Novel radiolabeled antibodies

Abstract

The present invention relates to radiolabeled gemtuzumab ozogamicin antibodies and humanized versions conjugated with the radionuclide astatine 211 The antibody conjugates are used in radiotherapy for the treatment of urothelial cancer, including bladder cancer.

Description

Technical Field

[0001] This invention relates to radiolabeled molecules, preferably antibodies, for the treatment of urothelial carcinoma, and methods of using them.

[0002] Cross-reference to related applications

[0003] This application claims priority to Australian Provisional Application AU 2023903890, the entire contents of which are hereby incorporated by reference. Background Technology

[0004] Molecularly targeted radiotherapy (MTR) is an emerging cancer treatment method that involves using radiolabeled agents to bind to molecular targets.

[0005] However, the mere presence of molecular targets on tumors does not always indicate that molecularly targeted radiotherapy can be used to effectively treat cancer.

[0006] Therefore, there is a need for new drugs and methods for treating cancer, for which there is currently an unmet need or where previous use of MTR has not been successful in targeting.

[0007] References to any prior art in this specification are not an admission or implication that such prior art constitutes part of common common sense in any jurisdiction, nor are they an admission or implication that such prior art can be reasonably expected or understood by a person skilled in the art, or be regarded as relevant and / or combined with other prior art. Summary of the Invention

[0008] This invention relates to molecules and methods for treating urothelial carcinoma, particularly urothelial carcinoma expressing the antigen carbonic anhydrase IX (CAIX).

[0009] Therefore, the present invention provides a radiolabeled girentuximab antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is conjugated with the radioisotope 211-astatine.

[0010] As used in this article, such radiolabeled antibodies can be referred to as... 211 At-geutzumab or 211-At-GmAb.

[0011] In any aspect or embodiment, the radioactive isotope can be directly conjugated with gemtuximab, for example, by halogenation of amino acid residues.

[0012] Preferably, the radioisotope is indirectly linked to gemutuximab or its antigen-binding fragment, for example, via a prosthetic group. The gemutuximab or its antigen-binding fragment may be conjugated to a bifunctional linker (e.g., bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, aryl iodide cation salt, or any amine or thiol-modified chemical known in the art) and to a radioisotope conjugated to the bifunctional linker.

[0013] In an alternative embodiment, the bifunctional linker (prosthetic group) may first be radiolabeled, and then the radiolabeled prosthetic group may be conjugated with gemutuximab or its antigen-binding fragment.

[0014] Examples of methods for astatination of auxiliary groups (e.g., diaryliodocation salts) are known in the art (e.g., in WO 2019027059 and WO2017 / 089492, which are incorporated herein by cross-reference in their entirety).

[0015] Optionally, the radioactive isotope can be indirectly linked to gemutuximab or its antigen-binding fragment via a chelating agent. In one example, the gemutuximab is conjugated with a chelating portion selected from the group consisting of: TMT (6,6"-bis[N,N",N"'-tetra(carboxymethyl)aminomethyl]-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOA, Diamsar, DTPA, CHX-A"-DTPA, TETE, Te2A, HBED, DFO, DFOsq, DFO-NCS and HOPO, chelating agents as described in WO 2022 / 133537 (incorporated herein by cross-reference), or other chelating agents as described herein or known to those skilled in the art, to provide a gemutuximab bioconjugate. A radioisotope may then be conjugated with the chelating portion of the conjugate.

[0016] As used herein, the term "chelate" or "chelating agent" refers to an organic compound or part thereof that can bond to a central metal or radioactive metal atom at two or more sites.

[0017] As used herein, the term "conjugate" or "bioconjugate" refers to a molecule containing a chelating group or its metal complex, a linking group, and optionally a therapeutic portion, a targeting portion, or a crosslinking group.

[0018] On the other hand, a method for obtaining the radiolabeled gemtuximab of the present invention is provided, wherein the method comprises astatinization of a cofactor, followed by conjugation of the cofactor to gemtuximab. In any embodiment, astatinization of the cofactor comprises the method described in WO2019 / 027059 or WO2017 / 089492 (incorporated herein by reference).

[0019] In alternative embodiments, the method comprises directly radiolabeling gemutuximab with the radioisotope 211-At. Alternatively, the method comprises radiolabeling a gemutuximab bioconjugate with the radioisotope 211-At. Optionally, the gemutuximab bioconjugate comprises a gemutuximab antibody conjugated with a prosthetic group (as described in WO2019 / 027059 or WO2017 / 089492) or any chelating or linking group suitable for further conjugation with a radioisotope.

[0020] On the other hand, a pharmaceutical composition is provided comprising a 211-At-geutzumab antibody or an antigen-binding fragment thereof as described herein, optionally in combination with a pharmaceutically acceptable excipient.

[0021] The present invention also provides a method for treating or preventing urothelial carcinoma in a subject or minimizing the progression of said urothelial carcinoma, the method comprising: - Administer 211-At-gegituximab antibody or its antigen-binding fragment to subjects in need. This can treat, prevent, or minimize the progression of the urothelial carcinoma in the subject.

[0022] On the other hand, the present invention provides a method for minimizing, reducing, or preventing the growth of urothelial carcinoma tumors in a subject, the method comprising: - Administer 211-At-gegituximab antibody or its antigen-binding fragment to subjects in need. This minimizes, reduces, or prevents the growth of urothelial carcinoma tumors in the subject.

[0023] On the other hand, the present invention provides a method for minimizing, reducing, or preventing urothelial carcinoma metastasis in a subject, the method comprising: - Administer 211-At-gegituximab antibody or its antigen-binding fragment to subjects in need. This minimizes, reduces, or prevents urothelial carcinoma metastasis in the subject.

[0024] On the other hand, the present invention provides a method for increasing the survival of subjects suffering from urothelial carcinoma, the method comprising: - Administer 211-At-gegituximab antibody or its antigen-binding fragment to subjects in need. This increases the survival time of the subjects.

[0025] On the other hand, the present invention provides the use of a 211-At-gegituximab antibody or its antigen-binding fragment thereof for the preparation of a medicament to achieve the following: - To treat, prevent, or minimize the progression of urothelial carcinoma in a subject. - To minimize, reduce, or prevent the growth of urothelial carcinoma tumors in the subject. - To minimize, reduce, or prevent urothelial carcinoma metastasis in the subject, or - Increased survival in subjects with urothelial carcinoma.

[0026] On the other hand, the present invention provides the use of a gemtuximab antibody bioconjugate conjugated with 211-At for the preparation of a drug to achieve the following: - To treat, prevent, or minimize the progression of urothelial carcinoma in a subject. - To minimize, reduce, or prevent the growth of urothelial carcinoma tumors in the subject. - To minimize, reduce, or prevent urothelial carcinoma metastasis in the subject, or - Increased survival in subjects with urothelial carcinoma.

[0027] On the other hand, the present invention provides the use of a 211-At-gebituximab antibody or its antigen-binding fragment thereof for: - To treat, prevent, or minimize the progression of urothelial carcinoma in a subject. - To minimize, reduce, or prevent the growth of urothelial carcinoma tumors in the subject. - To minimize, reduce, or prevent urothelial carcinoma metastasis in the subject, or - Increased survival in subjects with urothelial carcinoma.

[0028] On the other hand, the present invention provides a 211-At-gebituximab antibody or its antigen-binding fragment, which is used for: - To treat, prevent, or minimize the progression of urothelial carcinoma in a subject. - To minimize, reduce, or prevent the growth of urothelial carcinoma tumors in the subject. - To minimize, reduce, or prevent urothelial carcinoma metastasis in the subject, or - Increased survival in subjects with urothelial carcinoma.

[0029] In any of the methods or uses described herein, the urothelial carcinoma may be selected from bladder cancer, urethral cancer, ureteral cancer, or renal pelvis cancer. Preferably, the cancer is bladder cancer. Optionally, the subject may have bladder cancer and may be a candidate for radical cystectomy.

[0030] In any aspect or embodiment of the invention, it should be understood that the amount of 211-At-geutuzumab antibody or its antigen-binding fragment administered to the subject is a therapeutically effective amount of 211-At-geutuzumab antibody or its antigen-binding fragment.

[0031] In any aspect of this invention, any medicament or pharmaceutical composition described herein may be suitable for intraperitoneal, intratumoral, topical, oral, intravenous, or respiratory administration, preferably by inhalation or intranasal, subcutaneous, intramuscular, intracavitary, or intravesical administration. Typically, any medicament or pharmaceutical composition described herein is suitable for intravenous administration.

[0032] In any of the methods or uses described herein, the 211-At-geutuximab antibody or its antigen-binding fragment may be administered as the sole treatment for urothelial carcinoma or may be administered in combination with one or more other treatments. The 211-At-geutuximab antibody or its antigen-binding fragment may be administered following prior urothelial carcinoma treatment (optionally, said prior treatment includes radiosensitization therapy, surgery, chemotherapy, immunotherapy (such as Bacillus Calmette-Guerin, BCG), external beam radiation, autologous stem cell therapy, or combinations thereof). The 211-At-geutuximab antibody or its antigen-binding fragment may be administered concurrently or sequentially with other cancer treatments (optionally, said other treatments include surgery, chemotherapy, immunotherapy, external beam radiation, autologous stem cell therapy, or combinations thereof).

[0033] In any of the methods or uses described herein, the subject requiring treatment may have received prior treatment for urothelial carcinoma (such as surgery, chemotherapy, immunotherapy (such as BCG), external beam radiation, autologous stem cell therapy, or combinations thereof) but has not responded to the prior treatment. In a particularly preferred embodiment, the subject requiring treatment may have received prior treatment with BCG but is considered to have not responded to said treatment (in other words, the subject is a "non-responder to BCG treatment").

[0034] In yet another embodiment, a subject requiring treatment may be considered unlikely to respond to BCG treatment (e.g., as a result of previous screening or diagnostic methods).

[0035] It should be understood that, preferably according to the above aspects and embodiments, urothelial carcinoma is preferably a cancer that expresses or overexpresses carbonic anhydrase IX (CAIX).

[0036] In any embodiment, the urothelial carcinoma may be non-metastatic. In any embodiment, the urothelial carcinoma may be malignant or metastatic.

[0037] In any embodiment, the method further includes identifying a subject who requires the treatment described herein. The method may include identifying the subject as having urothelial carcinoma that expresses or overexpresses CAIX.

[0038] In any embodiment or aspect, the gemtuximab antibody or its antigen-binding fragment comprises: (a) a heavy chain variable domain (VH) comprising three complementarity-determining regions (CDRs) of an amino acid sequence as shown in any of SEQ ID NO: 4, 20, 36, 52 or 68; and / or (b) A light chain variable domain (VL) comprising three complementarity-determining regions (CDRs) of an amino acid sequence as shown in any of SEQ ID NO: 84, 100, 116, 132, 148 or 164.

[0039] In any embodiment, the antibody or its antigen-binding fragment comprises an antigen-binding domain that specifically binds to carbonic anhydrase IX (CAIX), and includes: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 -Linker - FR1a - CDR1a - FR2a - CDR2a - FR3a - CDR3a - FR4a in: FR1, FR2, FR3, and FR4 are each a frame region; CDR1, CDR2 and CDR3 are each complementary determinant regions; FR1a, FR2a, FR3a and FR4a are each a frame region; CDR1a, CDR2a and CDR3a are each complementarity-determining regions; The sequence of any of the complementarity-determining regions (CDRs) has the amino acid sequence described in Table 1 below. Preferably, the frame regions also have the amino acid sequence described in Table 1 below, including amino acid variations at specific residues, which can be determined by comparing various frame regions derived from each antibody. CDR1, CDR2, and CDR3 may be sequences from the VH, and CDR1a, CDR2a, and CDR3a may be sequences from the VL; alternatively, CDR1, CDR2, and CDR3 may be sequences from the VL, and CDR1a, CDR2a, and CDR3a may be sequences from the VH.

