Non-invasive methods using anti-CLDN18.2 antibody-radionuclide conjugates

Abstract

The present disclosure provides herein the use of a conjugate of an anti-CLDN18.2 antibody or an antigen-binding fragment thereof with a radionuclide in imaging, patient screening, treatment procedure monitoring, and efficacy assessment.

Description

Technical Field

[0001] The present invention generally relates to non-invasive imaging methods. Background Art

[0002] Claudin 18.2 (CLDN18.2) is a member of the Claudins protein family, which was discovered by Shoichiro Tsukita and his colleagues in 1998 (1). Claudin is an important molecule that constitutes tight junctions between cells, which can determine the permeability of epithelial cells and block the diffusion of proteins and lipids on the cell membrane surface (2, 3).

[0003] However, at present, there are still great limitations in the accurate detection of CLDN18.2 in lesions (13). Since CLDN18.2 and CLDN18.1 differ only in their first exon, they have highly homologous amino acid sequences (91% of the amino acid sequences are the same, and there are only 69 different amino acids), and most of the different amino acids are neutral, which makes it difficult to develop detection antibodies specifically for detecting CLDN18.2.

[0004] Currently, the detection antibodies for CLDN18.2 are only applicable to immunohistochemistry (IHC) of paraffin tissue sections and frozen tissue sections, which is invasive and cumbersome, and has high requirements for the general condition of patients. In addition, the IHC cannot reflect tumor heterogeneity in vivo and cannot perform repeated and dynamic detection. Therefore, exploring non-invasive, real-time and dynamic detection techniques is crucial for achieving a comprehensive assessment of all lesions. Summary of the Invention

[0005] On the one hand, the present disclosure provides the use of such anti-CLDN18.2 radionuclide conjugates provided herein (specifically, those conjugated to diagnostic radionuclides, and preferably 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177A method for detecting or visualizing CLDN18.2 protein, for diagnosing a subject with a CLDN18.2-related disease, for identifying a subject in need who may respond to a CLDN18.2-targeted therapy, for monitoring the progression of a CLDN18.2-related disease in a subject during a monitoring period, and for monitoring the therapeutic efficacy of a subject with a CLDN18.2-related disease who has been treated with a therapy for a certain treatment period, etc. The methods provided herein include administering to the subject a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate provided herein. In certain embodiments, the methods provided herein further include performing radionuclide imaging on the subject to obtain an image. In certain embodiments, the methods provided herein further include determining or visualizing the presence of the CLDN18.2 protein in the site of interest of the subject based on the image, wherein the presence and / or location of the radionuclide conjugate above background indicates the presence and / or location of the CLDN18.2 protein.

[0006] In one aspect, the present disclosure provides a method for detecting or visualizing CLDN18.2 protein at a site of interest of a subject, the method comprising:

[0007] a) administering to the subject a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate provided herein; and

[0008] b) performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain an image; and

[0009] c) determining or visualizing the presence of the CLDN18.2 protein in the site of interest of the subject based on the image,

[0010] wherein the presence and / or location of radionuclide uptake above background indicates the presence and / or location of the CLDN18.2 protein, and wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0011] In some embodiments, the subject has not been treated with a CLDN18.2-targeted therapy or has not received the CLDN18.2-targeted therapy.

[0012] In another aspect, the present disclosure provides a method for diagnosing a subject with a CLDN18.2-related disease, the method comprising:

[0013] a) administering to the subject a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate provided herein; and

[0014] b) At a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate, perform radionuclide imaging on the subject to obtain an image;

[0015] c) Diagnose the subject's CLDN18.2-related disease based on the presence and / or location of CLDN18.2 protein in the site of interest of the subject as determined from the image,

[0016] wherein the presence and / or location of radionuclide uptake above background indicates the presence and / or location of the CLDN18.2 protein, and

[0017] wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0018] In certain embodiments, the subject has not been treated with or has not received a CLDN18.2-targeted therapy.

[0019] In another aspect, the present disclosure provides a method for identifying a subject in need who may respond to a CLDN18.2-targeted therapy, the method comprising:

[0020] a) Administer to the subject a detectable effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate provided herein; and

[0021] b) At a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate, perform radionuclide imaging on the subject to obtain an image;

[0022] c) Identify that the subject may respond to the CLDN18.2-targeted therapy based on the presence and / or location of CLDN18.2 protein in the site of interest of the subject as determined from the image,

[0023] wherein the presence and / or location of radionuclide uptake above background indicates the presence and / or location of the CLDN18.2 protein, and

[0024] wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0025] In certain embodiments, the subject has not been treated with or has not received a CLDN18.2-targeted therapy.

[0026] In some embodiments, the diagnostic radionuclide is selected from the group consisting of: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 l, 124 l, 125 l, 131 l, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 lr, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac.

[0027] In one embodiment, the method further comprises:

[0028] d) Administer a therapeutically effective amount of a CLDN18.2 - targeted therapy to the subject identified as having the CLDN18.2 protein at the site of interest, diagnosed with a CLDN18.2 - related disease, and / or identified as likely to respond to a CLDN18.2 - targeted therapy.

[0029] In certain embodiments, the CLDN18.2 - targeted therapy comprises a therapy selected from the group consisting of: anti - CLDN18.2 antibody therapy, CLDN18.2 - targeted compounds, CLDN18.2 - targeted nucleic acid therapy, CLDN18.2 - targeted peptides, CLDN18.2 - targeted cell therapy, and CLDN18.2 - targeted gene therapy.

[0030] In certain embodiments, the anti - CLDN18.2 antibody therapy comprises an anti - CLDN18.2 antibody - therapeutic radionuclide conjugate, wherein the therapeutic radionuclide is selected from the group consisting of: 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au,199 Au and 211 Pb.

[0031] In certain embodiments, the therapeutic radionuclide is 177 Lu or 124 I.

[0032] In certain embodiments, the therapeutic radionuclide is the same as the diagnostic radionuclide.

[0033] In certain embodiments, both the therapeutic radionuclide and the diagnostic radionuclide are 177 Lu or 124 I.

[0034] In certain embodiments, the anti-CLDN18.2 antibody-diagnostic radionuclide conjugate is the same as the anti-CLDN18.2 antibody-therapeutic radionuclide.

