AMP-224, pegol-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, NSCD736012, 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 (e.g., alemtuzumab), anti-IL-10, anti-IL-35, anti-TGF-β (e.g., Fresolumimab), anti-CSF1R (e.g., FPA008), anti-NKG2A (e.g., monalizumab), anti-MICA (e.g., IPH43), and anti-CD39.

[0473] Anti-PD-1 and anti-PD-L1 antibodies

[0474] Examples of suitable PD-1 inhibitors that can be used according to 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. Pat. No. 8,008,449. Other anti-PD-1 mAbs have been described in, for example, U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757 and 8,354,509 and PCT Publication No. WO 2012 / 145493 (which are incorporated herein by reference).

[0475] Nivolumab (also known as 5C4 (formerly named 5C4, BMS-936558, MDX-1106, or ONO4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor Ab that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449, which is incorporated herein by reference).

[0476] Pidilizumab (also known as Lambortuzumab and MK-3475) are humanized monoclonal IgG4 antibodies for binding to the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pidilizumab is described, for example, in U.S. Pat. Nos. 8,354,509 and 8,900,587, which are incorporated herein by reference. The FDA has approved Pidilizumab for the treatment of relapsed or refractory melanoma.

[0477] Other suitable PD-1 inhibitors include Libtayo (cemiplimab), Blincyto (blinatumomab), Dostarlimab, Spartalizumab, Cetrelimab, Pidilizumab, and BI-754091.

[0478] The anti-PD-1Ab suitable for the disclosed methods or uses of the present invention is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and or PD-L2, and inhibits the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, the anti-PD-1 antibody includes an antigen binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to the whole Ab in inhibiting ligand binding and upregulating the immune system.

[0479] In certain embodiments, the anti-PD-1 antibody that can be used according to the present invention can be replaced with another PD-1 or anti-PD-L1 antagonist. For example, because the anti-PD-L1 antibody prevents the interaction between PD-1 and PD-L1, thereby exerting a similar effect on the signaling pathway of PD-1, in the methods disclosed herein, the anti-PD-L1 antibody can replace the use of the anti-PD-1 antibody. In any embodiment, suitable PD-L1 inhibitors include Imfinzi (durvalumab or MEDI4736), Tecentriq (atezumab or MPDL3280A), Bavencio (avelumab; MSB0010718C), MS-936559 (12A4 or MDX-1105) and CX-072.

[0480] Anti-CTLA-4 antibodies

[0481] The anti-CTLA-4 antibodies that can be used according to the present invention bind to human CTLA-4, thereby disrupting the interaction of CTLA-4 with the human B7 receptor. It should be understood that because the interaction of CTLA-4 with B7 transmits a signal that causes the inactivation of T cells with CTLA-4 receptors, the disruption of the interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging the immune response.

[0482] Suitable CTLA-4 inhibitors that can be used according to the present invention include Yervoy (ipilimumab), tremelimumab and AGEN 1884 or those disclosed in U.S. Pat. Nos. 6,984,720 and 7,605,238, which are 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.

[0483] Immune checkpoint inhibitors can be administered in the form of pharmaceutical compositions, including in combination with any pharmaceutically acceptable excipients, carriers and / or diluents described herein. Typically, immune checkpoint inhibitors are administered in the form of formulations known in the art.

[0484] Dosage and timing of administration

[0485] In any aspect or embodiment of the 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.

[0486] Administration may be simultaneous, separate or sequential.

[0487] Administration refers to the use of any of the various methods and delivery systems known to those skilled in the art, including those described herein, to physically introduce a composition comprising a therapeutic agent into a subject. The pharmaceutical composition can be formulated into any suitable route of administration by an active agent as described herein. Typically, in addition to a therapeutic agent (e.g., an antibody and / or a checkpoint inhibitor for binding to CAIX), the pharmaceutical composition comprises a pharmaceutically acceptable excipient, carrier, and / or diluent. Examples of suitable components included in the pharmaceutical composition are described in Martindale-The Extra Pharmacopoeia (Pharmaceutical Press, London, 1993) and Martin (ed.), Remington's Pharmaceutical Sciences.

[0488] The phrase 'therapeutically effective amount' or 'effective amount' generally refers to an amount of an antibody to CAIX and / or a checkpoint inhibitor of the present invention that (i) treats a particular disease, condition or disorder, (ii) alleviates, 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. Undesirable effects, such as side effects, may sometimes manifest along with the desired therapeutic effect; therefore, practitioners weigh potential benefits against potential risks when determining an appropriate "effective amount". In some embodiments, an antigen binding protein that binds to or specifically binds to carbonic anhydrase IX (CAIX) and is conjugated to a radionuclide and an immune checkpoint inhibitor are administered together to a subject in need thereof in a "therapeutically effective amount". The therapeutically effective amount may include an amount of one or both of the antigen binding protein or the immune checkpoint inhibitor, which itself will be less than the therapeutically effective amount. In some embodiments, an antigen binding protein that binds to or specifically binds to carbonic anhydrase IX (CAIX) and is conjugated to a radionuclide is administered in an amount less than the therapeutically effective amount in the absence of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is administered in an amount less than the therapeutically effective amount in the absence of an antigen binding protein that binds to or specifically binds to carbonic anhydrase IX (CAIX) and is conjugated to a radionuclide. In some embodiments, a "therapeutically effective amount" of a combination of an antigen binding protein that binds to or specifically binds to carbonic anhydrase IX (CAIX) and is conjugated to a radionuclide and an immune checkpoint inhibitor may be a synergistic amount. A synergistic amount may be synergistic relative to a monotherapy with an antigen binding protein that binds to or specifically binds to carbonic anhydrase IX (CAIX) and is conjugated to a radionuclide or an immune checkpoint inhibitor.

[0489] For example, for the treatment of tumors, a therapeutically effective amount of a compound or composition 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 relative to an untreated subject. Alternatively, the treatment described herein can result in complete regression of tumor mass. In other embodiments of the invention, tumor regression can be observed and lasts for a period of 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, or 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 more.

[0490] A therapeutically effective amount of a drug may also include a "prophylactic" or "prophylactically effective amount," which is any amount of an antibody and / or checkpoint inhibitor for binding to CAIX administered to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or with a recurrence of cancer that 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 development or recurrence of cancer means reducing the likelihood of the development or recurrence of cancer, or completely preventing the development or recurrence of cancer.

[0491] Suitable doses of antigen binding proteins and immune checkpoint inhibitors for use according to the present invention will vary depending on the specific active agent selected, the condition to be treated, and / or the subject to be treated. A skilled physician is capable of determining a suitable dose, for example, by starting with a suboptimal dose and incrementally modifying the dose to determine an optimal or useful dose. Alternatively, to determine an appropriate dose for treatment / prevention, data from cell culture assays or animal studies are used, where the appropriate dose is within a circulating concentration range that includes the ED of the active compound with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The therapeutically / prophylactically effective dose can be estimated initially from cell culture assays. The dose can be formulated in animal models to achieve an IC including as determined in cell culture. 50 (i.e., the concentration or amount of the compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0492] In some examples, the methods of the invention comprise administering a prophylactically or therapeutically effective amount of a protein described herein.

[0493] For the administration of checkpoint inhibitors including PD-1, PD-L1 or CTLA-4 inhibitors, the range of dosage can be 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. Based on the typical pharmacokinetic properties of Ab, the dosing regimen is generally designed to achieve exposure that causes sustained receptor occupancy (RO). Exemplary treatment regimens require about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once a month, about every 3-6 months or longer. In certain embodiments, the checkpoint inhibitor is administered to the subject about once every 2 weeks. In other embodiments, Ab is administered approximately once every 3 weeks. During the course of treatment, the dosage and schedule may vary. For example, the dosage regimen of anti-PD-1 therapy may include administering Ab: (i) approximately once every 2 weeks in an approximately 6-week cycle; (ii) approximately six doses approximately every 4 weeks, then approximately once every three months; (iii) approximately once every 3 weeks; (iv) approximately 3- to approximately 10 mg / kg once, then approximately 1 mg / kg every approximately 2-3 weeks. Considering that IgG4 antibodies typically have a half-life of 2-3 weeks, the dosage regimen of the anti-PD-1 Ab used in accordance with the present invention comprises approximately 0.3-1 to approximately 0 mg / kg body weight, 1-5 mg / kg body weight, or approximately 1- to approximately 3 mg / kg body weight administered intravenously, wherein Ab is administered once every approximately 14-21 days, up to approximately 6 weeks or approximately 12 weeks of circulation, until complete response or confirmation of progressive disease.