[0040] In any embodiment, the antigen or its antigen-binding fragment comprises: (i) VH, wherein VH comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR 1 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 1; wherein CDR 2 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 2; and wherein CDR 3 ... The sequence shown in 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%, or at least 99% identical; (ii) VH, wherein the VH comprises at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence shown in any of SEQ ID NO: 4, 20, 36, 52, or 68; (iii) VL, wherein VL comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 81, wherein CDR2 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 82, and CDR3 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 99% identical to the sequence shown in SEQ ID NO: 82, wherein CDR3 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, 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, wherein CDR3 comprises a sequence that is at least about 80%, at least 85%, at least The sequence shown in 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%, or at least 99% identical; (iv) VL, wherein the VL comprises at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence shown in SEQ ID NO: 84, 100, 116, 132, 148, or 164; (v) VH, which includes CDR1, CDR2 and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO: 1, CDR2 includes the sequence shown in SEQ ID NO: 2 and CDR3 includes the sequence shown in SEQ ID NO: 3; (vi) VH, wherein the VH comprises the sequence shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68; (vii) VL, which includes CDR1, CDR2 and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO: 81, CDR2 includes the sequence shown in SEQ ID NO: 82 and CDR3 includes the sequence shown in SEQ ID NO: 83; (viii) VL, wherein the VL comprises the sequence shown in any one of SEQ ID NO: 84, 100, 116, 132, 148 or 164; (ix) VH, wherein VH comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 comprises the sequence shown in SEQ ID NO: 2, and CDR3 comprises the sequence shown in SEQ ID NO: 3; and VL, wherein VL comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 81, CDR2 comprises the sequence shown in SEQ ID NO: 82, and CDR3 comprises the sequence shown in SEQ ID NO: 83; or (x) VH, which comprises the sequence shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68; and VL, which comprises the sequence shown in any one of SEQ ID NO: 84, 100, 116, 132, 148 or 164.

[0041] In another embodiment, the antibody or its antigen-binding fragment comprises: (i) VH, which comprises frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 comprises or is composed 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 or constitutes thereof, and FR2 comprises a sequence that is identical to or constitutes thereof as shown in SEQ ID NO: 9, 25, 41, 57, or 73. FR3 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, as shown in any of SEQ ID NO: 11, 27, 43, 59, or 75. FR4 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, as shown in any of SEQ ID NO: 11, 27, 43, 59, or 75. The sequences shown in any of 12, 28, 44, 60, or 76 are 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 or constitute a sequence thereof; and (ii) VL, wherein VL comprises frame regions (FR) 1, FR2, FR3, FR4, wherein FR1 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, and FR2 comprises a sequence that is identical to or consists of ... The FR3 contains at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences or constitutes sequences thereof, as shown in any of SEQ ID NO: The FR4 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof 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 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 or constitutes thereof a sequence that is at least about 80%, at least 81%, at least 82%, at least 8 ...

[0042] In another embodiment, the antibody or its antigen-binding fragment comprises: (i) VH, wherein VH comprises frame regions (FR) 1, FR2, FR3, FR4, wherein FR1 comprises or consists of a sequence as shown in or constitutes a sequence as shown in SEQ ID NO: 9, 25, 41, 57, or 73, wherein FR2 comprises or consists of a sequence as shown in or constitutes ... (ii) VL, which includes frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 includes or consists of a sequence as shown in SEQ ID NO: 89, 105, 121, 137, 153, or 169, FR2 includes or consists of a sequence as shown in any one of SEQ ID NO: 90, 106, 122, 138, 154, or 170, FR3 includes or consists of a sequence as shown in SEQ ID NO: 91, 107, 123, 139, 155, or 171, and FR4 includes or consists of a sequence as shown in SEQ ID NO: 92, 108, 124, 140, 156, or 172.

[0043] In any embodiment, the antibody or its antigen-binding fragment that specifically binds to CAIX comprises an amino acid sequence consisting of or composed of (in the order of N-terminus to C-terminus or C-terminus to N-terminus) essentially any one of SEQ ID NO: 4, 20, 36, 52 or 68 and / or any one of SEQ ID NO: 84, 100, 116, 132, 148, 164.

[0044] In any embodiment, the antibody or its antigen-binding fragment 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%, or at least 99% identical to or constitutes the sequence shown in SEQ ID NO: 4, 20, 36, 52, or 68; and / or (b) A light chain variable domain (VL) comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof.

[0045] As described herein, the gemtuximab antibody or its antigen-binding fragment may be in the following forms: (i) Single-domain antibodies (sdAb); (ii) Single-stranded Fv fragments (scFv); (iii) Dimer scFv (di-scFv); or (iv) One of (ii) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

[0046] Additionally, as described herein, the gemtuximab or its antigen-binding fragment may be in the following form: (i) Microantibodies (e.g., scFv-CH3); (ii) Bifunctional antibodies; (iii) Trifunctional antibodies; (iv) Four-function antibodies; (v)Fab; (vi)F(ab')2; (vii)Fv; (viii) Bispecific antibodies or other forms of multispecific antibodies; or (ix) One of (i) to (viii) connected to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

[0047] Optionally, the variable heavy region and variable light region of the antigen-binding domain are connected by a connector.

[0048] In any embodiment, the antigen-binding domain may include: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 -linker - FR1a - CDR1a - FR2a - CDR2a - FR3a - CDR3a - FR4a.

[0049] As defined herein, the linker may be chemical, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0050] Preferably, the gemutuximab is in the form of a complete IgG antibody.

[0051] In any aspect or embodiment of the invention, the gemutuximab or its antigen-binding fragment may comprise a human constant region, such as an IgG constant region, like IgG1, IgG2, IgG3, or IgG4 constant regions or mixtures thereof. When the antibody or protein contains V... H and V L In the case of V H It can be connected to the heavy chain constant region, and the V L It can be connected to the constant region of light chains.

[0052] In one instance, the gemutuximab or its antigen-binding fragment contains a constant region of an IgG4 antibody or a stable constant region of an IgG4 antibody. In one instance, the gemutuximab or its antigen-binding fragment contains an IgG4 constant region having a proline residue at position 241 (according to Kabat's numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991)).

[0053] In one instance, the gemutuximab or its antigen-binding fragment comprises a heavy chain constant region, the heavy chain constant region comprising a stable heavy chain constant region comprising a mixture of sequences having, or not having, a C-terminal lysine residue.

[0054] In any further embodiment of this document, the gemutuximab or its antigen-binding fragment comprises an Fc region, wherein the Fc region is engineered to have an enhanced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the enhanced ability to induce ADCC is conferred by mutation, deletion, or modification of amino acids in the Fc region that interact with the Fc receptor.

[0055] In another embodiment, the gemutuximab or its antigen-binding fragment comprises an Fc region, which is engineered to: - Has an increased in vitro or in vivo half-life; - Possesses an increased ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity; and / or - It has reduced effector functionality.

[0056] Preferably, the gemtuximab antibody is in the form of an antibody (i.e., containing a variable light chain and a variable heavy chain, linked to a constant region of an antibody including heavy chain CH2 and / or CH3).

[0057] In another embodiment, the gemutuximab may include a heavy chain constant region as defined in SEQ ID NO: 177, 178, 179 or 180 and / or a light chain constant region as defined in SEQ ID NO: 181.

[0058] In some embodiments, the gemutuximab may comprise a heavy chain as shown in any of SEQ ID NO: 182, 183, 184 or 185 and / or a light chain as shown in SEQ ID NO: 186.

[0059] In yet another embodiment, the gemutuximab is in the form of an antibody and may contain one or more amino acid substitutions in the constant region to reduce the antibody's in vivo half-life. Therefore, the present invention provides a gemutuximab antibody having substitutions in the CH2 and / or CH3 domains of the constant region and including substitutions at one or more of residues His310, His435, Tyr436, and Ile253 (Kabat number), thereby altering the FcRn binding affinity and / or serum half-life of the antibody (e.g., relative to gemutuximab containing native CH2 and CH3 domains).

[0060] In some instances, the amino acid at positions 310 and / or 435 of the antibody may be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine.

[0061] Preferably, the residue at position 310 is selected from alanine, glutamic acid, or glutamine; or amino acid residue 435 from the heavy chain constant region is selected from arginine, glutamine, or alanine. In other preferred embodiments, the antibody has an alanine residue at position 310 and a glutamine residue at position 435.

[0062] In another embodiment, the antibody further comprises an amino acid substitution at residue Lys322. Preferably, the substitution is K322A.

[0063] In a particularly preferred embodiment, the gemutuximab antibody comprises substituted K322A, H310A, and H435Q.

[0064] In preferred embodiments, the binding affinity of the modified antibody to FcRn and / or the serum half-life of the modified antibody is reduced by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times. In preferred embodiments of the invention, the binding affinity of the modified antibody to FcRn and / or the serum half-life of the modified antibody is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0065] In any aspect or embodiment, the antibody may also contain amino acid substitutions at residues corresponding to Ser228 and Leu235 in the constant heavy chain region, such as Ser228Pro and / or Leu235Glu.

[0066] As used herein, unless the context otherwise requires, the term “comprise” and variations thereof, such as “comprising,” “comprises,” and “comprised,” are not intended to exclude additional additions, components, wholes, or steps.

[0067] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. Attached Figure Description

[0068] Figure 1 RT112 cells exposed to 177 Lu-geutzximab ( 177 Lu-GmAb) or 211 At-geutzximab ( 211 Survival rate after At-GmAb.

[0069] Figure 2 Intrabladder instillation in mice at 30 minutes, 1 hour, and 4 hours (from left to right) 211 ID% / g after At-geutzumab.

[0070] Figure 3 Intrabladder instillation in mice at 30 minutes, 1 hour, and 4 hours (from left to right) 211 ID% / organ after At-geutzumab.

[0071] Figure 4 : A schematic diagram of the measurement schedule used in internalization studies.

[0072] Figure 5 : 125 Kinetics of I-geutuximab internalization in RT112 cells.

[0073] Figure 6 After a 2-hour washing step, RT112 cells were evaluated. 125 Uptake and elution kinetics of I-gegituximab. Detailed Implementation

[0074] This invention relates to the treatment of urothelial carcinomas (such as bladder cancer, urethral cancer, ureteral cancer, rectal cancer, renal pelvis cancer, and other cancers), for which effective oncology management options are currently lacking. Treatment innovation is needed to improve the morbidity and mortality rates of this indication.

[0075] Current treatments for urothelial carcinoma typically involve surgery, which may require subsequent chemotherapy and / or radiation therapy to prevent recurrence. In the case of bladder cancer, if the tumor cannot be easily removed, radical cystectomy may be necessary, but this is a significant surgical intervention that can have a profound impact on a patient's quality of life. For example, in men, radical cystectomy surgery often also involves the removal of the prostate and seminal vesicles. In women, radical cystectomy may also involve the removal of the ovaries, uterus, and part of the vagina.

[0076] The use of radiolabeled CAIX-binding antibodies in the treatment of renal cell carcinoma is known. However, prior to this invention, it was unknown whether molecular targeting of CAIX could be used to successfully treat other solid cancers that may express CAIX.

[0077] While CAIX is generally associated with advanced disease, it remains unclear whether molecular targeting of CAIX for radioimmunotherapy can be performed during the early stages of certain cancers, or whether cancer can only be targeted in advanced stages. Furthermore, some cancers exhibit reduced expression as the disease progresses, and therefore it is unclear whether CAIX targeting is a useful treatment for these types of cancer.

[0078] Furthermore, given the heterogeneity of many cancers, the mere presence of CAIX expression (as determined by immunohistochemistry) does not necessarily indicate that the cancer can be treated with molecularly targeted radioimmunotherapy. For example, in Huizing et al. (2021, Physics and Imaging in Radiation Oncology), Physics and Imaging in Radiation Oncology A recent study published in ( ) , 145-150) found 111In-labeled F(ab')2 form of the CAIX-binding antibody gemutuximab cannot distinguish between tumor cells and non-tumor cells, leading to the conclusion that this agent is unsuitable for imaging head and neck cancer. In contrast, the inventors have demonstrated that radiolabeled gemutuximab can be used to treat the same cancer cell type; demonstrating that results in diagnostic studies do not necessarily predict results in therapeutic studies.

[0079] Therefore, this invention is based on the discovery that radiolabeled antibodies binding to CAIX can indeed successfully treat specific cancers expressing CAIX. Furthermore, the inventors demonstrated a surprising therapeutic advantage when using 211-At-labeled gemutuximab to bind to CAIX, compared to antibodies labeled with alternative radioisotopes.

[0080] General definition

[0081] Throughout this specification, unless expressly stated otherwise or the context otherwise requires, references to a single step, a composition of substances, a group of steps, or a group of compositions of substances shall be deemed to cover one or more (i.e., one or more) of such steps, compositions of substances, groups of steps, or groups of compositions of substances. Therefore, as used herein, unless the context expressly indicates otherwise, the singular forms “a,” “an,” and “the” include the plural aspect, and vice versa. For example, reference to “a” includes one and two or more; reference to “an” includes one and two or more; reference to “the” includes one and two or more, and so on.