[0035] In some embodiments, the therapeutically effective amount is at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% of the detectable effective amount.

[0036] In yet another aspect, the present disclosure provides a method of monitoring the progression of a CLDN18.2-related disease in a subject during a monitoring period, the method comprising:

[0037] a) administering to the subject a detectable effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate provided herein after the monitoring period;

[0038] b) performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain a post-monitoring image; and

[0039] c) comparing the post-monitoring image with a pre-monitoring image to determine a change in the level of CLDN18.2 protein during the monitoring period based on the images, wherein the level of the radionuclide conjugate above background indicates the level of the CLDN18.2 protein,

[0040] wherein the change indicates the presence or absence of disease progression,

[0041] wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0042] In some embodiments, the pre-monitoring image is obtained from the subject prior to the monitoring period by:

[0043] Administer an effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate, and then perform radionuclide imaging on the subject at a subsequent time point after the administration of the anti-CLDN18.2 antibody-radionuclide conjugate to obtain the pre-monitoring image.

[0044] In some embodiments, the change includes an increase in CLDN18.2 level or the absence of an increase in CLDN18.2 level. In some embodiments, an increase in CLDN18.2 level during the monitoring period indicates disease progression, and the absence of the increase in CLDN18.2 level during the monitoring period indicates the absence of disease progression.

[0045] In some embodiments, the subject has not been treated with a CLDN18.2-targeted therapy or has not received the CLDN18.2-targeted therapy.

[0046] In some embodiments, the disease is a tumor.

[0047] In some embodiments, the progression is metastasis of the tumor.

[0048] In some embodiments, the level of the CLDN18.2 protein includes the amount, distribution, and / or location of the CLDN18.2 protein.

[0049] In some embodiments, the subject is at risk of metastasis.

[0050] In yet another aspect, the present disclosure provides a method for monitoring the therapeutic efficacy of a subject having a CLDN18.2-related disease and who has been treated with a therapy for a certain treatment period, the method comprising:

[0051] a) Administer a detectable effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate provided herein to the subject after the treatment period;

[0052] b) Perform radionuclide imaging on the subject at a subsequent time point after the administration of the anti-CLDN18.2 antibody-radionuclide conjugate to obtain a post-treatment image; and

[0053] c) Compare the post-treatment image with a pre-treatment image to determine a change in the level of the CLDN18.2 protein during the treatment period based on the images, wherein the level of radionuclide uptake above background indicates the level of the CLDN18.2 protein,

[0054] wherein the change indicates the presence or absence of the therapeutic efficacy,

[0055] Wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0056] In some embodiments, the pre-treatment image is obtained from the subject prior to the treatment period by:

[0057] administering an effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate, and subsequently performing radionuclide imaging on the subject at a subsequent time point after the administration of the anti-CLDN18.2 antibody-radionuclide conjugate to obtain the pre-treatment image.

[0058] In some embodiments, the change includes an increase in CLDN18.2 level, a decrease in CLDN18.2 level, or an absence of an increase in CLDN18.2 level. In some embodiments, an increase in CLDN18.2 level during the treatment period indicates a lack of therapeutic efficacy or poor therapeutic efficacy, and / or wherein a decrease or absence of the increase in CLDN18.2 level during the treatment period indicates the presence of therapeutic efficacy or positive therapeutic efficacy.

[0059] In some embodiments, the method further comprises:

[0060] a) when determining poor therapeutic efficacy, increasing the dose of the therapy or stopping the therapy; or

[0061] b) when determining positive therapeutic efficacy, advising the subject to continue the therapy.

[0062] In some embodiments, the therapy comprises a CLDN18.2-targeted therapy.

[0063] In some embodiments, the CLDN18.2-targeted therapy comprises a therapy selected from the group consisting of: anti-CLDN18.2 antibody therapy, CLDN18.2-targeted compound, CLDN18.2-targeted nucleic acid therapy, CLDN18.2-targeted peptide, CLDN18.2-targeted cell therapy, and CLDN18.2-targeted gene therapy.

[0064] In some embodiments, the method further comprises advising the subject to switch to a therapy other than the CLDN18.2-targeted therapy when the post-treatment CLDN18.2 level is lower than a corresponding reference level. In certain embodiments, the method further comprises advising the subject to switch to a therapy other than the CLDN18.2-targeted therapy when the post-treatment CLDN18.2 level is reduced by at least 40% (or at least 50%, 60%, 70%, 80%, 90%, or 95%) relative to the pre-treatment CLDN18.2 level.

[0065] In some embodiments, the therapy is not a CLDN18.2-targeted therapy.

[0066] In any of the above embodiments, the diagnostic radionuclide is selected from the group consisting of: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154" 1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac. In some of these embodiments, the diagnostic radionuclide is 124 I,123 I, 131 I, 89 Zr or 177 Lu.

[0067] In any of the above embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises an 124 I-labeled anti-CLDN18.2 antibody provided herein. In any of the above embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises 124 I-labeled 18B10.

[0068] In any of the above embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate is administered at a dose ranging from 0.5 mCi to 10 mCi (18.5 MBq to 370 MBq).

[0069] In any of the above embodiments, radionuclide imaging is performed 2 hours after administering the anti-CLDN18.2 antibody-radionuclide conjugate to the subject, or at a time point between 2 hours and 144 hours, between 12 hours and 144 hours, or preferably between 24 hours and 144 hours, between 24 hours and 120 hours, or between 24 hours and 96 hours after administering the anti-CLDN18.2 antibody-radionuclide conjugate to the subject. In any of the above embodiments, radionuclide imaging is performed one, two, three, or more times at different time points between 2 hours and 144 hours, between 12 hours and 144 hours, or preferably between 24 hours and 144 hours, between 24 hours and 120 hours, or between 24 hours and 96 hours after administering the anti-CLDN18.2 antibody-radionuclide conjugate to the subject.