[0494] In some embodiments, the check point inhibitors and / or antibodies disclosed herein for use in conjunction with CAIX treatment last for a minimum duration of 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, the check point inhibitors and / or antibodies disclosed herein for use in conjunction with CAIX treatment last for a maximum duration of up to about 18 months, 16 months, 14 months, 12 months, 10 months, 8 months, or 6 months. The duration of treatment can be any of these minimum durations to any of these maximum durations, for example, 1 to 18 months.

[0495] It should be understood that the specific dosage level for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, weight, health status, sex, diet, time of administration, route of administration and rate of excretion, drug combination (i.e., other drugs used to treat the patient) and the severity of the specific condition being treated.

[0496] The term "treatment" or "treating" a subject includes applying or administering a compound described herein to a subject for the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, mitigating, altering, remedying, reducing deterioration, ameliorating, improving, or affecting a disease or condition, symptoms of a disease or condition, or the risk (or susceptibility) of a disease or condition. The term "treat" refers to any indicator of successful treatment or improvement of an injury, pathology, or condition, including any objective or subjective parameter, such as alleviation; remission; reducing the rate of deterioration; reducing the severity of the disease; stabilizing, reducing symptoms, or making an injury, pathology, or condition more tolerable to the subject; slowing the rate of deterioration or decline; making the end of a deterioration less debilitating; or improving the physical or mental health of the subject.

[0497] As used herein, minimizing the progress of cancer or preventing cancer means treating a subject to prevent or delay the recurrence or metastasis of a tumor, or preventing the growth of an existing tumor. Minimizing the progress of cancer or preventing cancer includes preventing or delaying the recurrence of cancer after cancer treatment, or preventing the growth of an existing tumor. Prevented recurrence includes, for example, the recurrence of a tumor bed after surgical resection. Alternatively, recurrence includes the metastasis of cancer in another part of the body. As used herein, the terms "preventing recurrence (preventing recurrence)" and "preventing relapse (preventing relapse)" are interchangeable.

[0498] The present invention also includes methods for preventing the development of cancer in individuals. For example, an individual in need of cancer prevention may be considered to be at risk of developing cancer, but does not yet have detectable cancer. An individual at risk of developing cancer may be an individual with a family history of cancer, and / or an individual with a high risk or high probability of developing cancer as indicated by genetic testing or other tests. An individual may have cancer stem cells, but does not yet have any detectable tumors. It should be understood that methods for preventing the development of cancer include methods for delaying the onset of cancer in a subject.

[0499] The terms "subject" and "patient" will be understood to be interchangeable. Although the present invention can be used with humans, the present invention can also be used for veterinary treatment purposes. The present invention can be used with livestock or farm animals, such as cattle, sheep, horses, and poultry; with companion animals such as cats, dogs, etc.; and with zoo animals.

[0500] Reagent test kit

[0501] Also provided is a kit for use according to the invention, the kit comprising one or more of the following:

[0502] (i) An agent for use according to the invention or an expression construct encoding said agent.

[0503] In some embodiments, the kit may further comprise an immune checkpoint inhibitor, which is optionally formulated into a pharmaceutical composition together with one or more pharmaceutically acceptable excipients, carriers and / or diluents.

[0504] In the case of a kit for detecting cancer, the kit may additionally comprise detection means, for example linked to an antigen binding protein for use according to the present invention.

[0505] In the case of a kit for therapeutic / prophylactic use, the kit may further comprise a pharmaceutically acceptable carrier.

[0506] Optionally, according to any embodiment, the kits of the invention are packaged with instructions for use in the methods described herein.

[0507] Table 1: Summary of amino acid and nucleotide sequences for CAIX binding antibodies

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520] Examples

[0521] Example 1: Antibodies for binding to CAIX

[0522] Summary

[0523] The humanized antibody variable region gene is cloned into the vector encoding the unmodified human IgG1 heavy chain constant domain and the human κ light chain constant domain. The chimeric antibody is additionally cloned into the vector encoding the modified human IgG1 heavy chain constant domain or the modified human IgG4 heavy chain construct, the domain contains the mutation H310A and H435Q (Andersen et al., 2012) that eliminates FcRn and protein A binding, the construct contains the mutation S228P (to stabilize the hinge (Angal et al., 1993)) and L235E (to remove the effector function (Reddy et al., 2000)) and the mutation eliminating FcR described above. Chimeric and humanized antibodies are transiently expressed in HEK EBNA cells, and the combination with human carbonic anhydrase IX (CAIX) is tested using Biacore single cycle kinetic analysis. Lead antibody is selected by protein A or protein G, and analyzed by analyzing SEC, competitive ELISA and Biacore multi-cycle kinetic analysis.

[0524] Identify three kinds of leading candidate humanized antibodies with similar combination with chimeric antibody.These are cloned into the carrier of coding human IgG1 heavy chain constant domain (H310A, H435Q) and IgG4 heavy chain constant domain (S241P, L235E, H310A, H435Q) as described above subsequently.Chimeric and leading humanized antibody are transiently expressed in Chinese hamster ovary celI, and use Biacore single cycle kinetic analysis to test its combination with people's carbonic anhydrase IX (CAIX).By protein A or protein G purifying antibody, and analyze by analyzing SEC and Biacore multi-cycle kinetic analysis.

[0525] method

[0526] Composite Human Antibody TM Design of variable region sequences

[0527] The structural model of the Girentuximab antibody V region was generated using the Swiss PDB and analyzed to identify important "restrictive" amino acids that may be critical for the binding properties of the antibody. Most residues contained within the CDR (using both the Kabat and Chothia definitions) as well as many framework residues are considered important. The VH and Vκ sequences of Girentuximab contain typical framework residues and CDR 1, 2, and 3 motifs comparable to many mouse antibodies.

[0528] Based on the above analysis, it can be considered that the composite human sequence of girentuximab can have a large degree of freedom in alternative residues outside the CDR, but only a small number of possible residues within the CDR sequence. Preliminary analysis indicates that corresponding sequence segments from several human antibodies can be combined to generate CDRs similar or identical to the CDRs in the mouse sequence. For regions outside the CDR and flanking, a wide selection of human sequence segments were identified as possible components of the new humanized V region.

[0529] CD4+ T cell epitope avoidance

[0530] Based on structural analysis, a large set of preliminary sequence segments were identified that could be used to generate humanized variants of girentuximab. TM These segments were selected and analyzed using the TCED of T cell epitopes associated with known antibody sequences using in silico analysis of peptides binding to human MHC class II alleles (Perry et al., 2008). TM (Bryson et al., 2010). Identified as an important non-human germline binder of human MHC class II or targeting TCED TM The sequence segments scored as significant hits are discarded. This causes the segment group to reduce, as described above, and analyzes these combinations again, to ensure that the connection between the segments does not contain potential T cell epitopes. The sequence segments selected are assembled into complete V region sequences lacking significant T cell epitopes. Five heavy chains (VH0 (chimeric), VH1, VH3, VH4 and VH5) and six light chains (Vκ0 (chimeric), Vκ1 to Vκ5) sequences are then selected for gene synthesis and expression in mammalian cells (referring to Table 1).

[0531] Construction of chimeric antibodies and humanized variants

[0532] Chimeric VH0 and Vκ0 girentuximab sequences and humanized variants thereof were synthesized, flanked by restriction endonuclease sites, for cloning into Abzena's pANT expression vector system for unmodified human IgG1 heavy chain (allotype G1m1,17; equivalent to 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.

[0533] The mutations H310A and H435Q that abolished FcRn were incorporated into expression vectors for human IgG1 heavy chain (equivalent to the allotype of 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 (S228P, L235E) encoding IgG4.