[0082] Those skilled in the art will understand that variations and modifications are readily possible beyond those specifically described. It should be understood that the invention encompasses all such changes and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, and any and all combinations of any two or more of said steps or features.

[0083] Those skilled in the art will recognize that many methods and materials can be similar to or equivalent to those described herein, and can be used to practice this invention. This invention is by no means limited to the methods and materials described.

[0084] All patents and publications mentioned in this article are incorporated herein by reference in their entirety.

[0085] This invention is not limited to the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are obviously within the scope of this invention.

[0086] Unless otherwise expressly stated, any instance or embodiment of the invention described herein, with necessary modifications, should be considered applicable to any other instance or embodiment of the invention.

[0087] Unless otherwise specified, all technical and scientific terms used herein should be regarded as having the same meaning as commonly understood by a person skilled in the art (e.g., in diagnostic techniques, radiographic imaging, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0088] The term “and / or” (e.g., “X and / or Y”) should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both or either of these meanings.

[0089] As used herein, the complementarity-determining region (CDR) sequence of an antigen-binding protein or antibody can be defined according to the IMGT, Chothia, or Kabat numbering system or any other CDR numbering system known to those skilled in the art or described herein.

[0090] As used herein, the term "complementarity-determining region" (synonymous with CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable region, the presence of which is a major factor in specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2, and CDR3. The CDRs of VH are also referred to herein as CDR H1, CDR H2, and CDR H3, respectively, where 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. (Alternatively, CDRs may be named HCDR1, HCDR2, and HCDR3). Similarly, the CDRs of VL are referred to herein as CDR L1, CDR L2, and CDR L3, respectively, where 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. (Alternatively, the CDRs in the light variable region can be named LCDR1, LCDR2, and LCDR3).

[0091] “Frame” or “FR” residues are those variable domain residues other than the hypervariable regions or CDR residues defined herein. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, where FR H1 corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 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 FR L1, FR L2, FR L3, and FR L4, respectively, where 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.

[0092] In any instance, the amino acid positions assigned to CDR and FR can be defined according to Kabat, Sequences of Immunologically Significant Proteins, National Institutes of Health, Bethesda, Maryland, 1987 and 1991 (also referred to herein as the “Kabat Numbering System”). It should be understood that, according to the present invention, the system used to define FR and CDR is not limited to the Kabat numbering system, but includes all numbering systems, including the normative numbering systems in the following literature: the numbering systems of Chothia and Lesk, Journal of Molecular Biology 196: 901-917, 1987; Chothia et al., Nature 342: 877-883, 1989; and / or Al-Lazikani et al., Journal of Molecular Biology 273: 927-948, 1997; Honnegher and Plükthun, Journal of Molecular Biology 309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211 1997. In another instance, the amino acid positions assigned to CDRs and FRs can be defined according to the Martin (Enhanced Chothia) numbering scheme (http: / / www.bioinfo.org.uk / abs / info.html). In some instances, CDRs can be defined according to the AbM numbering system. The AbM system represents a compromise between the Kabat and Chothia structural loops and is used through Oxford Molecular's AbM antibody modeling software. In some instances, a CDR can be a "contact" CDR. A "contact" CDR is based on the analysis of available complex crystal structures.

[0093] In one instance, the CDR is defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not contain 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 of Experimental Biology (FASEB J.), 9: 133-139, 1995, demonstrated that the five C-terminal amino acids of heavy chain CDR2 are generally not involved in antigen binding.

[0094] The different systems used for dispensing CDRs and FRs of antigen-binding domains are well known to those skilled in the art and are outlined in the table below:

[0095] 1 Some of these definitions (especially the definition of the Chothia ring) vary depending on the individual publications examined.

[0096] 2 When using the Kabat numbering convention, the end of the Chothia CDR H1 ring varies between H32 and H34 depending on the ring length. This is because the Kabat numbering scheme places insertions at H35A and H35B: if neither H35A nor H35B exists, the ring ends at H32; if only H35A exists, the ring ends at H33; and if both H35A and H35B exist, the ring ends at H34.

[0097] Urothelial carcinoma

[0098] This invention relates to a method for treating urothelial carcinoma and a radiolabeled gemtuximab antibody.

[0099] Urothelial carcinoma can be bladder cancer, urethral cancer, ureteral cancer, or renal pelvis cancer.

[0100] In a preferred embodiment, urothelial carcinoma is bladder cancer. In any embodiment, bladder cancer can be: - Urothelial carcinoma (80-90% of bladder cancers) - begins with urothelial cells lining the bladder wall and is sometimes called transitional cell carcinoma.

[0101] Squamous cell carcinoma (which accounts for about 5% of all bladder cancers) begins with thin, flat squamous cells in the bladder lining and is more likely to be invasive; this type of cancer can occur in people with a history of bladder inflammation and irritation.

[0102] - Adenocarcinoma (accounting for about 1% of all bladder cancers) - develops from glandular cells in the bladder and can be invasive.

[0103] - Sarcoma (usually found in the bladder muscle) or invasive forms of bladder cancer, such as small cell carcinoma, plasmacytoid carcinoma, or micropapillary carcinoma.

[0104] Bladder cancer is also typically classified as non-invasive, non-muscle-invasive, or muscle-invasive.

[0105] Noninvasive bladder cancer typically comprises a tumor located only in or near a small portion of the tissue on or near the surface of the bladder. In any embodiment of the invention, bladder cancer can be noninvasive bladder cancer.

[0106] Non-muscle-invasive bladder cancer (NMIBC) refers to bladder cancer that has metastasized deep into the bladder but has not yet spread to the muscles. In any embodiment of the invention, bladder cancer can be non-muscle-invasive bladder cancer (NMIBC). NMIBC is a cancer found in the urothelium (the tissue lining the inner surface of the bladder).

[0107] Muscle-invasive bladder cancer is bladder cancer that has grown into the muscles of the bladder wall and may have spread to the fatty layer or tissue of organs outside the bladder. In any embodiment of the invention, the bladder cancer may be muscle-invasive bladder cancer.

[0108] Technicians will be familiar with the typical procedures used to determine whether a subject has bladder cancer, and therefore the subject will require treatment according to the present invention. Diagnostic methods for detecting bladder cancer include observing the initial symptoms of bladder cancer, such as hematuria, changes in bladder habits, or other symptoms (such as pain) or persistent bladder infection, and / or determining whether the subject is at risk of developing bladder cancer. Risk factors for bladder cancer include smoking, radiation exposure, chemotherapy, exposure to certain chemicals (dyes, rubber and textiles, and hair care products), frequent bladder infections, and prolonged catheterization.

[0109] Diagnosis of bladder cancer typically involves urinalysis, cytology, endoscopic procedures (such as cystoscopy), or imaging procedures such as ultrasound, CT, PET / SPECT, or MRI. Transurethral resection of bladder tumor (TURBT) can be used to remove part or all of the bladder tumor for subsequent testing.

[0110] Carbonic anhydrase IX

[0111] As used in this article, carbonic anhydrase is also referred to as: CA-IX, CA9, CAIX, carbonic anhydrase IX, carbonic anhydrase 9, carbonic anhydrase IX, carbonic anhydrase, G250, membrane antigen MN, P54 / 58N, pMW1, RCC-associated antigen G250, RCC-associated protein G250, and renal cell carcinoma-associated antigen G250.

[0112] Cancer cells primarily express 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 because the expression of this isoform in healthy tissues is limited to epithelial cells of the stomach and intestines, but it is strongly upregulated in renal cell carcinoma.

[0113] CAIX expression is controlled by hypoxia-inducible factor 1 (HIF-1), which is mainly located in chronically hypoxic tumor areas. However, CAIX can also be found in mildly hypoxic or even normoxic areas because CAIX expression can be activated by components of the mitogen-activated protein kinase (MAPK) pathway.

[0114] Getuximab

[0115] This invention relates to 211-astatine-labeled gemtuximab or its antigen-binding fragment, and its use in the methods described herein.

[0116] Gelatinumab is an antibody that binds specifically to CAIX and is sometimes referred to as an anti-G250 antibody (and sometimes as a G250 antibody). Anti-G250 antibodies are described, for example, in EP-B-0 637 336. The antibody or a fragment thereof can be a chimeric (cG250) or humanized G250 (hG250) antibody. The cG250 antibody is a chimeric version of the IgG1κ light chain of the proto-murretic monoclonal antibody mG250.

[0117] In some embodiments, antigen-binding proteins that bind to or specifically bind to CAIX, such as those described in WO 2002 / 062972A2 (US 2004 / 0219633 A1), WO 2004 / 002526 A1 (US 7,632,496 B2), WO 2006 / 002889 A2 (US 7,691,375 B2), WO 2009 / 056342 A1 (US 2014 / 0017252 A1), WO 2011 / 032973 A1 (US2012 / 0207672 A1), and WO 2014 / 128258 A1 (US 10,620,208 B2) or WO 2021 / 000017 A1, the entire contents of each of these publications are incorporated herein by reference.

[0118] The antibodies used in this invention can be produced by any suitable method known in the art, including but not limited to the methods described in PCT / EP02 / 01282 and PCT / EP02 / 01283, which are incorporated herein by reference.

[0119] Variants of the original chimeric G250 (cG250) antibody, including WX-G250 and WX-G250RIT (Janssen Global Services LLC), are known.

[0120] In any embodiment, the gemutuximab antibody or its antigen-binding fragment comprises: (a) a heavy chain variable domain (VH) comprising three complementarity-determining regions (CDRs) of an amino acid sequence as shown in any of SEQ ID NO: 4, 20, 36, 52 or 68; and / or (b) A light chain variable domain (VL) comprising three complementarity-determining regions (CDRs) of an amino acid sequence as shown in any of SEQ ID NO: 84, 100, 116, 132, 148 or 164.

[0121] In any embodiment, the antibody or its antigen-binding fragment comprises an antigen-binding domain that specifically binds to carbonic anhydrase IX (CAIX), and includes: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 -Linker - FR1a - CDR1a - FR2a - CDR2a - FR3a - CDR3a - FR4a in: FR1, FR2, FR3, and FR4 are each a frame region; CDR1, CDR2 and CDR3 are each complementary determinant regions; FR1a, FR2a, FR3a and FR4a are each a frame region; CDR1a, CDR2a and CDR3a are each complementarity-determining regions; The sequence of any of the complementarity-determining regions (CDRs) has the amino acid sequence described in Table 1 below. Preferably, the frame regions also have the amino acid sequence described in Table 1 below, including amino acid variations at specific residues, which can be determined by comparing various frame regions derived from each antibody. CDR1, CDR2, and CDR3 may be sequences from the VH, and CDR1a, CDR2a, and CDR3a may be sequences from the VL; alternatively, CDR1, CDR2, and CDR3 may be sequences from the VL, and CDR1a, CDR2a, and CDR3a may be sequences from the VH.

[0122] In any embodiment, the antigen or its antigen-binding fragment comprises: (i) VH, wherein VH comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR 1 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 1; wherein CDR 2 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 2; and wherein CDR 3 ... The sequence shown in 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%, or at least 99% identical; (ii) VH, wherein the VH comprises at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence shown in any of SEQ ID NO: 4, 20, 36, 52, or 68; (iii) VL, wherein VL comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 81, wherein CDR2 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 82, and CDR3 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 99% identical to the sequence shown in SEQ ID NO: 82, wherein CDR3 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, 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, wherein CDR3 comprises a sequence that is at least about 80%, at least 85%, at least The sequence shown in 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%, or at least 99% identical; (iv) VL, wherein the VL comprises at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence shown in SEQ ID NO: 84, 100, 116, 132, 148, or 164; (v) VH, which includes CDR1, CDR2 and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO: 1, CDR2 includes the sequence shown in SEQ ID NO: 2 and CDR3 includes the sequence shown in SEQ ID NO: 3; (vi) VH, wherein the VH comprises the sequence shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68; (vii) VL, which includes CDR1, CDR2 and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO: 81, CDR2 includes the sequence shown in SEQ ID NO: 82 and CDR3 includes the sequence shown in SEQ ID NO: 83; (viii) VL, wherein the VL comprises the sequence shown in any one of SEQ ID NO: 84, 100, 116, 132, 148 or 164; (ix) VH, wherein VH comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 comprises the sequence shown in SEQ ID NO: 2, and CDR3 comprises the sequence shown in SEQ ID NO: 3; and VL, wherein VL comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 81, CDR2 comprises the sequence shown in SEQ ID NO: 82, and CDR3 comprises the sequence shown in SEQ ID NO: 83; or (x) VH, which comprises the sequence shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68; and VL, which comprises the sequence shown in any one of SEQ ID NO: 84, 100, 116, 132, 148 or 164.