[0070] In any of the above embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises heavy chain HCDR1, HCDR2, and HCDR3 and / or light chain LCDR1, LCDR2, and LCDR3 sequences, wherein:

[0071] The HCDR1 sequence comprises GYNMN (SEQ ID NO:1) or a homologous sequence having at least 80% sequence identity thereto;

[0072] The HCDR2 sequence comprises X1IDPYYX2X3TX4YNQKFX5G (SEQ ID NO:32) or a homologous sequence having at least 80% sequence identity thereto;

[0073] The HCDR3 sequence includes X6X7X8GNAFDY (SEQ ID NO: 33) or a homologous sequence having at least 80% sequence identity thereto;

[0074] The LCDR1 sequence includes KSSQX9LX10NX11GNX12KNYLT (SEQ ID NO: 34) or a homologous sequence having at least 80% sequence identity thereto;

[0075] The LCDR2 sequence includes WASTRX13S (SEQ ID NO: 35) or a homologous sequence having at least 80% sequence identity thereto;

[0076] The LCDR3 sequence includes QNDYSX15PX16T (SEQ ID NO: 36) or a homologous sequence having at least 80% sequence identity thereto;

[0077] wherein X 1 is N or Y or H, X 2 is G or V, X 3 is A or G or T, X 4 is R or T or S, X 5 is K or R, X 6 is S or M, X 7 is Y or F, X 8 is Y or H, X 9 is S or N, X 10 is L or F, X 11 is S or N, X 12 is Q or L, X 13 is E or K, X 15 is F or Y and X 16 is F or L.

[0078] In any of the above embodiments, the CLDN18.2-related disease is a CLDN18.2-positive tumor or a CLDN18.2-positive non-cancerous lesion (e.g., gastric lesion).

[0079] In any of the above embodiments, the tumor is gastric cancer, ovarian cancer, pancreatic cancer, cholangiocarcinoma, colorectal cancer, lung cancer or esophageal adenocarcinoma.

[0080] In some embodiments, the gastric lesion is a gastric ulcer.

[0081] In any of the above embodiments, the CLDN18.2-targeted therapy includes a therapy that specifically targets CLDN18.2 or cells expressing CLDN18.2.

[0082] In some embodiments, the CLDN18.2 targeting therapy includes therapies selected from the group consisting of: anti-CLDN18.2 antibody therapy, CLDN18.2 targeting compounds, CLDN18.2 targeting nucleic acid therapy, CLDN18.2 targeting peptides, CLDN18.2 targeting cell therapy, and CLDN18.2 targeting gene therapy.

[0083] In some embodiments, the anti-CLDN18.2 antibody therapy includes an anti-CLDN18.2 antibody-therapeutic radionuclide conjugate, wherein the therapeutic radionuclide is selected from the group consisting of: 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb. In certain embodiments, the therapeutic radionuclide is 177 Lu.

[0084] In some embodiments, the CLDN18.2-targeted cell therapy includes CAR T cells, TCR T cells, or CAR NK cells that target CLDN18.2.

[0085] In any of the above embodiments, the site of interest has or is suspected of having a tumor or gastric lesion.

[0086] In some embodiments, the site of interest is the whole body.

[0087] In any of the above embodiments, the radionuclide imaging includes positron emission tomography (PET) or SPECT. In some embodiments, the radionuclide imaging is combined with CT, MR, or ultrasound.

[0088] In yet another aspect, the present disclosure provides a kit that includes an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0089] In yet another aspect, the present disclosure provides a kit for: 1) diagnosing a subject with a CLDN18.2-related disease; 2) identifying a subject in need who may respond to CLDN18.2-targeted therapy; 3) monitoring the progression of a CLDN18.2-related disease in a subject during a monitoring period; and / or 4) monitoring the therapeutic efficacy of a subject with a CLDN18.2-related disease who has been treated with a therapy for a certain treatment period, the kit including an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0090] In certain embodiments, the kit further includes a therapeutically effective amount of an anti-CLDN18.2 antibody-therapeutic radionuclide conjugate.

[0091] In certain embodiments, the therapeutic radionuclide is 177 Lu or 124 I.

[0092] In certain embodiments, the therapeutic radionuclide is the same as the diagnostic radionuclide.

[0093] In certain embodiments, both the therapeutic radionuclide and the diagnostic radionuclide are 177 Lu or 124 I.

[0094] In certain embodiments, the kit further includes instructions for providing a detectable effective amount of the anti-CLDN18.2 antibody-diagnostic radionuclide conjugate and a therapeutically effective amount of the anti-CLDN18.2 antibody-therapeutic radionuclide conjugate.

[0095] In certain embodiments, the therapeutically effective amount is at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% of the detectable effective amount.

[0096] In any of the above embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises:

[0097] A heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:3, and an HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6;

[0098] A heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:7, and an HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:8;

[0099] A heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:9, and an HCDR3 comprising the sequence of SEQ ID NO:11; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:10, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6;

[0100] A heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:19, and an HCDR3 comprising the sequence of SEQ ID NO:21; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:16, and an LCDR3 comprising the sequence of SEQ ID NO:18; or

[0101] Heavy chain variable region and light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:22, and HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprises LCDR1 comprising the sequence of SEQ ID NO:20, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:6.

[0102] In some embodiments of the anti-CLDN18.2 antibody-radionuclide conjugate, the heavy chain variable region further comprises one or more of heavy chain HFR1, HFR2, HFR3, and HFR4, and / or the light chain variable region further comprises one or more of light chain LFR1, LFR2, LFR3, and LFR4, wherein:

[0103] The HFR1 comprises QVQLVQSGAEVKKPGASVKVSCKASGYX 17 FT (SEQ ID NO:54) or a homologous sequence having at least 80% sequence identity therewith;

[0104] The HFR2 comprises WVX 18 QAPGQGLEWX 19 G (SEQ ID NO:55) or a homologous sequence having at least 80% sequence identity therewith;

[0105] The HFR3 sequence comprises RVTX 20 TIDKSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO:56) or a homologous sequence having at least 80% sequence identity therewith;

[0106] The HFR4 comprises WGQGTTVTVSS (SEQ ID NO:57) or a homologous sequence having at least 80% sequence identity therewith;

[0107] The LFR1 comprises DIVMTQSPDSLAVSLGERATX 21 NC (SEQ ID NO:58) or a homologous sequence having at least 80% sequence identity therewith;

[0108] The LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO:59) or a homologous sequence having at least 80% sequence identity therewith;

[0109] The LFR3 comprises GVPDRFX 22GSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:60) or a homologous sequence having at least 80% sequence identity thereto; and

[0110] the LFR4 comprises FGGGTKVEIK (SEQ ID NO:61) or a homologous sequence having at least 80% sequence identity thereto;

[0111] wherein X 17 is T or S, X 18 is R or K, X 19 is M or I, X 20 is M or L, X 21 is I or M, and X 22 is S or T.