[0534] Antibody Expression in HEK293 EBNA Cells

[0535] Chimeric girentuximab (VH0 / Vκ0), two control antibodies (VH0 / Vκ1, VH1 / Vκ0) and combinations of VH and Vκ chains (20 pairs in total) were transiently expressed as IgG1 in HEK EBNA cells (LGC Standards, Teddington, UK) using the PEI transfection method. Cells were incubated for seven days after transfection. Additionally, chimeric girentuximab VH0 and Vκ0 in IgG1 (H310A, H435Q) heavy chain vectors, IgG4 (S228P, L235E) heavy chain vectors, and IgG4 (S228P, L235E H310A, H435Q) heavy chain vectors were similarly transfected with HEK EBNA cells. Supernatant antibody titers were determined by ELISA.

[0536] Kinetic analysis of chimeric and humanized variants binding to carbonic anhydrase IX

[0537] To evaluate all Girentuximab Composite Human Antibody TM The combination of variants, single cycle kinetic analysis of 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 performed at 25 ° C with 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).

[0538] Based on the concentration assessed by ELISA titer, the antibody was diluted to a final concentration of 0.5 μg / ml in running buffer. At the beginning of each cycle, the antibody was loaded onto the Fc2, Fc3 and Fc4 of the protein G chip (GE Healthcare, Little Chalfont, UK). IgG was captured at a flow rate of 10 microliters / minute to obtain an immobilization level (RL) of about 79RU, which is a theoretical value for obtaining an Rmax of about 50RU. The surface was then stabilized. Carbonic anhydrase IX (CAIX, Stratech, Newmarket, UK) was used as analyte to obtain single-cycle kinetic data at a flow rate of 30 microliters / minute to minimize any potential mass transfer restrictions. Repeated multiple times with reference chimeric antibodies (VH0 / Vκ0IgG1) to check the stability of the surface and analyte in the kinetic cycle. The signal from reference channel Fc1 (no antibody) was subtracted from the signal of Fc2, Fc3 and Fc4 to correct the difference with reference surface nonspecific binding. A four-point two-fold dilution range from 3.125 nM to 25 nM CAIX was used with no regeneration between each concentration. The association phase of four injections of increasing CAIX concentrations was monitored for 200 seconds each, 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 injections of 10 mM glycine-HCL pH 1.5 followed by injections of 10 mM glycine-HCL pH 1.5 containing 0.5% P20.

[0539] Variants were analyzed using a reference chimera (VH0 / Vκ0IgG1) for calculation of relative KD. Signals from blank runs of each antibody were subtracted to correct for differences in surface stability. Single cycle kinetic data indicated that binding of all humanized variants to CAIX was within two-fold of the reference chimeric antibody, except for those containing VH5 and / or VK5, as these variants showed lower affinity for CAIX.

[0540] Purification of chimeric and lead humanized antibodies

[0541] Six IgG1 lead antibodies, VH3 / Vκ2, VH3 / Vκ3, VH3 / Vκ4, VH4 / Vκ2, VH4 / Vκ3, and VH4 / Vκ4, were purified based on humanization 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 protein A agarose columns (GE Healthcare, Little Chalfont, UK). TMChimeric IgG1 (H310A, H435Q) and IgG4 (S228P, L325E, H310A, H435Q) antibodies were purified from cell culture supernatants using a protein G HP column (GE Healthcare, Little Chalfont, UK) because the H310A H435Q double mutation has been shown to have an adverse effect on binding to some protein A resins. All antibodies were buffer exchanged into 1xPBS 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 SDS-PAGE reducing 2 μg of loaded antibody and bands corresponding to typical antibody profiles were observed.

[0542] Multi-cycle kinetic analysis of chimeric and lead antibodies

[0543] Purified chimeric antibodies and six leading IgG1 antibodies were subjected to multi-cycle kinetic analysis using a Biacore T200 (serial number 1909913) instrument running Biacore T200 evaluation software V3.0.1 (Uppsala, Sweden) to confirm the accurate affinity for carbonic anhydrase IX. The purified antibodies were diluted to a concentration of 1 μg / ml in HBS-P+. At the beginning of each cycle, each antibody was captured on the G protein surface to produce an RL of approximately 79RU. After capture, the surface was stabilized. Kinetic data were obtained using a flow rate of 30 microliters / minute to minimize any potential mass transfer effects. For kinetic analysis, carbonic anhydrase IX (CAIX) was used. Multiple repetitions of blank (CAIX) and repetitions of a single concentration of analyte were programmed into the kinetic run to check the stability of the surface and analyte in the kinetic cycle. For kinetic analysis, a two-fold dilution range was selected from 50nM to 0.078nM 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 nonspecific binding to the reference surface, and a global Rmax parameter was used in a 1 to 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 main chains showed relative KDs within 2-fold of the chimeric IgG1 antibody.

[0544] Carbonic Anhydrase IX Competition ELISA

[0545] 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 against 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.

[0546] CAIX was diluted to 0.5 μg / ml in 1x PBS and 100 μl / well was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed 3 times with 1x PBS / 0.05% Tween (PBST) and blocked with 200 μl of 2% BSA / PBS for one hour at room temperature. In a 96-well dilution plate, a fixed concentration of biotinylated girentuximab chimeric IgG1 antibody (0.3 μg / ml final concentration) was added in equal volumes to a three-fold titration series of test antibodies (starting from 20 μg / ml (10 μg / ml final concentration) diluted in blocking buffer).

[0547] After washing the plate 3 times with PBS-T, 100 μl of the chimeric / test antibody mixture was added to the ELISA plate.

[0548] After incubation for one hour at room temperature, the plate was washed 3 times with PBS-T and 100 μl of streptavidin-peroxidase conjugated secondary antibody (Sigma, Dorset, UK) diluted 1:1000 in PBS-T was applied at room temperature for one hour to detect the bound biotinylated girentuximab chimeric IgG1 antibody. For color development, the plate was washed 3 times with PBS-T, then 100 μl of TMB substrate was added and incubated at room temperature for about five minutes. The reaction was stopped with 50 μl of 3.0 M hydrochloric acid and the absorbance was immediately read at 450 nm using a Dynex plate reader.

[0549] Calculate the IC for each variant 50 The IC50 values ​​were compared and the relative IC50 values ​​were calculated by dividing the IC50 of the humanized variants by the IC50 of the girentuximab chimeric IgG1 antibody determined on the same plate. All lead humanized variants and chimeric antibodies on different backbones showed IC50 values ​​within two-fold of the girentuximab chimeric IgG1 antibody.

[0550] By analyzing all the data generated (including iTope TMBased on the analysis of the expression patterns, percentage of humans, multi-cycle kinetic data, and competition ELISA data), three antibodies, VH3 / Vκ4, VH4 / Vκ3, and VH4 / VK4 (having sequences as provided in Table 1 ), were selected as leads for IgG1, IgG1 (H310A, H435Q), and IgG4 (S228P, L235E, H310A, H435Q) for further expression and analysis.

[0551] Expression of the lead humanized Girentuximab antibody in CHO cells

[0552] 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.

[0553] Using the corresponding plasmid and using MaxCyte FreeStyle was transiently transfected using an electroporation system (MaxCyte Inc., Gaithersburg, USA). TM CHO-S cells (Thermo Fisher, Loughborough, UK) were transfected with chimeric (VH0 / Vκ0), VH3 / VK4, VH4 / VK3 and VH4 / VK4 as IgG1, IgG1(H310A, H435Q) and IgG4(S228, L235E, H310A, H435Q) (12 combinations in total). After cell recovery, cells were diluted to 3×106 cells / ml in CD Opti-CHO medium (Thermo Fisher, Loughborough, UK) containing 8 mM L-glutamine (Thermo Fisher, Loughborough, UK) and 1× hypoxanthine-thymidine (Thermo Fisher, Loughborough, UK). 24 hours after transfection, the culture temperature was reduced to 32°C and 1 mM sodium butyrate (Sigma, Dorset, UK) was added. On day 1, 30% CD High Efficiency Feed B (Thermo Fisher Scientific, Loughborough, UK) and 3.3% FunctionMAX TM The cultures were fed with TiterEnhancer (Thermo Fisher Scientific, Loughborough, UK) and again on day 7 with 15% CD High Efficiency Feed B (Thermo Fisher Scientific, Loughborough, UK) and 1.65% FunctionMAX TMCultures were fed (percentage based on starting culture volume) with TiterEnhancer (Thermo Fisher Scientific, Loughborough, UK). IgG supernatant titers were monitored by IgG ELISA and transfected cells were cultured for up to 14 days before supernatants were harvested.