[0123] In another embodiment, the antibody or its antigen-binding fragment comprises: (i) VH, which comprises frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 comprises or is composed 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 or constitutes thereof, and FR2 comprises a sequence that is identical to or constitutes thereof as shown in SEQ ID NO: 9, 25, 41, 57, or 73. FR3 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, as shown in any of SEQ ID NO: 11, 27, 43, 59, or 75. FR4 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, as shown in any of SEQ ID NO: 11, 27, 43, 59, or 75. The sequences shown in any of 12, 28, 44, 60, or 76 are 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 or constitute a sequence thereof; and (ii) VL, wherein VL comprises frame regions (FR) 1, FR2, FR3, FR4, wherein FR1 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, and FR2 comprises a sequence that is identical to or consists of ... The FR3 contains at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences or constitutes sequences thereof, as shown in any of SEQ ID NO: The FR4 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof 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 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 or constitutes thereof a sequence that is at least about 80%, at least 81%, at least 82%, at least 8 ...

[0124] In another embodiment, the antibody or its antigen-binding fragment comprises: (i) VH, wherein VH comprises frame regions (FR) 1, FR2, FR3, FR4, wherein FR1 comprises or consists of a sequence as shown in or constitutes a sequence as shown in SEQ ID NO: 9, 25, 41, 57, or 73, wherein FR2 comprises or consists of a sequence as shown in or constitutes ... (ii) VL, which includes frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 includes or consists of a sequence as shown in SEQ ID NO: 89, 105, 121, 137, 153, or 169, FR2 includes or consists of a sequence as shown in any one of SEQ ID NO: 90, 106, 122, 138, 154, or 170, FR3 includes or consists of a sequence as shown in SEQ ID NO: 91, 107, 123, 139, 155, or 171, and FR4 includes or consists of a sequence as shown in SEQ ID NO: 92, 108, 124, 140, 156, or 172.

[0125] In any embodiment, the antibody or its antigen-binding fragment that specifically binds to CAIX comprises an amino acid sequence consisting of or composed of (in the order of N-terminus to C-terminus or C-terminus to N-terminus) essentially any one of SEQ ID NO: 4, 20, 36, 52 or 68 and / or any one of SEQ ID NO: 84, 100, 116, 132, 148, 164.

[0126] In any embodiment, the antibody or its antigen-binding fragment 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%, or at least 99% identical to or constitutes the sequence shown in SEQ ID NO: 4, 20, 36, 52, or 68; and / or (b) A light chain variable domain (VL) comprising or consisting of at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence shown in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

[0127] In any embodiment, the antibody or its antigen-binding fragment for binding to CAIX may be in the following form: (i) Single-chain Fv fragments (scFv); (ii) Dimer scFv (di-scFv); or (iii) One of (i) or (ii) connected to the constant region of the antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0128] In any embodiment, the antibody or its antigen-binding fragment for binding to CAIX may be in the following form: (i) Bifunctional antibody; (ii) Trifunctional antibodies; (iii) Four-functional antibodies; (iv) Fab; (v)F(ab')2; (vi) Fv; or (vii) One of (i) to (vi) connected to the constant region of the antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0129] In any embodiment, the antibody or antigen-binding fragment thereof used according to the present invention may be a fusion protein comprising, as described herein, an antigen-binding protein, an immunoglobulin variable domain, an antibody, a dab (single-domain antibody), a bis-scFv, scFv, Fab, Fab', F(ab')2, an Fv fragment, a bifunctional antibody, a trifunctional antibody, a tetrafunctional antibody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody.

[0130] Antigen-binding fragments, immunoglobulin variable domains, antibodies, dab, bis-scFv, scFv, Fab, Fab', F(ab')2, Fv fragments, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, linear antibodies, single-chain antibody molecules or multispecific antibodies, fusion proteins or conjugates, as described herein, can be obtained by expressing the nucleic acid encoding them.

[0131] Antibodies or antigen-binding fragments thereof, as described herein, may contain human constant regions, such as IgG constant regions, such as IgG1, IgG2, IgG3, or IgG4 constant regions, or mixtures thereof. In cases where the antibody or protein contains VH and VL, the VH may be linked to the heavy chain constant region, and the VL may be linked to the light chain constant region.

[0132] In one instance, an antibody or its antigen-binding fragment as described herein comprises a heavy chain constant region, which includes a stable heavy chain constant region comprising a mixture of sequences having, or not having, a C-terminal lysine residue.

[0133] In one instance, an antibody or antigen-binding fragment thereof as described herein comprises a VH disclosed herein linked or fused to an IgG4 constant region or a stable IgG4 constant region (e.g., as discussed above), and a VL linked or fused to a κ light chain constant region.

[0134] The functional properties of the antigen-binding fragments described herein will be considered applicable to antibodies as described herein after necessary modifications.

[0135] Antibodies or antigen-binding fragments thereof used for the purposes described herein may be purified, substantially purified, isolated and / or recombinant.

[0136] Table 1: Overview of the preferred amino acid and nucleotide sequences of CAIX-binding antibodies

[0137] constant region

[0138] In a preferred embodiment, the antibody and / or its antigen-binding fragment used in the present invention as described herein may comprise a constant region of the antibody. This includes the antigen-binding fragment of an antibody fused to an Fc cell.

[0139] Sequences that can be used to generate constant regions of antibodies or antigen-binding fragments thereof as described herein can be obtained from many different sources. In some instances, the constant region of a protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one instance, the constant region is human isotype IgG4 or the stable IgG4 constant region.

[0140] The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. The function of FcRn is to rescue IgG from lysosomal degradation pathways, resulting in reduced clearance and prolonged half-life. It is a heterodimeric protein composed of two polypeptides: a 50 kDa major histocompatibility complex-like protein of class I (α-FcRn) and a 15 kDa p2-microglobulin (β2η). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG antibodies. The interaction between IgG antibodies and FcRn is pH-dependent and occurs at a stoichiometric ratio of 1:2, meaning that one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc region polypeptides (see, for example, Huber, AH et al., Journal of Molecular Biology 230(1993) 1077-1083).

[0141] Therefore, the in vitro FcRn binding properties / characteristics of IgG indicate its in vivo pharmacokinetic properties in blood circulation. Different amino acid residues of the heavy chain CH2- and CH3- domains are involved in the interaction between FcRn and the Fc region of IgG antibodies.

[0142] Different mutations affecting FcRn binding and half-life in the bloodstream are known. Fc region residues that are crucial for mouse Fc region-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF et al., Journal of Immunology 169 (2002) 5171-5180). Residues Ile253, His310, His433, Asn434, and His435 (numbered according to the EU indexing system) are involved in interactions (Medesan, C et al., *Eur. J. Immunol.* 26 (1996) 2533-2536; Firan, M. et al., *Int. Immunol.* 13 (2001) 993-1002; Kim, JK et al., *Eur. J. Immunol.* 24 (1994) 542-548). (Using the Kabat system, the relevant residues are Ile266, His329, His464, Asn465, and His466). The residues Ile253, His310, and His435 were found to be crucial for the interaction between the human Fc region and the mouse FcRn (Kim, JK et al., European Journal of Immunology 29 (1999) 2819-2885).

[0143] More specifically, antibodies or antigen-binding proteins may contain one or more amino acid substitutions that reduce the protein's half-life. For example, an antibody or its antigen-binding fragment may contain an Fc region containing one or more amino acid substitutions that reduce the affinity of the Fc region for the neonatal Fc region (FcRn).

[0144] Preferred modifications

[0145] In any embodiment, the antibody or its antigen-binding fragment (e.g., the G250 antibody or a variant thereof as described herein) is a modified IgG antibody or fragment thereof that, compared to wild-type IgG antibodies, comprises a heavy chain constant region having one or more amino acid substitutions, wherein the one or more amino acid substitutions reduce the antibody's affinity for neonatal Fc receptors (FcRn) compared to wild-type IgG antibodies, thereby reducing the serum half-life of the modified antibody.

[0146] In one embodiment, the one or more amino acid substitutions are selected from substitutions in the heavy chain constant region 2 (CH2) of the IgG molecule, thereby reducing the affinity of the IgG molecule for 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 for FcRn. Still further, the amino acid substitutions may include at least one substitution in the CH2 region and at least one substitution in the CH3 region of the IgG molecule, thereby reducing the affinity of IgG for FcRn.

[0147] In some preferred embodiments, the one or more amino acid substitutions may be located at one or more of the 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 antibody's affinity for FcRn are located at both His310 and His435.

[0148] In other preferred embodiments, the antibody and / or its antigen-binding fragment has a constant region substantially identical to that of naturally occurring IgG antibodies, wherein at least one amino acid residue selected from the group consisting of residues His310, His435, and Ile253 differs from amino acid residues present in naturally occurring IgG antibodies, thereby altering the FcRn binding affinity and / or serum half-life of the antibody relative to naturally occurring antibodies. In preferred embodiments, the naturally occurring IgG antibodies comprise the heavy chain constant region of human IgG1, IgG2, IgG2M3, IgG3, or IgG4 molecules.

[0149] Also in a preferred embodiment, amino acid residues 310 and / or 435 of the heavy chain constant region of an antibody having a constant region substantially identical to that of naturally occurring IgG antibodies are any amino acids that are not histidine and reduce the affinity of the constant region for FcRn. For example, the amino acids at residues 310 and / or 435 can be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine.

[0150] Amino acid substitutions may include substitutions from histidine residues to alanine, glutamine, glutamic acid, or aspartic acid. Preferably, the amino acid substitution at His310 is alanine. Preferably, the amino acid substitution at His435 is glutamine. Preferably, the amino acid substitution at Ile253 is alanine.

[0151] In a preferred embodiment of the invention, the binding affinity of the modified antibody to FcRn and / or its serum half-life is reduced by at least about 30%, 50%, 80%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold. In a preferred embodiment of the invention, the binding affinity of the modified antibody to FcRn and / or its serum half-life is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0152] Furthermore, the antibodies or fragments thereof used according to the present invention can be modified to include one or more mutations that alter the antibody's affinity for any or more Fcγ receptors. For example, the one or more amino acid modifications alter the affinity of the antibody's constant domain, Fc region, or Fcγ receptor-binding fragment for any or more Fcγ receptors.

[0153] In some embodiments, the modified antibody or its antigen-binding fragment retains the ability to bind to one or more Fc-γ receptors, and therefore in some embodiments, the modified antibody retains the ability to stimulate effector responses (including ADCC). In one example, the Fc region of the constant region contains one or more amino acid substitutions that regulate effector function, including increased effector function compared to wild-type IgG.

[0154] In one instance, for example, the Fc region of a constant region may have a reduced ability to induce effector function compared to the Fc region of natural or wild-type human IgG1 or IgG3. In one instance, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the effector function levels of Fc-containing proteins are known in the art and / or described herein.

[0155] In one example, the amino acid substitution that alters the ability of the antibody-induced effector to function is an amino acid substitution at residue Ile253 in the heavy chain constant region. In one example, the substitution is any amino acid selected from alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine, wherein the substitution reduces the ability of the antibody-induced effector to function. In a preferred embodiment, the substitution of Ile at residue 253 is arginine, proline, glutamic acid, or aspartic acid, more preferably alanine.

[0156] In one instance, the Fc region is the IgG4 Fc region (i.e., derived from the IgG4 constant region), such as the human IgG4 Fc region. The sequence of a suitable IgG4 Fc region is obvious to a person skilled in the art and / or available from publicly available databases (e.g., from the National Center for Biotechnology Information).

[0157] In one instance, the constant region is the stable IgG4 constant region. The term "stable IgG4 constant region" will be understood as referring to an IgG4 constant region that has been modified to reduce or undergo Fab arm exchange, or to have a tendency to form a hapten. "Fab arm exchange" refers to a type of protein modification against human IgG4 in which the IgG4 heavy chain and its linked light chain (half a molecule) are exchanged for a heavy-light chain pair from another IgG4 molecule. Thus, the IgG4 molecule can acquire two different Fab arms that recognize two different antigens (producing a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro using purified blood cells or reducing agents such as reduced glutathione. A "hapten" is formed when an IgG4 antibody dissociates to form two molecules, each containing a heavy chain and a light chain.