[0112] In some embodiments of the anti-CLDN18.2 antibody-radionuclide conjugate, HFR1 comprises a sequence selected from the group consisting of SEQ ID NO:62 and 63, HFR2 comprises a sequence selected from the group consisting of SEQ ID NO:64 and 65, HFR3 comprises a sequence selected from the group consisting of SEQ ID NO:66 and 67, HFR4 comprises the sequence of SEQ ID NO:57, LFR1 comprises a sequence consisting of the group of SEQ ID NO:68 and 69, LFR2 comprises the sequence of SEQ ID NO:59, LFR3 comprises a sequence selected from the group consisting of SEQ ID NO:70 and 71, and LFR4 comprises the sequence of SEQ ID NO:61.

[0113] In some embodiments of the anti-CLDN18.2 antibody-radionuclide conjugate, the heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:45 and SEQ ID NO:47, and homologous sequences having at least 80% sequence identity thereto but retaining the specific binding affinity for CLDN18.2.

[0114] In some embodiments of the anti-CLDN18.2 antibody-radionuclide conjugate, the light chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, and homologous sequences having at least 80% sequence identity thereto but retaining the specific binding affinity for CLDN18.2.

[0115] In some embodiments of the anti-CLDN18.2 antibody-radionuclide conjugate, the heavy chain variable region comprises the sequence of SEQ ID NO:23, and the light chain variable region comprises the sequence of SEQ ID NO:24;

[0116] The heavy chain variable region comprises the sequence of SEQ ID NO:25, and the light chain variable region comprises the sequence of SEQ ID NO:26;

[0117] The heavy chain variable region comprises the sequence of SEQ ID NO:27, and the light chain variable region comprises the sequence of SEQ ID NO:28;

[0118] The heavy chain variable region comprises the sequence of SEQ ID NO:29, and the light chain variable region comprises the sequence of SEQ ID NO:26 or 28;

[0119] The heavy chain variable region comprises the sequence of SEQ ID NO:37, and the light chain variable region comprises the sequence of SEQ ID NO:38;

[0120] The heavy chain variable region comprises the sequence of SEQ ID NO:39, and the light chain variable region comprises the sequence of SEQ ID NO:40;

[0121] The heavy chain variable region comprises the sequence of SEQ ID NO:41, and the light chain variable region comprises the sequence of SEQ ID NO:42;

[0122] The heavy chain variable region comprises the sequence of SEQ ID NO:45, and the light chain variable region comprises the sequence of SEQ ID NO:46; or

[0123] The heavy chain variable region comprises the sequence of SEQ ID NO:47, and the light chain variable region comprises the sequence of SEQ ID NO:48.

[0124] In some embodiments of the anti-CLDN18.2 antibody-radionuclide conjugate, the anti-CLDN18.2 antibody-radionuclide conjugate is humanized.

[0125] In some embodiments of the anti-CLDN18.2 antibody-radionuclide conjugate, the anti-CLDN18.2 antibody-radionuclide conjugate comprises a bifunctional antibody, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv'), disulfide-stable bifunctional antibody (ds bifunctional antibody), single-chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, and bivalent domain antibody.

[0126] In some embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate is in a composition having a specific activity of 3.0 - 6.0 GBq / μmol.

[0127] In some embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate is in a composition having at least 95% to at least 99% of 124 I-labeled anti-CLDN18.2 antibody (e.g., 124 I-labeled 18B10 or 124 I-SF106) or 89 Zr-labeled anti-CLDN18.2 antibody (e.g., 89 Zr-18B10) or 177 Lu-labeled anti-CLDN18.2 antibody (e.g., 177 Lu-18B10 or 177 Lu-DOTA-SF106), as measured by radio-thin layer chromatography (TLC) or as measured by radio-high performance liquid chromatography (HPLC).

[0128] In yet another aspect, the present disclosure provides a composition comprising the anti-CLDN18.2 antibody-radionuclide conjugate as defined herein and one or more pharmaceutically acceptable carriers, wherein the anti-CLDN18.2 antibody-radionuclide conjugate has at least one of the following properties:

[0129] a) having a radiochemical purity of at least 90% (e.g., 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%);

[0130] b) having a radiolabeling rate of at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%);

[0131] c) being able to specifically bind to CLDN18.2 with a Kd value of no more than 15 nM (e.g., no more than 14 nM, no more than 13 nM, no more than 12 nM, no more than 10 nM, no more than 8 nM, no more than 6 nM, no more than 4 nM, or no more than 2 nM); and

[0132] d) It can specifically bind to CLDN18.2 with an EC50 value of no more than 1.0 nM (such as no more than 0.8 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, or no more than 0.3 nM), as measured by ELISA.

[0133] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises a therapeutic radionuclide selected from the group consisting of: 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb or a diagnostic radionuclide selected from the group consisting of: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe,62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac.

[0134] In certain embodiments, the therapeutic radionuclide is 177 Lu or 124 I.

[0135] In certain embodiments, the diagnostic radionuclide is 124 I-18B10, 89 Zr or 177 Lu.

[0136] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate further comprises a chelator.

[0137] In certain embodiments, the chelating agent is DFO or DOTA.

[0138] In certain embodiments, the chelating agent is DFO, and the radionuclide is 89 Zr.

[0139] In certain embodiments, the chelating agent is DOTA, and the radionuclide is 177 Lu.

[0140] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises a compound of formula (I):

[0141] wherein

[0142] A is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof as defined in any one of the preceding claims, and k is from 1 to 40 (such as 3-5, 5-35, 10-30, 15-25 or 20).

[0143] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises a compound moiety of formula (II):

[0144] BRIEF DESCRIPTION OF THE DRAWINGS

[0145] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure. By referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein, the present disclosure can be better understood.

[0146] Figure 1 Shows 124 The study design of I-18B10 PET / CT and PET / MR imaging.

[0147] Figure 2A Shows the quantification of PET data by analyzing the ROI to demonstrate the dynamic changes of the tracer in the tumor at different post-injection time points. Figure 2B Shows paraffin-embedded tissue carrier slides of mice subjected to IHC staining. Figure 2C Shows maximum intensity projection (MIP) images.