[0554] Purification of chimeric and lead humanized antibodies expressed as different IgG

[0555] Chimeric (VH0 / Vκ0), VH3 / VK4, VH4 / VK3 and VH4 / VK4IgG1 antibodies and VH4 / Vκ4IgG4 (S228P, L235E) antibodies were purified from cell culture supernatants using protein A agarose columns. Chimeric (VH0 / Vκ0), VH3 / Vκ4, VH4 / Vκ3 and VH4 / Vκ4 expressed as IgG1 (H310A, H435Q) and IgG4 (S228P, L325E, H310A, H435Q) were purified using protein G columns. Purified antibodies were processed as outlined above. Antibodies were analyzed by SDS-PAGE of 2 μg of the loaded antibodies, and bands corresponding to typical IgG spectra were observed.

[0556] Multi-cycle kinetic analysis of chimeric antibodies and lead humanized antibodies expressed as different IgG

[0557] Chimeric (VH0 / Vκ0) VH3 / Vκ4, VH4 / Vκ3 and VH4 / VK4 antibodies on different IgG main chains were analyzed by multi-cycle kinetics using a Biacore T200 (serial number 1909913) instrument running Biacore T200 evaluation software V3.0.1 (Uppsala, Sweden) to confirm the accurate affinity for carbonic anhydrase IX. Multi-cycle kinetics were performed as outlined in Section 2.7. VH0 / VK0IgG1 was run on each Fc2 run as a reference. Relative KD was calculated by dividing the KD of the humanized variant by the KD of the chimeric antibody with the same IgG constant domain. In all three IgG main chains, the lead variants were combined with the same IgG main chain within two times of VH0 / VK0.

[0558] Conclusion and discussion

[0559] The V region genes from the girentuximab antibody were combined with four humanized VH regions and five humanized Vκ regions (using Composite Human Antibody TMThe chimera (VH0 / VK0) was cloned into the VH vectors IgG1 (H310A, H435Q) and IgG4 (S228P, L235E, H310A, H435Q), where the mutations H310A and H435Q abolished FcRn binding.

[0560] The 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 showed binding within two-fold of the IgG1 chimeric antibody.

[0561] Based on iTope TM Analysis, human percentage, multi-cycle kinetic data and competition ELISA data narrowed the six leads to three; VH3 / Vκ4 (SEQ ID NO: 36 / SEQ ID NO: 132), VH4 / Vκ3 (SEQ ID NO: 52 / SEQ ID NO: 132) and VH4 / VK4 (SEQ ID NO: 52 / SEQ ID NO: 148). These three lead variants were re-cloned and the antibodies were transiently expressed in CHO-S cells on three different main chains 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 main chains, the lead humanized variants showed binding within two times of VH0 / Vκ0 of the same IgG main chain.

[0562] Example 2 - Combination of targeted radionuclide therapy and immune checkpoint inhibition

[0563] Immune checkpoint inhibition (ICI) has dramatically changed cancer treatment, but most patients still do not respond (long-term). Key determinants of successful ICI treatment are high tumor mutational burden and pre-existing tumor-infiltrating lymphocytes (TILs). Ionizing radiation can trigger inflammatory signaling, increase TILs and induce mutations that can be recognized by T cells. Mouse models are being used to evaluate the therapeutic effects of targeted radionuclide therapy (TRT) and combined TRT+ICI therapy.

[0564] Preparation of radiolabeled girentuximab antibody conjugates

[0565] Table 2: Reagents used in Protocols 1 and 2

[0566]

[0567] Conjugate preparation scheme 1: 334 μL of 1.25M NaHCO3, pH 8.2 was added dropwise for every 20 mg of humanized girentuximab (GmAb) antibody. Next, 1.25 mg of NHS-DOTA was added for every 20 mg of GmAb while gently rotating the mixture until the NHS-DOTA was completely dissolved. The reaction mixture was incubated at 37 ° C for one hour and then cooled to room temperature. Using a 50 kDa MWCO membrane or sufficient PD10s, the GmAb-DOTA reaction solution was exchanged to 0.25M ammonium acetate pH 6.0 to a volume of >15x to ensure the removal of free NHS-DOTA in the solution. The GmAb-DOTA conjugate concentration was determined by UV-vis and calibrated to 5 mg / mL using 0.25M ammonium acetate, pH 6.0.

[0568] Conjugate Preparation Scheme 2: Alternatively, the conjugate can be prepared according to the following "Scheme 2" steps:

[0569] 1. Buffer exchange GmAb into reaction buffer: 100mM NaPO4 + 20mM EDTA pH 7.5, using a 50kDa MWCO membrane and approximately 10x volume change, and reach a concentration of approximately 5mg / mL. Ensure that the pH has changed to pH 7.5.

[0570] 2. No more than 30 minutes before use, i.e. immediately before use, prepare a 5 mg / ml NHS-DOTA stock solution by dissolving NHS-DOTA in cold phosphate buffered saline, pH 7.5. Add 12.1 molar equivalents of the NHS-DOTA stock solution to the buffer exchanged GmAb solution.

[0571] [Alternatively, 1.25 mg solid NHS-DOTA / 20 mg GmAb, while gently swirling to mix until the solid NHS-DOTA is completely dissolved.]

[0572] 3. Incubate the bioconjugation reaction at 22°C (21-24°C) for two hours to allow the DOTA conjugation reaction to complete.

[0573] 4. Buffer exchange the GmAb-DOTA reaction solution into 0.25M ammonium acetate pH 6.0 using a 50 kDa MWCO membrane; use >15x diafiltration volume to ensure removal of free NHS-DOTA in solution

[0574] 5. Check the final pH and protein concentration of GmAb-DOTA, then concentrate to 5 mg / ml using 0.25 M ammonium acetate (pH 6.0)

[0575] According to Scheme 1 or 2, the generated GmAb-DOTA is exposed to a source of 177-Lu for a period of time sufficient to chelate the radioisotope to the DOTA moiety. The source of 177-Lu can be any suitable source known in the art.

[0576] Methods: CT26 (murine colorectal cancer cell line) and Renca (murine renal epithelial) cells transfected with human CAIX were injected subcutaneously in the flank of Balb / C mice. 3 When the mice received lutetium-177-labeled CAIX-specific antibody 177 Lu-hG250 (iv) followed by ex vivo biodistribution studies (24 hours, ip). Antibodies were prepared as described in Examples 1 and 2.

[0577] 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).

[0578] Additionally, mice bearing Renca-CAIX tumors were injected with (1) 12, 18, or 24 MBq 177 Lu-hG250 monotherapy, (2) ICI consisting of anti-PD-1Ab (200 μg) + anti-CTLA-4Ab (200 μg), (3) 18 MBq 177 The combination of Lu-hG250 and ICI, (4) vehicle was used to determine tumor growth inhibition and survival rate. The body weight of the mouse subjects was monitored ( Figure 2 ). The mice were euthanized according to ethical principles ( Figure 3 Tumor growth curves were quantified using the area under the curve (AUC) corrected for animal survival ( Figure 1 , 5 and 6). Mice that had completely regressed were re-challenged with tumor cells.

[0579] Results: In CT26-CAIX and Renca-CAIX, 177Tumor uptake of Lu-hG250 was 17±2% and 32±9% ID / g, respectively. Immunohistochemistry (IHC) confirmed the expression of CAIX in both tumors. In addition, Renca-CAIX tumors showed high expression of PD-L1 and very little CD8 + TIL. + When TILs were heavily infiltrated, CT26-CAIX also had significant but uneven PD-L1 expression.

[0580] 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±78mm 3 / day, p<0.0001 and AUC=66±64 mm 3 / day, p<0.0001). Additionally, 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. Rechallenge of these animals with tumor cells resulted in 100% rejection of Renca-CAIX in all groups. In the TRT+ICI combination therapy group, 100% of the parental Renca tumors were also rejected, while in the 18 and 24 MBq TRT monotherapy groups, 40% and 75% were rejected, respectively.