[0158] In one instance, according to Kabat's system (Kabat et al., Sequences of Immunologically Significant Proteins, U.S. Department of Health and Human Services, District of Columbia, Washington, 1987 and / or 1991), the stable IgG4 constant region contains 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 typically serine. Following the substitution of proline with serine, the IgG4 hinge region contains the sequence CPPC (SEQ ID NO: 187). In this respect, those skilled in the art will recognize that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region connecting the Fc and Fab regions, which imparts fluidity to the two Fab arms of the antibody. The hinge region includes cysteine ​​residues involved in inter-heavy chain disulfide bonds. According to Kabat's numbering system, it is generally defined as extending from Glu226 to Pro243 of human IgG1 (or from Glu216 to Pro230 using the EU index). By placing the first and last cysteine ​​residues that form the heavy chain disulfide bond (SS) in the same position, the hinge region of other IgG isotypes can be aligned with the IgG1 sequence (see, for example, WO2010 / 080538).

[0159] 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 antibodies, the modified antibody or its antigen-binding fragment may further contain one or more amino acid substitutions, wherein the amino acid substitutions further reduce the antibody's affinity for one or more Fcγ receptors.

[0160] In another embodiment, compared to wild-type IgG antibodies, the modified antibody or its antigen-binding fragment further comprises one or more amino acid substitutions, wherein the amino acid substitutions increase the stability of the CH1-CH2 hinge region in the modified antibody compared to wild-type IgG antibodies.

[0161] In any embodiment, the constant region of the heavy chain of the antibody or antigen-binding protein includes amino acid substitutions at both His310 and His435. The antibody may also include amino acid substitutions at residues corresponding to Ser228 and Leu235 of the constant heavy chain region.

[0162] In any embodiment, the antibody or its antigen-binding fragment contains mutations at Ser228, Leu235, His310, and His435. Preferably, the amino acid modification is Ser228Pro, Leu235Glu, His310Ala, and His435Gln.

[0163] Another example of a stable IgG4 antibody is an antibody in which arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) is replaced by lysine, threonine, methionine, or leucine (e.g., as described in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively contain residues 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 contains proline at position 241 (i.e., the CPPC sequence, SEQ ID NO: 187) (as described above).

[0164] In another instance, the Fc region is a modified region with reduced effector function, i.e., a “non-immunostimulatory Fc region.” For example, the Fc region is an IgG1 Fc region containing substitutions at one or more sites selected from the group consisting of 268, 309, 330, and 331. In another instance, the Fc region is an IgG1 Fc region containing the deletion of one or more of the following variations: E233P, L234V, L235A, and G236, and / or one or more of the following variations: A327G, A330S, and P331S (Armour et al., *European Journal of Immunology*. 29:2613-2624, 1999; Shields et al., *Journal of Biochemistry*. 276(9):6591-604, 2001). Other examples of non-immunostimulatory Fc regions are described, for example, in the following literature: Dall'Acqua et al., Journal of Immunology. 177: 1129-1138 2006; and / or Hezareh, Journal of Virology (JVirol); 75: 12161-12168, 2001.

[0165] In another example, the Fc region is a chimeric Fc region, for example, comprising at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, wherein the Fc region comprises substitutions at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbers) (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.

[0166] Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region, said heavy chain constant region comprising a sequence as shown in any one of SEQ ID NO: 177 to 180, preferably as shown in SEQ ID NO: 178.

[0167] In yet another embodiment, the antibody or its antigen-binding fragment preferably comprises a heavy chain, said heavy chain comprising the sequence shown in any one of SEQ ID NO: 182 to 185, preferably as shown in SEQ ID NO: 183.

[0168] In any embodiment, the antibody or its antigen-binding fragment comprises a light chain constant region containing an amino acid sequence as shown in SEQ ID NO: 181. Preferably, the antibody or antigen-binding protein comprises a light chain containing an amino acid sequence as shown in SEQ ID NO: 186.

[0169] In any embodiment, the antibody or its antigen-binding fragment comprises the sequence shown in SEQ ID NO: 183 and the sequence shown in SEQ ID NO: 186.

[0170] In one embodiment, the antibody or its antigen-binding fragment comprises: VH, the VH comprising at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO: 36 or 52, or including the sequence shown therein; and VL, the VL comprising at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO: 116, 132 or 148, or including the sequence shown therein.

[0171] Preferably, VH contains at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO: 36 or 52, or contains the sequence shown therein, and VL contains at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO: 132 or 148, or contains the sequence shown therein.

[0172] More preferably, the VH contains at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO: 36, or contains the sequence shown therein, and the VL contains at least about 95%, or 96%, or 97%, or 98% or 99% of the sequence shown in SEQ ID NO: 148, or contains the sequence shown therein.

[0173] Alternatively, the VH comprises at least about 95%, or 96%, or 97%, or 98% or 99% identical to or includes the sequence shown therein of the sequence shown in SEQ ID NO: 52, and the VL comprises the sequence shown in SEQ ID NO: 132 or 148, preferably at least about 95%, or 96%, or 97%, or 98% or 99% identical to or includes the sequence shown therein of the sequence shown in SEQ ID NO: 148.

[0174] Radiolabeled antibodies

[0175] Technicians will be familiar with standard methods for conjugating detectable components (such as radionuclides (radiolabeled)) to antibodies or their antigen-binding fragments.

[0176] As used herein, the term radionuclide may be used interchangeably with the terms radioisotope and radiolabel.

[0177] In any embodiment of the invention, the radiolabel may be conjugated to gemutuximab or its antigen-binding fragment, directly by binding to a single or multiple amino acid residues in a protein (e.g., halogenation of tyrosine residues) or indirectly (through a chelating agent, prosthetic group, or linker) to a radioisotope (i.e., the radiolabel).

[0178] Examples of methods for astatylation of auxiliary groups (e.g., diaryliodocation salts) are known in the art (e.g., in WO2019027059 and WO2017 / 089492, which are incorporated herein by cross-reference in their entirety).

[0179] Examples of suitable chelating agents or linkers can be selected from the group consisting of: TMT (6,6"-bis[N,N",N"'-tetra(carboxymethyl)aminomethyl]-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid, also known as tetraxetan), TCMC (tetraamine of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), NOA (1,4,7-triazacyclononane-triacetic acid), Diam Sar (3,6,10,13,16,19-hexaazabicyclo[6,6,6]eicosano-1,8-diamine), DTPA (pentetate or diethylenetriaminepentaacetic acid), CHX-A"-DTPA ([(R)-2-amino-3-(4-isothiocyanophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid), TETA (1,4,8,11-tetraazabicyclotetradecane-1,4,8,11-tetraacetic acid), Te2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), HBED, DFO (deferroamine), DFOsq (DFO-squamamide), and HOPO (3,4,3-(LI-1,2-HOPO)) or other chelating agents as described herein. Other known chelating moieties include 3p- C-NETA ({4-[2-(bis-carboxymethylamino)-5-(4-nitrophenyl)pentyl]-7-carboxymethyl-[1,4,7]triazanon-1-yl}acetic acid), 5p- C -NETA(2-({1-[4,7- pair (carboxymethyl)-1,4,7-triazanon-1-yl]-7-(4-nitrophenyl)hept-2-yl}(carboxymethyl)amino)acetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and NODA (1,4,7-triazacyclononane-1,4-diacetic acid). Other suitable chelating agents (e.g., comprising DOTA and DFO) are disclosed in WO 2022 / 133537, which is incorporated herein by reference.

[0180] In any embodiment, radiolabeling of proteins or antibodies can be accomplished by covalent iodination, particularly using chloroglyurea reagent (1,3,4,6-tetrachloro-3a,6a-diphenylglyurea). Although chloroglyurea labeling is a solid-phase oxidation method similar to the chloramine-T method, it is generally considered milder because the reaction takes place on the oxidant surface, thus minimizing substrate exposure (Salacinzki, PRP et al., Analytical Biochemistry 117:136 (1981)).

[0181] Chelating agents containing radioactive metals and other halogenated radioisotopes can bind to antibodies through one or more amino acid residues or reactive moieties in proteins / antibodies, including but not limited to one or more lysine residues, tyrosine residues, or thiol moieties.

[0182] In another instance, the antibody may be conjugated to a bifunctional linker, such as bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, or any amine or thiol-modified chemical known in the art.

[0183] Those skilled in the art will be familiar with standard methods for conjugating chelating agents with antibodies and their derivatives or fragments. Additionally, those skilled in the art will be familiar with methods for selecting appropriate chelating agents for pairing with radioactive metals, for example, as described in *Chem. Soc. Rev.*, 2014, 43, 260, which is incorporated herein by reference.

[0184] Administration and treatment of radiolabeled gemtuximab

[0185] Methods for preparing antigen-binding proteins into forms suitable for administration to subjects (e.g., pharmaceutical compositions) are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th edition, Mack Publishing Co., Easton, PA, 1990) and US Pharmacopeia: National Formulary (Mack Publishing Co., Easton, PA, 1984).

[0186] Radiolabeled gemutuximab antibodies are typically administered as pharmaceutical compositions with pharmaceutically acceptable carriers (e.g., physiological saline) or other aqueous carriers, optionally containing protein stabilizers such as human serum albumin (HSA). The compositions may contain pharmaceutically acceptable excipients close to physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antigen-binding protein according to the invention in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., depending on the specific route of administration and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextran solution, and 5% human serum albumin. Non-aqueous mediators such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. Mediators may contain small amounts of additives to enhance isotonicity and chemical stability, such as buffers and preservatives.

[0187] Administration means the physical introduction of a composition containing a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art, including those described herein. The pharmaceutical composition may be formulated with active agents of the invention as described herein for use via any suitable route of administration.

[0188] Radiolabeled gemtuximab is preferably administered intravenously, preferably by infusion or intravenous injection. Infusion administration of the antibody is preferably performed over a period of up to about 30 minutes, more preferably within about 15 minutes. Of course, radiolabeled gemtuximab can also be administered via subcutaneous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, intracavitary, intravesical, and intracranial injection or infusion techniques.

[0189] The phrase 'therapeutic effective dose' or 'effective dose' generally refers to the amount of radiolabeled gemtuximab or its antigen-binding fragment thereof, which (i) treats a specific disease, symptom, or condition, (ii) reduces, improves, or eliminates one or more symptoms of a specific disease, symptom, or condition, or (iii) delays the onset of one or more symptoms of a specific disease, symptom, or condition described herein. Undesirable effects, such as side effects, sometimes occur alongside desired therapeutic effects; therefore, practitioners weigh potential benefits against potential risks when determining what constitutes an appropriate 'effective dose'.

[0190] For example, in the treatment of tumors, a therapeutically effective amount of the radiolabeled antibody 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 last for at least about 10 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, 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 longer.

[0191] Therapeutic effective amounts of the drug may also include “prophylactic” or “preventive effective amounts,” which are any amounts of radiolabeled gemutuximab or its antigen-binding fragments administered to subjects at risk of developing cancer (e.g., subjects with pre-malignant conditions) or with recurrent cancer that inhibit the development or recurrence of cancer. In some embodiments, preventive effective amounts completely prevent the development or recurrence of cancer. “Inhibit” or “prevent” the development or recurrence of cancer means reducing the likelihood of cancer development or recurrence, or completely preventing the development or recurrence of cancer.

[0192] The term "treatment" or "treating" refers to the application or administration of the compounds of the present invention to a subject with the aim of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, reducing deterioration, improving, enhancing, or influencing the disease or condition, symptoms of the disease or condition, or the risk (or susceptibility) to the disease or condition. The term "treatment" means any indicator of successful treatment or improvement of an injury, pathology, or condition, including any objective or subjective parameter such as reduction; relief; reduction of the rate of deterioration; reduction of the severity of the disease; stabilization, reduction of symptoms, or making the injury, pathology, or condition more tolerable for the subject; slowing the rate of deterioration or decline; making the late stages of deterioration less debilitating; or improving the physical or mental health of the subject.