[0148] Figure 3A Shows the maximum intensity projection (MIP) images of Patient 3 at 2h, 24h, 48h, and 72h. Figure 3B Shows 124 The biodistribution of I in humans. Figure 3C Shows 124There were significant differences in I-18B10 tumor uptake (P<0.0001), with ovarian metastases showing the highest uptake, where the mean SUV maximum was 23.65±2.05, and lung metastases showing the lowest uptake, where the SUV maximum was 1.5. Figure 3D It was shown that the uptake in lesions not treated with CLDN18.2-targeted therapy was significantly higher than that in treated lesions (SUV maximum at 24 h: 6.00±7.38 vs. 2.37 vs. 1.43, P = 0.042).

[0149] Figure 4 It was shown that 124 I-18B10 PET / CT results, where inset A indicates that the maximum intensity projection (MIP) images of patient 4 at 2 h, 24 h, 48 h, and 120 h showed two abnormally highly uptake masses in the bilateral ovaries; insets B-D indicate 124 I-18B10 PET / CT showed increased uptake in some liver and bone metastases 124 with SUV maxima of 3.2 and 4.4 respectively, and two soft tissue masses with abnormally high uptake were observed in the bilateral ovaries, where the SUV maxima on the left and right were 12.5 and 17.6 respectively. Figure 4 Inset E shows the pathological examination results (IHC) of the patient's primary lesion. Figure 4 Inset F is 124 the time-dependent curve of I-18B10 uptake in each organ.

[0150] Figure 5 It was shown that 124 I-18B10 PET results, where insets A-C indicate 18F-FDG PET / CT at 72 h, 124 I-18B10 PET / CT and 124 I-18B10 PET / MR at 96 h (patient 8) showed high uptake in the lymph nodes beside the left iliac vessels, where the SUV maxima were 4, 6.1, and 6.6 respectively; inset D is the pathological examination result (IHC) of the patient's primary lesion in patient 8, which showed a CLDN18.2 expression level of 3+, 90%; insets E-G indicate 18F-FDG PET / CT at 72 h, 124 I-18B10 PET / CT and 124 I-18B10 PET / MR at 96 h (patient 10) showed high uptake in the retroperitoneal metastatic lymph nodes, where the SUV maxima were 3.5, 8.1, and 2.5 respectively; and inset H is the pathological examination result of the patient's primary lesion in patient 10, which also showed a CLDN18.2 expression level of 3+, 90%.

[0151] Figure 6 shows 124 I-18B10 PET results, where insets A-C indicate that before treatment 124 on I-18B10 PET, several peritoneal metastases showed 124 high uptake of I-18B10, with SUVmax values of 3.1, 3.2, and 4.2; insets D-F indicate that after receiving CLDN18.2-targeted therapy 124 on I-18B10 PET, the original highly-uptaken peritoneal metastases did not show obvious uptake; and inset G indicates the pathological examination results of the patient's primary lesion.

[0152] Figure 7A shows 124 the stability of I-18B10 in PBS within 48 h. Figure 7B shows that at each selected time point, the uptake of positive cells was significantly higher than that of negative cells or blocking controls (p < 0.0001). Figure 7C shows the 124 binding constant (Kd = 4.11 nM) of I-18B10 to the CLDN18.2 receptor determined by cell saturation binding assay using MKN45CLDN18.2+ cells. Figure 7D shows the biodistribution study in normal mice (n = 4), which shows a decrease over time in all tissues, indicating that the probe has no specificity in normal organs.

[0153] Figure 8A shows high gastric uptake within 4 h after gavage (2.53 ± 0.27 at 4 h and 2.98 ± 0.24 at 24 h), and the uptake gradually decreases as the gastric injury gradually heals. Figure 8B Indicates that after autopsy, the gastric body shows local redness, punctate bleeding of varying degrees, and congestion of the gastric body. Figure 8C Indicates the HE results, which show that except at 4 h after gavage, there is no obvious damage to the gastric tissue at all time points. Figure 8D Shows that at longer time points (1.81 ± 0.20 at 48 h vs. 0.74 ± 0.05, and 1.01 ± 0.10 at 96 h vs. 0.46 ± 0.07), higher uptake (p < 0.05) was observed in the gastric tissue approaching healing in the gastric ulcer model compared to normal mice.

[0154] Figure 9 shows information regarding 124 the quality control of I-18B10.

[0155] Figure 10Summarized the characteristics of 17 patients with pathological results included in the analysis during the period from May 2021 to April 2022, namely, 6 males and 11 females, with a median age of 51 (29 - 65) years. CLDN18.2 expression was determined by IHC on primary lesions resected prior to PET imaging.

[0156] Figure 11 Dosimetry estimates indicating OLINDA showed that the three organs receiving the highest absorbed doses were the spleen, kidney, and liver, with average values of 1.20, 0.717, and 0.616 mGy / MBq, respectively. The average effective dose was 0.213 mSv / MBq (0.788 rem / mCi).

[0157] Figure 12 Indicated the amino acid sequences of certain proteins used in the present disclosure.

[0158] Figure 13 Showed 89 Study design of 89Zr-18B10 microPET imaging.

[0159] Figure 14A Showed the MALDI-TOF-MS of 18B10. Figure 14B Showed the MALDI-TOF-MS of DFO-18B10. Figure 14C Showed non-reducing SDS-PAGE characterization. Figure 14D Showed the binding of 18B10 and DFO-18B10 to human CLDN18.2 protein evaluated by ELISA.

[0160] Figure 15 Showed 89 Synthesis and radiolabeling, purity, and in vitro stability of 89Zr]89Zr-DFO-18B10, where inset A showed 89 Synthesis and radiolabeling of 89Zr]89Zr-DFO-18B10, inset B showed before and after purification 89 Radioactive TLC results of 89Zr]89Zr-DFO-18B10, and inset C showed 89 In vitro stability of 89Zr]89Zr-DFO-18B10.