[0581] Conclusion: TRT and 177 Lu-hG250 and its combination with ICI are therapeutically effective. Compared with TRT alone, the combination of TRT and ICI can achieve comparable antitumor efficacy at a lower dose of TRT. Tumor re-challenge experiments showed that tumor-specific T cell responses were induced after TRT.

[0582] Example 3 - Combination of targeted radionuclide therapy and immune checkpoint inhibition

[0583] Studies similar to those described in Example 2 were performed using lower doses of targeted radiation therapy (TRT), either alone or in combination with ICI treatment. Briefly, Renca cells transfected with human CAIX were injected subcutaneously into the flank of Balb / C mice. When tumors reached a volume of 50-100 mm 3 When the mice received lutetium-177-labeled CAIX-specific antibody 177Lu-hG250 (iv), followed by anti-PD-1 and anti-CTLA4 antibodies.

[0584] Experimental design in Figure 7 Briefly, mice were divided into the following treatment groups:

[0585] 1. Control (vehicle + vehicle)

[0586] 2. ICI monotherapy (vehicle + PD-1Ab + anti-CTLA-4Ab);

[0587] 3. TRT low-dose monotherapy (4MBq 177 Lu-hG250+mediator);

[0588] 4. TRT+ICI low-dose therapy (4MBq 177 Lu-hG250+PD-1Ab+anti-CTLA-4Ab)

[0589] 5. TRT medium-dose monotherapy (12MBq 177 Lu-hG250+mediator);

[0590] 6. TRT+ICI medium-dose therapy (12MBq 177 Lu-hG250+PD-1Ab+anti-CTLA-4Ab)

[0591] Tumor growth inhibition and survival were determined, and the results were Figure 8 and 9 In summary, the results are consistent with the observations in Example 2 and indicate that the combination of TRT and ICI, including a lower radiation dose than in Example 2, showed a significant reduction in tumor growth compared to the control or monotherapy (TRT or ICI alone).

[0592] deal with % survival at the end of the study Complete response % Comparison 0 0 ICI 20 10 4MBq TRT 0 0 4MBq TRT+ICI 80 80 12MBq TRT 20 10 12MBq TRT+ICI 90 80

[0593] Mice that had completely regressed were re-challenged with tumor cells - the "parental" Renca cell line on one flank and Renca-CAIX, which had been received in the preliminary experiments, on the other flank.

[0594] The table below shows that a high proportion of animals treated with TRT+ICI combination therapy had complete tumor regression, and nearly all of these animals rejected tumor rechallenge. These results indicate that the animals have induced tumor-specific memory T cell responses.

[0595] deal with Number of re-trigger times (total 10) Renca-CAIX Tumor Rejection Renca tumor rejection Comparison 0 ICI 1 1(100％) 1(100％) 4MBq TRT 0 4MBq TRT+ICI 8 8(100％) 8(100％) 12MBq TRT 1 1(100％) 1(100％) 12MBq TRT 8 8(100％) 7(87.5％)

[0596] Example 4 - Combination of targeted radionuclide therapy and immune checkpoint inhibition (CT26 model)

[0597] Studies similar to those described in Example 3 were performed using the CT26-huCAIX model. CT26 cells transfected with human CAIX were injected subcutaneously into the flank of Balb / C mice. When tumors reached a volume of 50-100 mm 3 When the mice received lutetium-177-labeled CAIX-specific antibody 177 Lu-hG250 (iv), followed by anti-PD-1 antibody.

[0598] Experimental design in Fig.10 Briefly, mice were divided into the following treatment groups:

[0599] 1. Control (vehicle + vehicle)

[0600] 2. ICI monotherapy (vehicle + anti-PD-1Ab)

[0601] 3. TRT low-dose monotherapy (4MBq 177 Lu-hG250+mediator)

[0602] 4. TRT+ICI low-dose therapy (4MBq 177 Lu-hG250+anti-PD-1Ab)

[0603] Tumor growth inhibition and survival were determined, and the results were Fig.11 and 12 In summary, the results are consistent with the observations in Example 2 and indicate that the combination of TRT and ICI, including a lower radiation dose than in Example 2, showed a significant reduction in tumor growth compared to the control or monotherapy (TRT or ICI alone).

[0604] Example 5 - Evaluation of changes in gene expression following 177-Lu-hG250 treatment

[0605] To better understand the mechanism of action by which 177-Lu-hG250 treatment modulates the tumor microenvironment to increase the efficacy of ICI therapy, the present inventors performed gene expression profiling of Renca-hCAIX tumor-bearing mice using the IO360 PanCancer panel from the nanostring platform.

[0606] The TRT group was treated with 177-Lu-hG250, 12 MBq of radiation. Tumors were harvested 7 days later for gene expression analysis.

[0607] The results are shown in Example 13 and indicate that 177-Lu-hG250 treatment induces the expression of several genes involved in processes that promote anti-tumor immunity and are associated with response to checkpoint inhibition, including genes associated with interferon (IFN) signaling, antigen presentation, cytotoxicity, and DNA damage repair.

[0608] The graph was derived from a list of genes that passed the filter of at least 1.5-fold differential expression and p-value < 0.05. N = 3 mice per group. Expression values ​​represent the mean minus the normalized log2 value.

[0609] Example 6 - Cellular profiling and assessment of changes in gene expression following 177-Lu-hG250 treatment

[0610] In order to better understand the mechanism of action of 177-Lu-hG250 treatment to regulate the tumor microenvironment to increase the efficacy of ICI treatment, further studies were conducted. In this series of experiments, a lower dose of 177-Lu-hG250 treatment was applied compared to Example 4 (e.g., 4 MBq 177-Lu-hG250 was applied compared to 12 MBq 177-Lu-hG250 in Example 4).

[0611] The experimental plan Fig.14 Briefly, mice expressing Renca-hCAIX were treated according to the following protocol:

[0612] Part 1:

[0613] Animal Group

[0614] Control (untreated)

[0615] 4MBq 177Lu-hG250

[0616] Anti-PD-1 + anti-CTLA-4 antibodies

[0617] 4MBq 177Lu-hG250+anti-PD-1+anti-CTLA-4 antibodies

[0618] Tumors were harvested on day 0, about day 5, and about day 8 after the start of treatment. Flow cytometry was 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.

[0619] The results will show that low-dose targeted radiation / combination of low-dose targeted radiation with checkpoint inhibition alters the types / ratios of immune cells in a way that promotes anti-tumor immunity. For example, we will see an increase in the activation state of effector T cells and a decrease in suppressive immune cell types.

[0620] Part 2:

[0621] Animal Group:

[0622] Control (untreated)

[0623] 4MBq 177Lu-hG250

[0624] Anti-PD-1 + anti-CTLA-4 antibodies

[0625] 4MBq 177Lu-hG250+anti-PD-1+anti-CTLA-4 antibodies

[0626] Tumors were harvested on day 0, about day 5, and about day 8 after treatment initiation, and gene expression profiling was performed using nanostring to assess expression changes of >700 immunological genes. In addition, immunohistochemistry for T cell and myeloid markers was performed to assess the effects of the combination of 177Lu-hG250, anti-PD-1 + anti-CTLA-4 antibodies on the tumor microenvironment.

[0627] The results will show that low-dose targeted radiation / a combination of low-dose targeted radiation and checkpoint inhibition alters the gene expression profile of cells in the tumor microenvironment, which promotes anti-tumor immunity, similar to the observations in Example 4. For example, the results will show increased expression of genes involved in IFN signaling, T cell chemoattraction, cytotoxicity, and antigen presentation.

[0628] Example

[0629] 1. A method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising:

[0630] administering to the subject a therapeutically effective amount of an antigen binding protein that binds or specifically binds to carbonic anhydrase IX (CAIX) in combination with an immune checkpoint inhibitor therapy, wherein the antigen binding protein is conjugated to a radionuclide,

[0631] Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

[0632] 2. The method of embodiment 1, wherein the antigen binding protein comprises an antigen binding domain that specifically binds to carbonic anhydrase IX (CAIX) and comprises:

[0633] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a

[0634] in:

[0635] FR1, FR2, FR3 and FR4 are each framework regions;

[0636] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0637] FR1a, FR2a, FR3a and FR4a are each framework regions;

[0638] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0639] The sequence of any of the complementarity determining regions has an amino acid sequence as described in Table 1.