[0193] As used herein, minimizing or preventing the progression of cancer means treating a subject to prevent or delay the recurrence or metastasis of a tumor, or to prevent the growth of an existing tumor. Minimizing or preventing the progression of cancer includes preventing or delaying cancer recurrence after cancer treatment, or preventing the growth of an existing tumor. Prevented recurrence includes, for example, recurrence of the tumor bed after surgical removal. Alternatively, recurrence includes metastasis of cancer to another part of the body. As used herein, the terms “preventing recurrence” and “preventing relapse” are interchangeable.

[0194] This invention also includes methods for preventing the development of cancer in an individual. For example, an individual who needs cancer prevention may be considered 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 whose genetic testing or other tests indicate a high risk or likelihood of developing cancer. An individual may have cancer stem cells but does not yet have any detectable tumor. It should be understood that methods for preventing cancer development include methods for delaying the onset of cancer in the subject.

[0195] In any aspect or embodiment of this document, radiolabeled gemutuximab may be administered in combination with (i.e., sequentially, simultaneously, or subsequently) another treatment for cancer. In any embodiment, the treatment may be any treatment for cancer, optionally selected from: radiosensitization therapy, surgery, immunotherapy (e.g., BCG), external beam radiation, chemotherapy, autologous stem cell therapy, or radioimmunotherapy.

[0196] Chemotherapy agents are chemical agents or drugs that selectively destroy cancer cells and tissues. Chemotherapy agents can include, but are not limited to, compounds such as taxane compounds, compounds that act through the taxane mechanism, platinum compounds, anthracycline compounds, antimetabolites, epipodophyllotoxin compounds, camptothecin compounds, or any combination thereof.

[0197] In any aspect of the invention, radiolabeled gemtuximab and additional cancer treatment may be administered simultaneously. Alternatively, they may be administered sequentially. For example, the additional therapeutic agent (e.g., a chemotherapy agent) may be administered before the radiolabeled gemtuximab, or the radiolabeled gemtuximab may be administered before the additional therapeutic agent (e.g., a chemotherapy agent). Alternatively, treatment with the additional therapeutic agent (e.g., a chemotherapy agent) and / or radiolabeled gemtuximab may be interleaved.

[0198] The 211-At-gelotuximab antibody or its antigen-binding fragment can be administered as the sole treatment for urothelial carcinoma, or it can be administered after prior urothelial carcinoma treatment (optionally, said prior treatment includes radiosensitization therapy, surgery, chemotherapy, immunotherapy (such as BCG), external beam radiation, autologous stem cell therapy, or combinations thereof). The 211-At-gelotuximab antibody or its antigen-binding fragment can be administered concurrently or sequentially with other cancer treatments (optionally, said other treatments include surgery, chemotherapy, immunotherapy, external beam radiation, autologous stem cell therapy, or combinations thereof).

[0199] In any of the methods or uses described herein, the subject requiring treatment may have received prior treatment for urothelial carcinoma (such as surgery, chemotherapy, immunotherapy (such as BCG), external beam radiation, autologous stem cell therapy, or combinations thereof) but has not responded to the prior treatment. In a particularly preferred embodiment, the subject requiring treatment may have received prior treatment with BCG but is considered to have not responded to said treatment (in other words, the subject is a "non-responder to BCG treatment").

[0200] In yet another embodiment, a subject requiring treatment may be considered unlikely to respond to BCG treatment (e.g., as a result of previous screening or diagnostic methods).

[0201] The appropriate dose of radiolabeled gemutuximab or its antigen-binding fragment used according to the present invention will vary depending on the condition to be treated and / or the subject being treated. A skilled physician is capable of determining the appropriate dose, for example, by starting with a suboptimal dose and incrementally modifying it to determine the optimal or useful dose. Alternatively, to determine the appropriate dose for treatment / prophylaxis, data from cell culture assays or animal studies are used, where the appropriate dose is within the range of circulating concentrations, including the ED of active compounds with low or no toxicity. 50 The dosage can vary within this range depending on the formulation and route of administration used. The effective therapeutic / prophylactic dose can initially be estimated based on cell culture assays. In animal models, the dose can be formulated to achieve the IC50 values ​​determined in cell culture. 50 The range of circulating plasma concentrations (i.e., the concentration or amount of a compound that achieves half-maximal inhibition of symptoms). This information can be used to more accurately determine the effective dose in the human body. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0202] Those skilled in the art will understand that the dosage of the antibody used in the method according to the invention will depend on various factors, including the age, sex, height, and weight of the subject to whom the antibody is administered, and also on the antibody itself.

[0203] In some embodiments, gemutuximab may be administered to or infused into a subject at a dose of about 1 mg to about 50 mg, preferably at a dose of about 5 mg to about 20 mg, and more preferably at a dose of about 10 mg. The specific activity of the radiolabeled antibody may be about 15 MBq / mg to about 1500 MBq / mg, more preferably about 25 MBq / mg to about 1250 MBq / mg, or about 50 MBq / mg to about 1000 MBq / mg, or about 75 MBq / mg to about 500 MBq / mg, or about 100 MBq / mg to about 250 MBq / mg. In a more preferred embodiment, the specific activity of the radiolabeled gemutuximab can be from about 50 MBq / mg to about 1000 MBq / mg, such as about 50 MBq / mg, 60 MBq / mg, 70 MBq / mg, 80 MBq / mg, 90 MBq / mg, or 100 MBq / mg, or about 700 MBq / mg, 800 MBq / mg, or about 900 MBq / mg. In a particularly preferred embodiment, the specific activity can be in the range of about 70 MBq / mg to about 900 MBq / mg (e.g., a specific activity of about 74 MBq / mg or about 888 MBq / mg to provide a dose of about 111 MBq or 444 MBq and an antibody mass of 0.5 mg to 1.5 mg).

[0204] In some embodiments, radiolabeled gemtuximab can be administered via intravesical infusion at a specific activity of about 60 to about 1400 MBq / mg, preferably about 80 MBq / mg to 1200 MBq / mg or about 90 MBq / mg to about 1000 MBq / mg, or most preferably about 70 MBq / mg to about 900 MBq / mg (e.g., about 74 MBq / mg or about 888 MBq / mg).

[0205] In a preferred embodiment, the dose of the radiolabeled gemutuximab (or its antigen-binding fragment) administered is in the range of about 100 MBq to about 1000 MBq, optionally about 150 MBq to about 900 MBq, optionally about 200 MBq to about 800 MBq, optionally about 300 MBq to about 700 MBq, or optionally about 400 MBq to about 600 MBq. In a particularly preferred embodiment, the radiolabeled antibody is administered at a dose of about 100 MBq, about 200 MBq, about 300 MBq, about 400 MBq, about 500 MBq, about 600 MBq, about 700 MBq, about 800 MBq, about 900 MBq, or about 1000 MBq (e.g., about 110 MBq, about 220 MBq, about 330 MBq, about 440 MBq, about 550 MBq, about 660 MBq, about 770 MBq, about 880 MBq, or about 990 MBq).

[0206] In some embodiments, radiolabeled gemtuximab is administered by slow infusion at a mass dose of about 0.25 to 20 mg, preferably about 0.5 mg to 10 mg, or 0.5 mg or 5 mg, most preferably about 0.5 mg or about 1.5 mg.

[0207] In some embodiments, an effective amount of the antibody or its antigen-binding fragment thereof is administered to a patient identified as having cancer at a loading dose of about 15 mg / kg, about 20 mg / kg, or about 25 mg / kg on each of days 1 and 8 of the first 21-day cycle or on each of days 1 and 8 of the first 28-day cycle, followed by a standard dose of the antibody or its antigen-binding fragment thereof being administered to the patient at about 15 mg / kg, about 20 mg / kg, or about 25 mg / kg on each of days 1 and 8 of the subsequent 21-day cycle or on each of days 1 and 8 of the subsequent 28-day cycle.

[0208] In some embodiments, an effective amount of radiolabeled antibody or its antigen-binding fragment thereof is administered to a patient identified as having cancer in a loading volume solution filling a cavity. Specifically, the volume filling the bladder is approximately 40 mL. Therefore, in a preferred embodiment, the volume of the radiolabeled antibody or its antigen-binding fragment administered is approximately 40 mL.

[0209] In some embodiments, the duration of treatment with the radiolabeled gemtuximab or its antigen-binding fragment disclosed herein is a minimum of about 1 month, a minimum of about 2 months, a minimum of about 3 months, a minimum of about 4 months, a minimum of about 5 months, a minimum of about 6 months, a minimum of about 7 months, a minimum of about 8 months, a minimum of about 9 months, a minimum of about 10 months, a minimum of about 11 months, a minimum of about 1 year, a minimum of about 18 months, a minimum of about 24 months, a minimum of about 3 years, a minimum of about 5 years, or a minimum of about 10 years. In some embodiments, treatment with the radiolabeled gemtuximab or its antigen-binding fragment disclosed herein is a maximum of about 18 months, a maximum of 16 months, a maximum of 14 months, a maximum of 12 months, a maximum of 10 months, a maximum of 8 months, or a maximum of 6 months. The treatment duration can be any of these minimum durations to any of these maximum durations, for example, from 1 to 18 months.

[0210] In some embodiments, the administration of the radiolabeled gemutuximab or its antigen-binding fragment disclosed herein is staggered, such that individual doses are administered at intervals of at least 1 day, 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 (1 week). Doses may be administered at intervals of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or at least 7 weeks. In a particularly preferred embodiment, the radiolabeled gemutuximab or its antigen-binding fragment is administered at 1-week intervals.

[0211] Therefore, in any embodiment, radiolabeled gemutuximab or its antigen-binding fragment can be administered at doses of about 100 MBq, 200 MBq, 300 MBq, 400 MBq, 500 MBq, 600 MBq, 700 MBq, 800 MBq, 900 MBq or 1000 MBq, such as about 110 MBq, 220 MBq, 330 MBq, 440 MBq, 550 MBq, 660 MBq, 770 MBq, 880 MBq or 990 MBq, preferably about 110 MBq or about 440 MBq, at intervals of at least 1 day, 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 (1 week).

[0212] More preferably, radiolabeled gemutuximab or its antigen-binding fragment can be administered at doses of about 100 MBq, 200 MBq, 300 MBq, 400 MBq, 500 MBq, 600 MBq, 700 MBq, 800 MBq, 900 MBq or 1000 MBq, such as about 110 MBq, 220 MBq, 330 MBq, 440 MBq, 550 MBq, 660 MBq, 770 MBq, 880 MBq or 990 MBq, preferably about 110 MBq or about 440 MBq, at intervals of at least 7 days (1 week).

[0213] Even more preferably, radiolabeled gemutuximab or its antigen-binding fragment can be administered at doses of about 110 MBq or about 440 MBq (e.g., 111 MBq or 444 MBq) at intervals of at least 7 days (1 week).

[0214] In some embodiments, the dose may be administered in fractions to provide the total dose (as described above) over a series of administrations. For example, in some embodiments, a dose of radiolabeled gemutuximab may be provided across two, three, four, or more administrations. In other words, for a total dose of 111 MBq, two separate infusions of 55.5 MBq each may be administered to the subject. Similarly, a dose of approximately 444 MBq may be administered via two separate infusions of approximately 222 MBq each. Fractionated doses are preferably administered at intervals of at least 1 day, 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 (1 week). Fractionated doses may be administered at intervals of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or at least 7 weeks. In a particularly preferred embodiment, fractionated doses of radiolabeled gemutuximab or its antigen-binding fragment are administered at 1-week intervals.

[0215] As used herein, the terms “subject,” “individual,” and “patient” will be understood to be interchangeable. Although the invention can be used in humans, it can also be used for veterinary therapeutic purposes. The invention can be used with livestock or farm animals such as cattle, sheep, horses, and poultry; with companion animals such as cats and dogs; and with zoo animals.

[0216] As used herein, the term “about” regarding dosage should be understood to include variations of at least 10% of the stated dosage. For example, a dosage of “about 400 MBq” should be understood to include a dosage of 400 Mbq plus or minus up to 40 MBq (in other words, a dosage of 360 MBq to 440 MBq, and any intermediate values ​​listed within that range). Similarly, a dosage of “about 440 MBq” should be understood to include a dosage of 400 MBq to 480 MBq, and any intermediate values ​​listed within that range.

[0217] Reagent test kit

[0218] In another embodiment, a kit or product comprising radiolabeled gemutuximab or antigen-binding fragments as described above is provided, preferably for use in the methods or uses outlined herein.

[0219] Optionally, the kit may further include a label or packaging insert with instructions for use.