[0161] Figure 16 Showed in vitro CLDN18.2 expression in cell lines, where inset A showed BGC823 CLDN18.2 and Western blot results of CLDN18.2 expression in BGC823 cell line, inset B showed BGC823 CLDN18.2 and relative expression of CLDN18.2 in BGC823 cells (results shown as mean ± SD, n = 3), inset C showed BGC823CLDN18.2 Flow cytometry histograms of BGC823 cells, and inset D shows BGC823 CLDN18.2 and 89 cellular uptake of [89Zr]Zr-DFO-18B10 in BGC823 cells. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0162] Figure 17A showing small animal PET images of four different groups at 2, 24, 48, 72, and 96 h. Figure 17B showing BGC823 CLDN18.2 average SUV of [89Zr]Zr-DFO-18B10 in mouse organs. 89 average SUV of [89Zr]Zr-DFO-18B10 in mouse organs of BGC823 without unlabeled 18B10 blocking. Figure 17C showing BGC823 CLDN18.2 average SUV of [89Zr]Zr-DFO-18B10 in mouse organs. 89 average SUV of [89Zr]Zr-DFO-IgG in mouse organs of BGC823. Figure 17D showing BGC823 89 average SUV of [89Zr]Zr-DFO-18B10 in mouse organs. Figure 17E showing BGC823 CLDN18.2 average SUV of [89Zr]Zr-DFO-IgG in mouse organs of BGC823.

[0163] Figure 18A showing BGC823 CLDN18.2 section images of tumor uptake at p.i. 48 h in BGC823 mice compared with section images of [18F]F-FDG at p.i. 1 h. 18 section images of tumor uptake at p.i. 48 h in BGC823 mice compared with section images of [18F]F-FDG at p.i. 1 h. Figure 18B showing average SUV in mouse organs of different experimental groups at 48 h. Figure 18C showing T / H at each time point of p.i. Figure 18D showing T / M at each time point of p.i. Figure 18E showing BGC823 CLDN18.2 (++) (e1) and BGC823 (-) (e2) IHC analysis of CLDN18.2 expression in tumors.

[0164] Figure 19A showing biodistribution in three different tumor models at p.i. 48 h. Figure 19B showing T / L at p.i. 48 h. Figure 19C showing T / S at p.i. 48 h. Figure 19D showing T / B at p.i. 48 h.

[0165] Figure 20A showing 89Biodistribution of [Zr]Zr-DFO-18B10 in normal mice. Figure 20B MicroPET / CT imaging of [Zr]Zr-DFO-18B10 at 2, 24, 48, 72 h and 120 h after tail vein injection in normal mice is shown. 89 Figure 20C The average SUV values of important organs at different time points are shown.

[0166] Figure 21A - 21E The average SUV values of organs in mice of different experimental groups at different time points are shown.

[0167] Figure 22 Stomach of BGC823 CLDN18.2 model mice (+++) (inset A); stomach of BGC823 model mice (+++) (inset B); BGC823 CLDN18.2 spleen of BGC823 model mice (-) (inset C); spleen of BGC823 model mice (-) (inset D); BGC823 CLDN18.2 liver of BGC823 model mice (-) (inset E); immunohistochemical results of liver of BGC823 model mice (-) (inset F).

[0168] Figure 23 Shown is 177 the steps and conditions of [Lu]Lu labeling.

[0169] Figure 24A Shown is the small animal PET image of BGC823 177 positive tumor-bearing mice after injection of 300 uCi CLDN18.2 of [Lu]Lu-DOTA-18B10. Figure 24B Shown is the small animal PET image of AGS 177 positive tumor-bearing mice after injection of 300 uCi CLDN18.2 of [Lu]Lu-DOTA-18B10.

[0170] Figure 25 Shown is the small animal PET image of BGC823 177 CLDN18.2 positive tumor-bearing mice, BGC823 CLDN18.2 negative tumor-bearing mice and blocked tumor-bearing mice after injection of 300 uCi

[0171] Figure 26 Shown is the small animal PET image of AGS 177 CLDN18.2 positive tumor-bearing mice, AGS CLDN18.2 negative tumor-bearing mice and blocked tumor-bearing mice after injection of 300 uCi

[0172] Figure 27 ​CLDN18.2 immunohistochemical staining of BGC823 CLDN18.2-positive tumor-bearing mice, AGS CLDN18.2-positive tumor-bearing mice, and BGC823 CLDN18.2-negative tumor-bearing mice is shown.

[0173] Figure 28 Shown are those in gastric cancer cell lines BGC823 and AGS 177 Lu-DOTA-18B10 uptake.

[0174] Figure 29A Shown are those in AGS cells 177 Kd constant in the saturation binding assay of Lu-DOTA-18B10.

[0175] Figure 29B Shown are those on normal KM mice 177 Pharmacokinetics of Lu-DOTA-18B10.

[0176] Figure 30 - 31 Shown is 177 Biodistribution of Lu-DOTA-18B10 in BGC823 CLDN18.2-positive tumor-bearing mice.

[0177] Figure 32 Shown is 177 Therapeutic effect of Lu-DOTA-18B10.

[0178] Figure 33 Shown is 177 Effect of Lu-DOTA-18B10 on mouse weight.

[0179] Figure 34 Shown are the HE stainings of tumor tissues of mice injected with 300 uCi or 150 uCi 177 Lu-DOTA-18B10 or the control group.

[0180] Figure 35 Shown are the HE stainings of different organs of mice injected with 300 uCi or 150 uCi 177 Lu-DOTA-18B10 or the control group.

[0181] Figure 36 Shown is the novel scFv-Fc (SF106) antibody targeting CLDN18.2 labeled with 124 I and 177 Lu. The uptake of 124 I-SF106 in targeted cells was analyzed. MicroPET imaging of 124 I-SF106 was performed on CLDN18.2-positive tumor-bearing mice, and by 177Preliminary treatment research on Lu-DOTA-SF106.

[0182] Figure 37 shows the construction process and biochemical characterization of the scFv-Fc antibody. Figure 37A An overview of the production of the scFv-Fc antibody is shown. Figure 37B It is shown that the average molecular weight of the SF106 antibody measured by MALDI-TOF-MS is 108303.124 Da. Figure 37C It is shown that FACS analysis of SF106 binding on HEK293-human CLDN18.2-hi cells was performed, and the EC50 was 11.31 nM.