[0640] 3. The method of embodiment 2, wherein the antigen binding domain comprises:

[0641] (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 1, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 2, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 3; and

[0642] (ii) a VL comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence shown in SEQ ID NO: 81, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 82, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 83;

[0643] or

[0644] (iii) a VH comprising a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 1, a CDR2 comprising, or consisting 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 shown in SEQ ID NO: 2, and a CDR3 comprising, or consisting 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 shown in SEQ ID NO: 3. NO:3 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 or consisting of the sequence shown in NO:3; and

[0645] (iv) a VH comprising a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO:81, a CDR2 comprising, or consisting of, a sequence 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 shown in SEQ ID NO:82, and a CDR3 comprising, or consisting of, a sequence 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 shown in SEQ ID NO:83. The sequence shown in NO:83 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 or consisting of the sequence shown in NO:83.

[0646] 4. The method of embodiment 2 or 3, wherein the antigen binding domain comprises:

[0647] (i) a VH comprising a framework region (FR) 1 comprising or consisting of a sequence as shown in SEQ ID NO: 9, 25, 41, 57 or 73; a FR2 comprising or consisting of a sequence as shown in SEQ ID NO: 10, 26, 42, 58 or 74; a FR3 comprising or consisting of a sequence as shown in SEQ ID NO: 11, 27, 43, 59 or 75; a FR4 comprising or consisting of a sequence as shown in SEQ ID NO: 12, 28, 44, 60 or 76; and

[0648] (ii) a VL comprising a framework region (FR) 1 comprising or consisting of a sequence as shown in SEQ ID NO: 89, 105, 121, 137, 153 or 169; a FR2 comprising or consisting of a sequence as shown in any one of SEQ ID NO: 90, 106, 122, 138, 154 or 170; a FR3 comprising or consisting of a sequence as shown in SEQ ID NO: 91, 107, 123, 139, 155 or 171; and a FR4 comprising or consisting of a sequence as shown in SEQ ID NO: 92, 108, 124, 140, 156 or 172;

[0649] or

[0650] (iii) a VH comprising a framework region (FR) 1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO: 9, 25, 41, 57 or 73; and a FR2 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%, at least 99% identical to the sequence shown in SEQ ID NO: 9, 25, 41, 57 or 73. NO:10, 26, 42, 58 or 74 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 or consisting of a FR3; the FR3 comprising a sequence as set forth in SEQ ID NO:11, 27, 43, 59 or 75 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 or consisting of a sequence; FR4, the FR4 comprising a sequence as set forth in SEQ ID NO:12, 28, 44, 60 or 76 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 or consisting of a sequence as set forth in NO:12, 28, 44, 60 or 76; and

[0651] (iv) a VL comprising a framework region (FR) 1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO: 89, 105, 121, 137, 153 or 169; and a FR2 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%, at least 99% identical to the sequence shown in SEQ ID NO: NO:90, 106, 122, 138, 154 or 170 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 or consisting of a sequence; FR3 comprising a FR3 having a sequence as set forth in SEQ ID NO:91, 107, 123, 139, 155 or 171 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 or consisting of a sequence; FR4, the FR4 comprising a sequence as set forth in SEQ ID NO:92, 108, 124, 140, 156 or 1722 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 or consisting of the sequence shown in NO:92, 108, 124, 140, 156 or 1722.

[0652] 5. The method according to any one of claims 1 to 4, wherein the antigen binding protein comprises:

[0653] (a) a heavy chain variable domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 4, 20, 36, 52 or 68 and / or (b) a light chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 84, 100, 116, 132, 148 or 164;

[0654] or

[0655] (c) a heavy chain variable domain (VH) comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO:4, 20, 36, 52 or 68 and / or (d) a light chain variable domain (VL) comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO:4, 20, 36, 52 or 68 NO:84, 100, 116, 132, 148 or 164 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 or consisting of the sequence shown in NO:84, 100, 116, 132, 148 or 164.

[0656] 6. The method of any one of embodiments 1 to 5, wherein the antigen binding protein is an antibody or an antigen binding fragment thereof.

[0657] 7. The method of embodiment 1, wherein said antigen binding protein is said antibody Girentuximab.

[0658] 8. The method of any one of embodiments 1 to 6, wherein the antigen binding protein is a modified IgG class antibody, wherein the modified IgG class antibody comprises a heavy chain constant region having one or more amino acid substitutions compared to the wild-type IgG class antibody, wherein the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn) compared to the wild-type IgG class antibody, thereby shortening the serum half-life of the modified monoclonal antibody.

[0659] 9. The method of 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, Ile253.

[0660] 10. The method according to embodiment 9, wherein the one or more amino acid substitutions comprise a substitution in the heavy chain constant region at position His310 or at His435, preferably wherein the amino acid substitutions are at both His310 and His435.

[0661] 11. The method of any one of embodiments 8 to 10, wherein the modified antibody further comprises: (a) one or more amino acid substitutions that reduce the affinity of the antibody for one or more Fcγ receptors compared to a wild-type IgG antibody; 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 a wild-type IgG antibody.

[0662] 12. The method of embodiment 11, wherein the one or more amino acid substitutions that reduce the affinity of the antibody for an Fcγ receptor comprise an amino acid substitution at position Leu235.

[0663] 13. The method of 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 comprise a substitution at Ser228.

[0664] 14. The method of any one of embodiments 8 to 13, wherein the modified antibody is conjugated to the radionuclide via a linker or chelator moiety.

[0665] 15. The method of any one of embodiments 1 to 14, wherein the radioisotope is 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), Samarium-153 ( 153 Sm), Scandium-47 ( 47 Sc), Strontium-90 ( 90 Sr) and Yttrium-90 ( 90 Y).

[0666] 16. The method of any one of embodiments 1 to 15, wherein the antigen binding protein is a modified antibody having reduced FcRn binding affinity compared to the FcRn binding affinity of an unmodified form of the antibody or a wild-type IgG class antibody, the modified antibody comprising:

[0667] - a heavy chain constant region, wherein one or more of the amino acid residues at positions His310, His433, His435, His 436 and Ile253 are different from the residues present in said unmodified antibody or in a wild-type IgG class antibody.

[0668] 17. The method of any one of embodiments 1 to 16, wherein the antigen binding protein comprises a heavy chain comprising the sequence shown in any one of SEQ ID NOs: 182 to 185; and / or a light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 181 or SEQ ID NO: 186.

[0669] 18. The method of embodiment 16, wherein the antibody comprises the amino acid sequence shown in SEQ ID NO: 183 and 186.

[0670] 19. The method of embodiment 1, wherein the antigen binding protein is a molecule comprising an immunoglobulin portion and a non-protein agent conjugated thereto,

[0671] Wherein, the immunoglobulin portion specifically binds to CAIX and comprises:

[0672] An antigen binding protein consisting essentially of or consisting of the amino acid sequence 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 N to C terminus or C to N terminus), wherein the immunoglobulin portion reduces or eliminates affinity for the FcRn receptor compared to a wild-type immunoglobulin; and

[0673] Wherein the non-protein agent comprises a radioactive element.

[0674] 20. The method of any one of the preceding embodiments, wherein the radionuclide is selected from the group consisting of: actinium-225 ( 225 Ac), astatine-211( 211 At) and iodine-123( 123 I).

[0675] 21. The method of any one of the preceding embodiments, wherein the antigen binding protein is a therapeutic IgG molecule comprising:

[0676] - heavy and light chains, wherein each chain comprises a variable region and a constant region;

[0677] - wherein the constant region of the heavy chain comprises one or more amino acid substitutions compared to a wild-type IgG class antibody, wherein the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn) compared to a wild-type IgG class antibody, thereby shortening the serum half-life of the modified monoclonal antibody; and

[0678] - a radioactive isotope, which is conjugated to one or more amino acid residues of the heavy chain or the light chain.

[0679] 22. The method according to any one of the preceding embodiments, wherein the antigen binding protein is an antibody, preferably a naked antibody.

[0680] 23. The method of any one of the preceding embodiments, wherein the radionuclide is 177 Lu.