[0220] The kit or "article" may include a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container can be formed from a variety of materials, such as glass or plastic. The container contains a therapeutic composition for effectively treating the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper for puncture by a subcutaneous needle). The label or packaging insert indicates that the therapeutic composition is intended for treating the selected condition. In one embodiment, the label or packaging insert includes instructions for use.

[0221] The kit may comprise (a) a therapeutic composition; and (b) a second container containing a second active ingredient. In this embodiment of the invention, the kit may further include a packaging insert indicating that the active ingredient and other active ingredients can be used to treat a condition or prevent complications arising from cancer. Alternatively or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. The kit may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0222] In some embodiments, the diagnostic composition may be provided in the form of a disposable or reusable device, including a receiver for containing the diagnostic composition. In one embodiment, the device is a syringe. The device may contain 1-2 mL of the therapeutic composition. The therapeutic composition may be provided in the device in a ready-to-use state or in a state where mixing or adding additional components is required.

[0223] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0224] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0225] The following examples are intended to illustrate, but not in any way limit, the invention.

[0226] Example

[0227] Example 1: Preparation of radiolabeled gemtuximab

[0228] 211 At the Arronax Cyclone Accelerator facility 209 Bi(α, 2n) 211 At was generated and recovered in chloroform using a distillation scheme adapted from the procedure previously reported by Lindegren et al. (2001) Applied Radioactivity and Isotopes, 55: 157-160.

[0229] Radiolabeling of the anti-CAIX gemutuximab antibody (TLX-250) was performed using a bifunctional aryl iodide cation precursor and according to the procedure described by Guerard et al., 2017, *Bioorg. Med. Chem. Letters*, 25:5975-5980. The aryl iodide cation selected for the study was 3-(succinimideoxycarbonyl)phenyl(4-methoxyphenyl)iodide trifluoromethanesulfonate. It was prepared according to the synthetic protocol developed in the following literature. Radiolabeling was then performed in two steps as described by Guerard et al.: a) radioisotope labeling of the iodide cation salt to obtain […]. 211 At]SAB, and 2) conjugated with gemtuximab. The radiochemical purity of both is higher than 95%.

[0230] The following table provides example batch results: Table 2: GMP Batch Results for 211At-Gitoximab

[0231] Example 2: Selection of cell lines

[0232] Western blot analysis was used to assess CAIX antigen expression in a cohort of eight bladder cancer cell lines.

[0233] Table 3: Cell line expression results

[0234] Treatment with 200 mM CoCl2 was used to induce hypoxia (as determined by overexpression of HIF-α).

[0235] With the exception of one cell, all tested urothelial carcinoma cells expressed the CAIX antigen under both normoxic and hypoxic conditions.

[0236] The RT112 / 84 cell line was selected for subsequent experiments and obtained from ECACC (ECACC reference number: 85061106). After thawing, the cells were grown in EMEM supplemented with 10% fetal bovine serum and 1% L-glutamine at 37°C and 5% CO2. Accutase was used. TM Cells were separated and dissociated in solution for binding and cytotoxicity assays.

[0237] Example 3: 211 The binding of At-geutzumab to RT112 cells.

[0238] Prepare 20 × 10⁻⁶ samples in 1X PBS pH 7.4 containing 0.5% (w / v) BSA (sample buffer). 6 RT112 cell suspension at 1 cell / mL. To determine total binding, samples were prepared in a sample buffer covering concentrations from 25 nM to 0.10 nM. 211 Serial dilutions of At-gemituximab solution were performed. To determine nonspecific binding, a sample buffer containing 40 nM was prepared. 211 A solution of At-geutzumab and 100-fold molar excess of unlabeled antibody (4 μM) was prepared and then serially diluted 4-fold in 0.5% PBS / BSA, including 5 dilutions.

[0239] For the saturation binding assay, RT112 cells (1 × 10⁶ cells in 50 μL PBS / BSA 0.5%) were used. 6 (100 cells) were incubated with an increased concentration of radiolabeled antibody solution (final volume 150 μL) at room temperature with stirring for 90 minutes.

[0240] Nonspecific binding was determined in parallel by incubating five different concentrations of radiolabeled antibody containing an excess of unlabeled antibody (100-fold molar excess). After incubation, the reaction mixture was coated onto a 200 μL dibutyl phthalate oil pad and centrifuged at 12,000 rpm for 3 minutes in a microcentrifuge tube. The tubes were frozen in liquid nitrogen, and the tips containing cell pellets were cut off for radioactivity determination using a gamma counter.

[0241] The results shown in the table below indicate the high binding affinity of 211-At-GmAb to RT112 cells, where K D > 0.8 nM. These results also indicate that the radiolabeling of GmAb has virtually no effect on binding affinity, as literature reports that GmAb has an affinity of approximately 8 nM for CAIX.

[0242] Table 4: Analysis of binding assays for 211At-GmAb to RT112 cells

[0243] Example 4: 177 Lu-GmAb and 211 Comparative analysis of At-GmbAb

[0244] In the CAIX-positive cell line RT112, comparisons were made. 211 At-Gitoximab and 177 In vitro cytotoxicity of Lu-DOTA-gegituximab.

[0245] method

[0246] By diluting in complete culture medium 211 At-gemetuximab (136 MBq / mg; 31.8 MBq / mL) was used to prepare a 1000 kBq / mL working solution. The 1000 kBq / mL working solution was then diluted in complete culture medium by making two-fold serial dilutions to cover a concentration range from 1000 kBq / mL to 15.6 kBq / mL.

[0247] By diluting in complete culture medium 177 Lu-DOTA-gemetuximab (1005 MBq / mg; 812 MBq / mL) was used to prepare a 40,000 kBq / mL working solution. The 40,000 kBq / mL working solution was then diluted in complete culture medium by performing two-fold serial dilutions to cover a concentration range from 40,000 kBq / mL to 625 kBq / mL.

[0248] 211 At-Gitoximab and 177The cytotoxicity of Lu-DOTA-geutuximab was assessed in RT112 cells after exposure to seven different concentrations for 2 hours. Cell viability was then assessed at two different time points (day 2 and day 5) using a colorimetric MTS assay (a colorimetric assay for assessing cellular metabolic activity). The method is based on the reduction of the MTS compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole) by live cells to generate a colored formazan compound soluble in the cell culture medium. The formazan dye produced by live cells can be directly quantified by measuring the absorbance at OD = 490 nm.

[0249] result

[0250] Exposure to different concentrations of alpha emission 211 At-geutzximab and β-emission 177 The survival curve of RT112 cells obtained after the effect of Lu-DOTA-geutuximab on RT112 cell survival Figure 1 The diagram shows the concentration (volume activity, in MBq / mL) on the x-axis and the survival fraction on the y-axis.

[0251] exist 211 Significant toxicity was observed when the active concentration of At-GmAb exceeded 100 kBq / mL. From this point onward, a dose-dependent response was observed, leading to a significant decrease in cell viability; after exposure to a concentration of 1000 kBq / mL, over 80% of cells failed to survive by day 5. Furthermore, the difference in survival observed on days 2 and 5, respectively, indicates that… 211 The cytotoxic effect of At-geutzumab on RT112 cells increased significantly between day 2 and day 5.

[0252] for 177 Cells treated with Lu-DOTA-GmAb showed very moderate toxicity at activity concentrations exceeding 20,000 kBq / mL, and no significant decrease in cell viability was observed even at the highest concentrations. Exposure to 40,000 kBq / mL... 177 After Lu-DOTA-gelotuximab treatment, cell survival remained above 65%.

[0253] Show 211 At-gegituximab has significantly higher cytotoxicity than 177 Lu-DOTA-gelotuximab. RT112 cells were exposed to 1 MBq / mL of... 211 At-gegituximab caused 85% cell death, while exposure to 40 MBq / mL... 177Lu-gemetuximab resulted in very moderate cytotoxicity, with an estimated cell death rate of only about 45%. The results suggest that the alpha-emitter radioisotope astatine-211 is better suited than the beta-emitter radioisotope Lu-177 for delivering higher doses to the RT112 cell target and is most likely to approach the cell nucleus, as the short range of alpha particles favors more pronounced linear energy transfer.

[0254] Example 5: Study on biological distribution

[0255] Intrabladder administration in mice 211 At-geutzumab was administered, and subsequent tissue distribution was evaluated.

[0256] Nine female CD-1 mice were divided into three groups (each group representing three different analysis time points: 30 minutes, 1 hour, and 4 hours). Approximately 1200 kBq of [a specific substance / material] was administered via catheter. 211 At-geutzsumab was administered to the bladders of mice. Mice were sacrificed at 30 minutes, 1 hour, and 4 hours, and serum levels in different organs and tissues were measured. 211 At activity.

[0257] Radioactivity concentration is expressed as a percentage of the injected dose per gram of organ (ID% / g) and as a percentage of the ID of the whole organ.

[0258] Figure 2 and 3 The results shown indicate 211 At-geutzumab did not spread across the bladder wall to surrounding organs.

[0259] Example 6: Histological Study

[0260] Local tolerance following intravesical administration was assessed by histological examination of the bladder and kidneys of mice. 211 At-geutzsumab was administered via catheter to the bladders of five female CD-1 mice. The mice were held in captivity for 15 days before sacrifice.

[0261] Histological examination revealed no abnormal findings attributable to the experimental procedure. These results confirm that the intrabladder infusion of approximately 700 kBq... 211 At-geutzximab does not induce toxic side effects. This dose estimate for mice (700 kBq / 30 µL) is equivalent to approximately 933 MBq / 40 mL for intrabladder infusion in humans.

[0262] Example 7: Internalization Study

[0263] 125 The internalization kinetics of I-geutuximab in human bladder cancer cells RT 112 were used as... 211A model of the expected internalization properties of At-geutzximab.

[0264] Also assessed 125 After I-geutzximab internalization 125 I release from cells, and 125 I represents the time it remains bound to or internalized within target cells. The incubation time mimics the clinical protocol described in Example 8 below, where the incubation time is 2 hours. A schematic diagram of the assay design is shown in... Figure 4 As shown in the image.

[0265] Figure 5 and 6 The results are shown in the figure. The results of the two internalization studies demonstrate that... 125 I-Gibutuximab was internalized in the target cells, and even 22 hours after the elution step, significant amounts of activity remained in the cells. This suggests that the target cells will be exposed to significant radiation in a clinical setting.

[0266] Example 8: Clinical Trials

[0267] Orthotopic xenograft mouse models are commonly used to determine the likelihood of clinical success. However, in the context of intravesical alpha therapy, this orthotopic xenograft model does not optimally reflect the patient's clinical condition because it requires non-physiological electrocautery of the bladder mucosa to promote graft uptake.

[0268] Therefore, a clinical trial was conducted to evaluate 211 Safety and efficacy of At-geutzumab in the treatment of urothelial carcinoma.

[0269] Preliminary studies of CAIX antigen expression and localization were performed on biopsy samples from six patients using immunohistochemistry (IHC). Ten biopsy samples were examined from the six patients studied: eight were papillary tumors, one was inflammatory mucosa without tumor, and one was inflammatory and scarred bladder wall without tumor. The percentage of tumor cells expressing CAIX antigen was less than 20%, and in some cases even less than 10%. Despite the low overall percentage of CAIX-expressing tumor cells, immunohistochemical images showed significantly greater CAIX expression on the luminal surface of the papillary tumors, directly in contact with the bladder cavity (where radiolabeled antibodies would be instilled).

[0270] The dose calculations given in mGy / MBq are based on the MIRD formula and the calculation of cumulative activity in a single source volume (in this case, via instillation into the bladder, with the bladder wall as the single target volume). The calculations are based on reference geometry (a mathematical model of human females and / or males as defined in ICRP or ORNL) and involve dose coefficients S previously calculated for the model by simulation, and are listed in the calculation software used, OLINDA 1. (2003) and IDAC-Dose 2.1. (2017).

[0271] Table 5: OLIDA software estimation of the dose equivalent of isotope absorption to the bladder wall

[0272] OLINDA 1 software is used to derive the application of [methods] for male and female models. 211 The absorbed dose equivalent per unit of activity of At. The contribution of 211Po must be added to the calculation because OLIDA does not consider sub-elements.

[0273] Based on a patient with a maximum predicted uptake of 9 mSv / MBq (female model), even with a maximum recommended dose of 370 MBq 211At-GmAb, the estimated dose would be approximately 3.39 Gy. This figure is ten times lower than the tolerable dose of 40 Gy defined by Meredith et al. (2008) in Semin. Nucl. Med. 38: 347-357.