[0183] Figure 38 It is shown that 124 Construction and in vitro analysis of I-SF106, where inset A shows the structural model of the SF106 antibody and the schematic diagram of isotope labeling of the SF106 antibody with 124 I, inset B shows 124 the stability of I-SF106 in 0.01 M PBS and 5% HSA solution, inset C shows the expression of CLDN18.2 in BGC823 CLDN18.2 and BGC823 cells detected by flow cytometry, and inset D shows 124 the cellular uptake experiment of I-SF106 in BGC823 CLDN18.2 and BGC823 cells (***P < 0.001).

[0184] Figure 39 It is shown that in the tumor-bearing mouse model after injection (4, 24, 48, 72, 96, and 120 h) 124 microPET imaging of I-SF106 (arrows indicate tumors).

[0185] Figure 40 It is shown that the analysis of microPET imaging, where inset A shows the trend of the maximum SUV of I-SF106 in tumors of different groups from 4 to 120 h, inset B shows the tumor / muscle (T / M) of different groups at each time point after injection, inset C shows the tumor / liver (T / L) of different groups at each time point after injection, inset D shows the tumor / heart (T / H) of different groups at each time point after injection, inset E shows that the IHC of BGC823 124 tumors is positive (++), and inset F shows that the IHC of BGC823 tumors is negative (-). CLDN18.2 The IHC of BGC823

[0186] Figure 41 It is shown that 177Construction of Lu-DOTA-SF106 and research on radioimmunoimaging and radionuclide therapy, where small figure A shows the synthesis 177 stages of Lu-DOTA-SF106, and small figure B shows AGS CLDN18.2 in tumor-bearing mice 177 SPECT / CT imaging of Lu-DOTA-SF106, and small figure C shows in AGS tumor-bearing mice 177 small animal SPECT / CT imaging of Lu-DOTA-SF106, and small figure D shows by 177 tumor growth curves of mice after treatment with Lu-DOTA-SF106 and 0.01M PBS (***P<0.001), and small figure E shows by 177 weight curves of mice after treatment with Lu-DOTA-SF106 and 0.01M PBS.

[0187] Figure 42 shows nat mass spectrometry detection of I-SF106 and 124 in vitro study of I-SF106. Figure 42A shows nat MALDI-TOF-MS (108963Da) of I-SF106. Figure 42B shows SF106, nat I-SF106 and 124 microPET imaging and SDS-PAGE characterization of I-SF106. Figure 42C shows the radioactive TLC results of the experiment before purification. Figure 42D shows after purification 124 radioactive TLC results of I-SF106. Figure 42E shows BGC823 CLDN18.2 in cells 124 determination of the saturation binding constant (K d ) of I-SF106 in, and the K d value is 17.74 nmol / L. Figure 42F shows after intravenous (tail vein) injection 124 pharmacokinetics of I-SF106 in the blood of KM mice (n = 5). The half-life (slow) is 27.68 h, and the half-life (fast) is 0.6354 h.

[0188] Figure 43 shows the SUV maximum value analysis and biodistribution study of microPET / CT imaging, where small figure A shows BGC823 CLDN18.2The maximum SUV values of tumors, muscles, heart, and liver in the PET / CT imaging of the model. Small panel B shows the maximum SUV values of tumors, muscles, heart, and liver in the PET / CT imaging of the BGC823 model. Small panel C shows the BGC823 co-injected with SF106 CLDN18.2 The maximum SUV values of tumors, muscles, heart, and liver in the PET / CT imaging of the model. Small panel D shows the values at 4 h, 24 h, 48 h, and 96 h after tail vein injection 124 The biodistribution of I-SF106 in KM mice, and small panel E shows the values at 4 h, 24 h, 48 h, 96 h, and 120 h after tail vein injection 124 I-SF106 in BGC823 CLDN18.2 The biodistribution in mice.

[0189] Figure 44 Shows the results of co-injecting the anti-CLDN18.2 monoclonal antibody Hu18B10HaLa, where small panel A shows the co-injection 124 I-SF106 and Hu18B10HaLa and only injecting 124 I-IgG, and then the microPET imaging of tumor-bearing BGC823 CLDN18.2 mice. Small panel B shows the BGC823 CLDN18.2 The maximum SUV value of the tumor in the PET / CT imaging of tumor-bearing mice.

[0190] Figure 45 Shows the detection by DOTA-SF106 mass spectrometry and 177 the in vitro study of Lu-DOTA-SF106. Where small panel A shows the MALDI-TOF-MS study of DOTA-SF106, and small panel B shows 177 the stability of Lu-DOTA-SF106 in 0.01 M PBS and 5% HSA solution. Small panel C shows the radioactive TLC results of the experiment before purification, and small panel D shows the results after purification 124 of the radioactive TLC of I-SF106.

[0191] Figure 46 shows the radioisotope labeling, cell uptake assay, and saturation binding experiment. Figure 46A Shows 177 The flow chart of the radioisotope labeling of Lu-DOTA-18B10. Figure 46B Shows BGC823 CLDN18.2 / BGC823 and AGS CLDN18.2 / AGS cells 177 The cell uptake assay of Lu-DOTA-18B10. Figure 46C Shows the cell saturation binding experiment and 177K of Lu-DOTA-18B10 d Calculation.

[0192] Figure 47A - 47E SPECT imaging, ROI analysis, and histological verification are shown. Figure 47A BGC823 is shown CLDN18.2 / AGS CLDN18.2 in the xenograft mouse model 177 of the Lu-DOTA-18B10 probe. Figure 47B BGC823 is shown CLDN18.2 / AGS CLDN18.2 ROI analysis of the SPECT imaging of the model. Figure 47C BGC823 is shown CLDN18.2 Imaging comparison of xenografts between the mouse model and its negative / blocking groups, and AGS CLDN18.2 Comparison between the mouse model and its blocking group. Figure 47D Shows the comparison of radioactive uptake of tumors or spleens between the positive / negative / blocking groups in the BGC823 and AGS xenograft models. Figure 47E Shows from BGC823 CLDN18.2 / BGC823 / AGS CLDN18.2 CLDN18.2 IHC staining of tumor tissues isolated from the mouse model. (Scale = 100 μm).

[0193] Figure 48 Shows the biodistribution experiment, where inset A shows the biodistribution of Lu-DOTA-18B10 over time in the BGC823 CLDN18.2 mouse model 177 and inset B shows the biodistribution comparison between the positive, negative, and blocking groups of the BGC823 CLDN18.2 model 48 h after injection.