[0681] 24. The method of any of the preceding embodiments, wherein the radioactivity of the antigen binding protein used to bind to CAIX does not exceed about 18 MBq, and the antigen binding protein is conjugated to a radionuclide.

[0682] 25. The method of any preceding embodiment, wherein the radionuclide is conjugated to the antibody for binding to CAIX via a linker or a chelator.

[0683] 26. A method according to embodiment 25, wherein the chelating agent is selected from: TMT (6,6"-bis(N,N",N"'-tetrakis(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N" (N"'-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A"-DTPA, TETE, Te2A, HBED, DFO, DFOsq and HOPO.

[0684] 27. The method of any one of the preceding embodiments 1 to 68, further comprising conjugating the antigen binding protein to the radionuclide prior to the administering step.

[0685] 28. A method according to embodiment 27, wherein the antigen binding protein is a modified antibody for binding to CAIX, and the method further comprises, prior to the conjugation step, producing the modified antibody for binding to CAIX comprising a non-peptide portion.

[0686] 29. The method of embodiment 28, wherein the non-peptide moiety is a linker or a chelator.

[0687] 30. A method according to embodiment 29, wherein the chelating agent is selected from the group consisting of: TMT (6,6"-bis(N,N",N"'-tetrakis(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N" (N"'-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A"-DTPA, TETE, Te2A, HBED, DFO, DFOsq and HOPO.

[0688] 31. The method of any one of the preceding embodiments, wherein the immune checkpoint inhibitor therapy comprises targeting a protein selected from the group consisting of: PD-1, PD-L1, and CTLA-4, or a combination thereof.

[0689] 32. The method of embodiment 31, wherein the immune checkpoint inhibitor therapy comprises targeting PD-1 and CTLA4.

[0690] 33. The method of 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.

[0691] 34. The method of any of the preceding embodiments, further comprising administering one or more immune checkpoint inhibitors to the subject.

[0692] 35. The method of embodiment 34, wherein said administration of said antigen binding protein and said immune checkpoint inhibitor is separate, consecutive or sequential.

[0693] 36. A method of inducing a T cell response to a cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof against CAIX in combination with an immune checkpoint inhibitor, thereby inducing the T cell response to the cancer in the subject.

[0694] 37. A method for inducing an adaptive immune response to a cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof against CAIX in combination with an immune checkpoint inhibitor, thereby inducing a T cell response to the cancer in the subject.

[0695] 38. The method of any one of the preceding embodiments, wherein the cancer is renal cancer.

[0696] 39. Use of an antigen binding protein for binding to CAIX for the preparation of a medicament for use in combination with immune checkpoint inhibitor therapy to treat cancer, preferably renal cancer.

[0697] 40. Use of an immune checkpoint inhibitor for the preparation of a medicament for use in combination with an antigen binding protein that binds to CAIX to treat cancer, preferably renal cancer, wherein the antigen binding protein is conjugated to a radionuclide.

[0698] 41. An antigen binding protein for binding to CAIX for use in combination with immune checkpoint inhibitor therapy to treat cancer, preferably renal cancer.

[0699] 42. An immune checkpoint inhibitor for use in combination with an antigen binding protein for binding to CAIX to treat cancer, preferably renal cancer, wherein the antigen binding protein is conjugated to a radionuclide.

[0700] 43. A pharmaceutical composition comprising an antigen binding protein for binding to CAIX, wherein the pharmaceutical composition is for use in combination with an immune checkpoint inhibitor to treat cancer, preferably renal cancer.

[0701] 44. A pharmaceutical composition comprising an immune checkpoint inhibitor for use in combination with an antigen binding protein for binding to CAIX to treat cancer, preferably renal cancer, wherein the antigen binding protein is conjugated to a radionuclide.

[0702] 45. A kit for use or when used according to any method described herein, comprising in separate parts:

[0703] an antigen binding protein for binding to CAIX, optionally conjugated to a radionuclide; and

[0704] Instructions for use thereof, which are used in combination with an immune checkpoint inhibitor.

[0705] 46. ​​The kit of embodiment 87, further comprising one or more immune checkpoint inhibitors in a separate part.

[0706] 47. A kit for use according to or when used according to any method described herein, the kit comprising:

[0707] One or more immune checkpoint inhibitors; and

[0708] - Instructions for use thereof, said instructions for use in combination with an antigen binding protein for binding to CAIX, said antigen binding protein being conjugated to a radionuclide.

[0709] 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.

[0710] 49. A kit according to any one of embodiments 45 to 48, wherein the antigen binding protein for binding to CAIX and / or the immune checkpoint inhibitor are provided in the form of a pharmaceutical composition, which optionally contains one or more pharmaceutically acceptable excipients, carriers and / or diluents.

Claims

1. A method of treating, preventing, or minimizing the progression of cancer in a subject, the method comprising: administering to the subject: a radiolabeled agent that binds or specifically binds to carbonic anhydrase IX (CAIX) and an immune checkpoint inhibitor, Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

2. A method of inducing a T cell response to cancer in a subject, the method comprising: administering to the subject a radiolabeled agent in combination with an immune checkpoint inhibitor, wherein the radiolabeled agent binds or specifically binds to CAIX, Thereby inducing said T cell response to said cancer in said subject.

3. A method of inducing an adaptive immune response to cancer in a subject, the method comprising: administering to the subject a radiolabeled agent in combination with an immune checkpoint inhibitor, wherein the radiolabeled agent binds or specifically binds to CAIX, An adaptive immune response to said cancer in said subject is thereby induced.

4. A method of treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving treatment with an immune checkpoint inhibitor, the method comprising: administering to the subject a radiolabeled agent that binds or specifically binds to carbonic anhydrase IX (CAIX), Cancer in the subject is thereby treated, prevented, or progression of cancer in the subject is minimized.

5. The method according to any one of claims 1 to 4, wherein the agent is selected from a small molecule, a peptide or a protein that is capable of binding or specifically binding to CAIX.

6. The method of any one of claims 1 to 5, wherein the radiolabeled agent is a radiolabeled peptide.

7. The method according to any one of claims 1 to 5, wherein the radiolabeled agent is an antibody or antigen-binding fragment thereof capable of binding or specifically binding to CAIX, and wherein the antibody or antigen-binding fragment thereof is conjugated to a radionuclide.

8. The method of claim 7, wherein the radiolabeled antibody or antigen-binding fragment thereof is a radiolabeled girentuximab antibody or antigen-binding fragment thereof.

9. The method of any one of claims 1 to 5, 7 or 8, wherein the radiolabeled antibody or antigen binding protein comprises an antigen binding domain that binds or specifically binds to carbonic anhydrase IX (CAIX), and wherein the antigen binding domain comprises: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a in: 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; Preferably, the complementarity determining region has an amino acid sequence of any of the sequences described in Table 1.

10. The method of claim 6, wherein the antigen binding domain comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:1, and 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 shown in SEQ ID NO:

1. NO:2 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 shown in SEQ ID NO:2, and a CDR3 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:3; (ii) a VH comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in any one of SEQ ID NOs: 4, 20, 36, 52, or 68; (iii) a VL comprising 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 shown in SEQ ID NO:81, and 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 shown in SEQ ID NO:

81. 82 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 shown in SEQ ID NO:82, and a CDR3 comprising a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:83; (iv) a VL comprising a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:84, 100, 116, 132, 148, or 164; (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3; (vi) a VH comprising the sequence shown in any one 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 one of SEQ ID NOs: 84, 100, 116, 132, 148 or 164; (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3; and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 81, a CDR2 comprising the sequence set forth in SEQ ID NO: 82, and a CDR3 comprising the sequence set forth in SEQ ID NO: 83; or (x) a VH comprising the sequence shown in any one of SEQ ID NOs: 4, 20, 36, 52 or 68, and a VL comprising the sequence shown in any one of SEQ ID NOs: 84, 100, 116, 132, 148 and 164, preferably wherein the VH comprises the sequence as shown in SEQ ID NO: 4 and the VL comprises the sequence as shown in SEQ ID NO: 100, or wherein the VH comprises the sequence as shown in SEQ ID NO: 52 and the VL comprises the sequence as shown in SEQ ID NO:

148.