[0274] Conduct clinical trials to evaluate 211 The safety and efficacy of AT-gegituximab in the treatment of non-muscle-invasive bladder cancer that is unresponsive to standard-of-care BCG therapy were evaluated. This was a Phase I, first-in-human trial. 211 At-labeled GmAb ( 211 Safety, tolerability, and preliminary response of At-TLX250 in patients with refractory nonmuscle-invasive bladder cancer treated with standard therapy.

[0275] The applied radiation therapy dose is between 100 MBq and 1000 MBq, preferably about 111 MBq and about 444 MBq.

[0276] Preliminary results will indicate that patients respond well to the treatment. 211 At-geutzumab treatment resulted in good responses, with partial or complete responses, and the tumors responded to the treatment, with an overall reduction in tumor volume after treatment.

[0277] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

Claims

1. A radiolabeled girentuximab antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is conjugated to the radioisotope 211-astatine.

2. The radiolabeled gemutuximab antibody or its antigen-binding fragment according to claim 1, wherein the radioisotope is directly conjugated to the gemutuximab or its antigen-binding fragment, optionally via halogenation of amino acid residues.

3. The radiolabeled gemtuximab antibody or its antigen-binding fragment according to claim 1, wherein the radioisotope is indirectly conjugated to the gemtuximab or its antigen-binding fragment, for example, through a prosthetic group or other linker.

4. The radiolabeled gemutuximab according to any one of claims 1 to 3, wherein the antibody or its antigen-binding fragment comprises an antigen-binding domain that specifically binds to carbonic anhydrase IX (CAIX), and comprises: (a) A heavy chain variable domain (VH) comprising three complementarity-determining regions (CDRs) of an amino acid sequence as shown in any of SEQ ID NO: 4, 20, 36, 52, or 68; and / or, (b) A light chain variable domain (VL) comprising three complementarity-determining regions (CDRs) of an amino acid sequence as shown in any of SEQ ID NO: 84, 100, 116, 132, 148 or 164.

5. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 4, wherein the antibody or its antigen-binding fragment comprises any one of the following: (i) VH, wherein VH comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR 1 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 1, wherein CDR 2 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 2, and wherein CDR 3 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 2, and CDR 3 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least The sequence shown in 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%, or at least 99% identical; (ii) VH, wherein the VH comprises at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence shown in any of SEQ ID NO: 4, 20, 36, 52, or 68; (iii) VL, wherein VL comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 81, wherein CDR2 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 82, and CDR3 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 99% identical to the sequence shown in SEQ ID NO: 82, wherein CDR3 comprises a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, 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, wherein CDR3 comprises a sequence that is at least about 80%, at least 85%, at least The sequence shown in 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%, or at least 99% identical; (iv) VL, wherein the VL comprises at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence shown in SEQ ID NO: 84, 100, 116, 132, 148, or 164; (v) VH, which includes CDR1, CDR2 and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO: 1, CDR2 includes the sequence shown in SEQ ID NO: 2 and CDR3 includes the sequence shown in SEQ ID NO: 3; (vi) VH, wherein the VH comprises the sequence shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68; (vii) VL, which includes CDR1, CDR2 and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO: 81, CDR2 includes the sequence shown in SEQ ID NO: 82 and CDR3 includes the sequence shown in SEQ ID NO: 83; (viii) VL, wherein the VL comprises the sequence shown in any one of SEQ ID NO: 84, 100, 116, 132, 148 or 164; (ix) VH, wherein VH comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 comprises the sequence shown in SEQ ID NO: 2, and CDR3 comprises the sequence shown in SEQ ID NO: 3; and VL, wherein VL comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 81, CDR2 comprises the sequence shown in SEQ ID NO: 82, and CDR3 comprises the sequence shown in SEQ ID NO: 83; or, (x) VH, which comprises the sequence shown in any one of SEQ ID NO: 4, 20, 36, 52 or 68; and VL, which comprises the sequence shown in any one of SEQ ID NO: 84, 100, 116, 132, 148 or 164.

6. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or its antigen-binding fragment further comprises: (i) VH, which comprises frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 comprises or is composed 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 or constitutes thereof, and FR2 comprises a sequence that is identical to or constitutes thereof as shown in SEQ ID NO: 9, 25, 41, 57, or 73. FR3 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, as shown in any of SEQ ID NO: 11, 27, 43, 59, or 75. FR4 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, as shown in any of SEQ ID NO: 11, 27, 43, 59, or 75. The sequences shown in any of 12, 28, 44, 60, or 76 are 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 or constitute the same sequence; and, (ii) VL, wherein the VL comprises frame regions (FR) 1, FR2, FR3, FR4, wherein FR1 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof, and FR2 comprises a sequence that is identical to or consists of ... The FR3 contains at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences or constitutes sequences thereof, as shown in any of SEQ ID NO: The FR4 comprises or consists of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or constitutes thereof 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 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 or constitutes thereof a sequence that is at least about 80%, at least 81%, at least 82%, at least 8 ... Or include: (i) VH, wherein VH comprises frame regions (FR) 1, FR2, FR3, FR4, wherein FR1 comprises or consists of sequences as shown in SEQ ID NO: 9, 25, 41, 57 or 73, and FR2 comprises or consists of sequences as shown in SEQ ID NO: 10, 26, 42, 58 or 74. FR3 comprises or consists of sequences as shown in SEQ ID NO: 11, 27, 43, 59 or 75, and FR4 comprises or consists of sequences as shown in SEQ ID NO: 12, 28, 44, 60 or 76, and, (ii) VL, which includes frame regions (FR) 1, FR2, FR3, and FR4, wherein FR1 includes or consists of a sequence as shown in SEQ ID NO: 89, 105, 121, 137, 153, or 169, FR2 includes or consists of a sequence as shown in any one of SEQ ID NO: 90, 106, 122, 138, 154, or 170, FR3 includes or consists of a sequence as shown in SEQ ID NO: 91, 107, 123, 139, 155, or 171, and FR4 includes or consists of a sequence as shown in SEQ ID NO: 92, 108, 124, 140, 156, or 172.

7. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or its antigen-binding fragment 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%, or at least 99% identical to or constitutes the sequence shown in SEQ ID NO: 4, 20, 36, 52, or 68; and / or, (b) A light chain variable domain (VL) comprising or consisting of at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence shown in SEQ ID NO: 84, 100, 116, 132, 148, or 164.

8. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 7, wherein the antibody or its antigen-binding fragment comprises an amino acid sequence consisting substantially of or composed of any one of SEQ ID NO: 4, 20, 36, 52 or 68 and / or any one of SEQ ID NO: 84, 100, 116, 132, 148, 164 (in the order of N-terminus to C-terminus or C-terminus to N-terminus).

9. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or its antigen-binding fragment is in the following form: (i) Single-domain antibodies (sdAb); (ii) Single-stranded Fv fragments (scFv); (iii) Dimer scFv (di-scFv); or, (iv) One of (ii) or (iii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

10. The radiolabeled gemtuximab or its antigen-binding fragment according to any one of claims 1 to 8, wherein the gemtuximab or its antigen-binding fragment is in the following form: (i) Microantibodies; (ii) Bifunctional antibodies; (iii) Trifunctional antibodies; (iv) Four-function antibodies; (v)Fab; (vi)F(ab')2; (vii)Fv; (viii) Bispecific antibodies or other forms of multispecific antibodies; or, (ix) One of (i) to (viii) connected to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

11. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 4 to 10, wherein the linker, in the context of an antigen-binding domain having the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, is chemical, or a disulfide bond formed between two cysteine ​​residues.

12. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 11, wherein the gemutuximab is in the form of a complete IgG antibody.

13. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 12, wherein the gemutuximab or its antigen-binding fragment comprises an Fc region, the Fc region being engineered to: -Has an increased in vitro or in vivo half-life; - Possesses an increased ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity; and / or, - It has reduced effector functionality.

14. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 13, wherein the gemutuximab antibody comprises a variable light chain and a variable heavy chain, the variable light chain and the variable heavy chain being linked to a constant region of an antibody comprising heavy chain CH2 and / or CH3.

15. The radiolabeled gemutuximab or its antigen-binding fragment according to claim 14, wherein the gemutuximab comprises a heavy chain constant region as defined in any one of SEQ ID NO: 177, 178, 179 or 180 and / or a light chain constant region as defined in SEQ ID NO:

181.

16. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 15, wherein the gemutuximab comprises a heavy chain as shown in any one of SEQ ID NO: 182, 183, 184 or 185 and / or a light chain as shown in SEQ ID NO:

186.

17. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of claims 1 to 16, wherein the gemutuximab comprises one or more amino acid substitutions in the constant region to reduce the in vivo half-life of the antibody.

18. The radiolabeled gemutuximab or its antigen-binding fragment according to claim 17, wherein the gemutuximab antibody comprises a substitution at one or more residues of His310, His435, Tyr436 and Ile253 (Kabat number) in the heavy chain constant region, thereby altering the FcRn binding affinity and / or serum half-life of the antibody (e.g., relative to gemutuximab containing native CH2 and CH3 domains).

19. The radiolabeled gemutuximab or its antigen-binding fragment according to claim 18, wherein the substitution at amino acid position 310 and / or 435 of the antibody is alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine.

20. The radiolabeled gemutuximab or its antigen-binding fragment according to claim 19, wherein 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.

21. The radiolabeled gemutuximab or its antigen-binding fragment according to claim 20, wherein the antibody has an alanine residue at position 310 and a glutamine residue at position 435.

22. The radiolabeled gemutuximab or its antigen-binding fragment according to claim 21, wherein the antibody further comprises an amino acid substitution at residue Lys322, preferably wherein the substitution is Lys322Ala.

23. The radiolabeled gemutuximab or its antigen-binding fragment according to any one of the preceding claims, wherein the antibody comprises substituted K322A, H310A and H435Q in the constant region of the heavy chain.

24. A pharmaceutical composition comprising, optionally, a 211-At-geutzumab antibody or an antigen-binding fragment thereof according to any one of claims 1 to 23, in combination with a pharmaceutically acceptable excipient.

25. A method for treating, preventing, or minimizing the progression of urothelial carcinoma in a subject, the method comprising: -Administer the 211-At-geutuzumab antibody or its antigen-binding fragment or pharmaceutical composition according to any one of claims 1 to 24 to the subject in need. This can treat, prevent, or minimize the progression of the urothelial carcinoma in the subject.

26. A method for minimizing, reducing, or preventing the growth of urothelial carcinoma tumors in a subject, the method comprising: -Administer the 211-At-geutuzumab antibody or its antigen-binding fragment or pharmaceutical composition according to any one of claims 1 to 24 to the subject in need. This minimizes, reduces, or prevents the growth of urothelial carcinoma tumors in the subject.

27. A method for minimizing, reducing, or preventing urothelial carcinoma metastasis in a subject, the method comprising: -Administer the 211-At-geutuzumab antibody or its antigen-binding fragment or pharmaceutical composition according to any one of claims 1 to 24 to the subject in need. This minimizes, reduces, or prevents urothelial carcinoma metastasis in the subject.

28. A method for increasing the survival of a subject with urothelial carcinoma, the method comprising: -Administer the 211-At-geutuzumab antibody or its antigen-binding fragment or pharmaceutical composition according to any one of claims 1 to 24 to the subject in need. This increases the survival time of the subjects.

29. Use of the 211-At-geutzsumab antibody or its antigen-binding fragment according to any one of claims 1 to 23, wherein it is used to prepare a medicament for achieving the following: - To treat or prevent urothelial carcinoma in a subject or to minimize the progression of said urothelial carcinoma. - To minimize, reduce, or prevent the growth of urothelial carcinoma tumors in the subject. - To minimize, reduce, or prevent urothelial carcinoma metastasis in the subject, or, - Increase the survival rate of subjects with urothelial carcinoma.

30. The method or use according to any one of claims 25 to 29, wherein the urothelial carcinoma is bladder cancer, ureteral cancer or urethral cancer.

31. The method or use according to any one of claims 25 to 29, wherein the urothelial carcinoma is bladder cancer.

32. The method or use according to any one of claims 25 to 31, wherein the subject has been deemed unresponsive or likely unresponsive to treatment with Bacillus Calmette-Guerin (BCG).

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