11. The method of claim 9 or 610, wherein the antigen binding domain comprises: (i) a VH comprising a framework region (FR) 1 comprising or consisting of a sequence as shown in any one of SEQ ID NOs: 9, 25, 41, 57 or 73; a FR2 comprising or consisting of a sequence as shown in any one of SEQ ID NOs: 10, 26, 42, 58 or 74; a FR3 comprising or consisting of a sequence as shown in any one of SEQ ID NOs: 11, 27, 43, 59 or 75; a FR4 comprising or consisting of a sequence as shown in any one of SEQ ID NOs: 12, 28, 44, 60 or 76, and (ii) a VL comprising a framework region (FR) 1 comprising or consisting of a sequence as shown in any one of SEQ ID NOs: 89, 105, 121, 137, 153, or 169; a FR2 comprising or consisting of a sequence as shown in any one of SEQ ID NOs: 90, 106, 122, 138, 154, or 170; a FR3 comprising or consisting of a sequence as shown in any one of SEQ ID NOs: 91, 107, 123, 139, 155, or 171; and a FR4 comprising or consisting of a sequence as shown in any one of SEQ ID NOs: 92, 108, 124, 140, 156, or 172; or (i) a VH comprising a framework region (FR) 1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in any one of SEQ ID NOs: 9, 25, 41, 57 or 73; and a FR2 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%, at least 99% identical to the sequence shown in any one of SEQ ID NOs: 9, 25, 41, 57 or 73. NO:10, 26, 42, 58 or 74, wherein the sequence 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 or consisting of a sequence as set forth in any one of SEQ ID NO:10, 26, 42, 58 or 74; FR3, wherein the FR3 comprises a sequence as set forth in SEQ ID NO:11, 27, 43, 59 or 75, wherein the sequence 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 or consisting of a sequence as set forth in any one of SEQ ID NO:11, 27, 43, 59 or 75; FR4, wherein the FR4 comprises a sequence as set forth in SEQ ID NO:12, 28, 44, 60 or 76 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 or consisting of a sequence as set forth in any of NO:12, 28, 44, 60 or 76, and (ii) a VL comprising a framework region (FR) 1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in any one of SEQ ID NOs: 89, 105, 121, 137, 153, or 169; and a FR2 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%, at least 99% identical to the sequence shown in any one of SEQ ID NOs: NO:90, 106, 122, 138, 154 or 170, wherein the sequence 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 or consisting of a sequence as set forth in any one of SEQ ID NO:90, 106, 122, 138, 154 or 170; FR3, wherein the FR3 comprises a sequence as set forth in SEQ ID NO:90, 106, 122, 138, 154 or 170, wherein the sequence 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 or consisting of a sequence as set forth in SEQ ID NO: ID NO:91, 107, 123, 139, 155 or 171, wherein the sequence 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 or consisting of a sequence as set forth in any one of SEQ ID NO:91, 107, 123, 139, 155 or 171; FR4, wherein the FR4 comprises a sequence as set forth in SEQ ID NO:91, 107, 123, 139, 155 or 171, wherein the sequence 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 or consisting of a sequence as set forth in SEQ ID NO: NO:92, 108, 124, 140, 156 or 1722, or 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 or consisting of the sequence shown in any of NO:92, 108, 124, 140, 156 or 1722.

12. The method according to any one of claims 9 to 11, wherein the antigen binding protein comprises: (a) a heavy chain variable domain (VH) comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68; and / or (b) a light chain variable domain (VL) comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in any one of SEQ ID NO:84, 100, 116, 132, 148 or 164.

13. The method according to any one of claims 9 to 12, wherein the antigen binding protein comprises: (a) a heavy chain variable domain (VH), said VH comprising an amino acid sequence as shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68; and / or (b) a light chain variable domain (VL), said VL comprising the amino acid sequence as shown in any one of SEQ ID NO: 84, 100, 116, 132, 148 or 164, Preferably, the antigen binding protein comprises the amino acid sequences of VH and VL selected from the following: 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, more preferably, the antigen binding protein comprises the amino acid sequences of VH and VL as shown in SEQ ID NO:52 / SEQ ID NO:

148.

14. The method according to any one of claims 9 to 13, wherein the antigen binding protein is a modified IgG class antibody, comprising a heavy chain constant region having one or more amino acid substitutions compared to a wild-type IgG class antibody, wherein the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn) compared to the wild-type IgG class antibody, thereby shortening the serum half-life of the modified monoclonal antibody.

15. The method according to any one of claims 9 to 14, wherein the antigen binding protein is a modified antibody having reduced FcRn binding affinity compared to the FcRn binding affinity of an unmodified form of the antibody or a wild-type IgG class antibody, the modified antibody comprising: - a heavy chain constant region, wherein one or more of the amino acid residues at positions His310, His433, His435, His 436 and Ile253 are different from the residues present in said unmodified antibody or in a wild-type IgG class antibody.

16. The method of any one of claims 1 to 15, wherein the radioconjugated 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).

17. The method according to any one of claims 9 to 16, wherein the radionuclide is lutetium-177( 177 Lu) or actinium-225( 225 Ac).

18. The method according to any one of claims 12 to 16, wherein the radionuclide is 177 Lu.

19. The method of any one of claims 1 to 18, wherein the radioactivity of the agent conjugated to the radionuclide does not exceed about 18 MBq, optionally wherein the radioactivity does not exceed about 12 MBq.

20. The method according to any one of claims 6 to 19, wherein the radionuclide is conjugated to the peptide or the antigen binding protein via a linker or a chelator.

21. The method of claim 20, wherein the chelating agent is selected from: TMT (6,6"-bis(N,N",N"'-tetrakis(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N" (N"'-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A"-DTPA, TETE, Te2A, HBED, DFO, DFOsq, DFO-NCS, HOPO or a chelating agent as described in WO 2022 / 133537.

22. The method of any one of claims 1 to 21, further comprising conjugating the agent to the radionuclide prior to the administering step.

23. The method of any one of claims 1 to 22, wherein immune checkpoint inhibitor therapy comprises inhibiting a protein selected from the group consisting of: PD-1, PD-L1, and CTLA-4, or a combination thereof.

24. The method of claim 23, wherein the immune checkpoint inhibitor therapy comprises inhibition of PD-1 and / or CTLA4.

25. The method of claim 24, wherein the immune checkpoint inhibitor therapy comprises administering to the subject an antibody for binding to PD-1 and / or an antibody for binding to CTLA4.

26. The method of any one of claims 1 to 25, wherein the immune checkpoint inhibitor is administered after the radiolabeled agent for binding to CAIX.

27. The method of claim 26, wherein the immune checkpoint inhibitor is administered at least 24 hours after administration of the radiolabeled agent for binding to CAIX.

28. The method of any one of claims 1 to 27, wherein the cancer is renal cancer.

29. Use of a radiolabeled antigen binding protein that binds or specifically binds to CAIX for the preparation of a medicament for use in combination with immune checkpoint inhibitor therapy to treat cancer, preferably renal cancer.

30. Use of an immune checkpoint inhibitor for the preparation of a medicament for use in combination with a radiolabeled antigen binding protein for the treatment of cancer, preferably renal cancer, wherein the radiolabeled antigen binding protein binds or specifically binds to CAIX.

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

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

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

34. A pharmaceutical composition comprising an immune checkpoint inhibitor for use in combination with a radiolabeled antigen binding protein for binding to CAIX to treat cancer, preferably renal cancer.

35. A kit comprising, when used or intended for use according to the method of any one of claims 1 to 29, in separate parts: an agent that specifically binds to CAIX, optionally conjugated to a radionuclide; and ●Instructions for use thereof, which are used in combination with an immune checkpoint inhibitor.

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

37. A kit according to claim 35 or 36, further comprising one or more immune checkpoint inhibitors in a separate part.

38. A kit, when used or intended for use according to the method of any one of claims 1 to 29, comprising: ●One or more immune checkpoint inhibitors; and - Instructions for use thereof, said instructions for use in combination with an antigen binding protein for binding to CAIX, said antigen binding protein being conjugated to a radionuclide.

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

40. A kit according to any one of claims 35 to 39, wherein the agent for binding to CAIX and / or the immune checkpoint inhibitor is provided in the form of a pharmaceutical composition, which optionally comprises one or more pharmaceutically acceptable excipients, carriers and / or diluents.

Images