[0194] Figure 49 shows the therapeutic efficacy of Lu-DOTA-18B10 in the GC mouse model 177 Figure 49A BGC823 is shown CLDN18.2 / AGS CLDN18.2 in the model 177 of the Lu-DOTA-18B10 treatment curve. Figure 49B BGC823 is shown CLDN18.2 / AGS CLDN18.2 Tumor volume comparison between each treatment group of the model. Figure 49C shows the tumor H&E and Ki67 IHC staining comparison between each treatment group. (H&E scale = 50 μm; IHC scale = 100 μm; ***: p < 0.001). ​

[0195] Figure 50 Shows the results of the toxicity experiment. Complete blood count analysis and liver function tests were performed on male or female balb / c nude mice treated with 300 μCi 177 Lu-DOTA-18B10 or saline. The grey boxes represent the mean ± SD of the values collected from the entire toxicity experiment mouse cohort one day before treatment. The grey dashed lines represent the normal ranges for female or male balb / c mice. (*: p < 0.05, ***: p < 0.001).

[0196] Figure 51 Shows the stability test. 177 Stability test results generated by Lu-DOTA-18B10.

[0197] Figure 52 Shows the flow cytometry experiment, where inset A shows the CLDN18.2 expression levels in AGS CLDN18.2 and AGS cells, and inset B shows the CLDN18.2 expression levels in BGC823 CLDN18.2 and BGC823 cells.

[0198] Figure 53 Shows the spleen CLDN18.2 IHC staining. CLDN18.2 expression levels in the spleen tissue of the balb / c nude mouse model. (Scale = 100 μm).

[0199] Figure 54 Shows the T / NT values of the biodistribution of important organs over time in the BGC823 CLDN18.2 mouse model.

[0200] Figure 55 Shows the pharmacokinetic experiment. Pharmacokinetic results of 177 Lu-DOTA-18B10 in normal healthy KM mice.

[0201] Figure 56 Shows the body weight monitoring of BGC823 CLDN18.2 and AGS CLDN18.2 mice during the therapy experiment.

[0202] Figure 57 Shows the histological toxicity test. H&E staining of various organs in balb / c nude mice injected with 300 μCi 177 Lu-DOTA-18B10 probe or saline. (Scale = 50 μm). Detailed implementation

[0203] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and thus can of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, as the scope of the present disclosure will be defined only by the appended claims.

[0204] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0205] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and incorporated herein by reference to disclose and describe the methods and / or materials. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the provided publication dates may be different from the actual publication dates, which may need to be independently confirmed.

[0206] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and shown herein has discrete components and features that can be readily separated from or combined with the features of any of the several other embodiments without departing from the scope or spirit of the present disclosure. Any of the recited methods can be performed in the recited order of events or in any other order that is logically possible.

[0207] Definition

[0208] The following definitions are provided to assist the reader. Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms used herein are intended to have the meaning commonly understood by those skilled in the art of chemistry and medicine. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from the definitions commonly understood in the art.

[0209] As used herein, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include plural referents.

[0210] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. A naturally occurring intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and each heavy chain is composed of a variable region (V H ), as well as first, second, and third constant regions (C H1 , C H2 , and C H3 ), respectively; mammalian light chains are classified as λ or κ, and each light chain is composed of a variable region (V L) and a constant region. An antibody has a "Y" shape, where the stem of the Y is composed of the second and third constant regions of two heavy chains joined together by disulfide bonds. Each arm of the Y includes a variable region and a first constant region of a single heavy chain that binds to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain typically contains three highly variable loops called complementarity-determining regions (CDRs) (the light-chain CDRs include LCDR1, LCDR2, LCDR3, and the heavy-chain CDRs include HCDR1, HCDR2, HCDR3). The CDR boundaries of the antibodies and antigen-binding domains disclosed herein can be defined or identified by the Kabat, IMGT, AbM, Chothia, or Al-Lazikani rules (Al-Lazikani, B., Chothia, C., Lesk, A.M., Journal of Molecular Biology (J. Mol. Biol.), 273(4), 927 (1997); Chothia, C. et al., Journal of Molecular Biology, December 5; 186(3): 651-63 (1985); Chothia, C. and Lesk, A.M., Journal of Molecular Biology, 196, 901 (1987); N.R. Whitelegg et al., Protein Engineering, v13(12), 819-824 (2000); Chothia, C. et al., Nature, December 21-28; 342(6252): 877-83 (1989); Kabat E.A. et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (Second Edition), Chapter 26, 481-514, (2015)). The three CDRs are interposed between flanking extensions called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified into multiple classes based on the amino acid sequence of their heavy-chain constant regions.The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the main antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In certain embodiments, the antibodies provided herein encompass any antigen-binding fragment thereof.

[0211] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed by an antibody fragment containing one or more CDRs or any other antibody moiety that binds to an antigen but does not contain the complete native antibody structure. Examples of antigen-binding fragments include, but are not limited to, bispecific antibodies, Fab, Fab', F(ab') 2 , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bispecific antibodies (ds bispecific antibodies), single-chain antibody molecules (scFv), scFv-Fc, scFv dimers (bivalent bispecific antibodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment may include one or more CDRs from a particular human antibody.

[0212] "Fab" with respect to an antibody refers to the monovalent antigen-binding fragment of the antibody consisting of a single light chain (both variable and constant regions) and the variable region and first constant region of a single heavy chain joined by a disulfide bond. Fab can be obtained by pepsin digestion of the antibody at the residues N-terminal to the disulfide bond between the heavy chains near the hinge region.

[0213] "Fab'" refers to a Fab fragment that includes a portion of the hinge region and can be obtained by pepsin digestion of the antibody at the residues C-terminal to the disulfide bond between the heavy chains near the hinge region and thus differs from Fab in terms of a small number of residues (including one or more cysteines) in the hinge region.

[0214] "F(ab') 2 " refers to a dimer of Fab' that includes two light chains and a portion of two heavy chains.

[0215] "Fc" with respect to an antibody refers to the portion of the antibody consisting of the second constant region and the third constant region of the first heavy chain bound by disulfide bonds to the second constant region and the third constant region of the second heavy chain. The IgG and IgM Fc regions include three heavy chain constant regions (the second, third, and fourth heavy chain constant regions in each chain). It can be obtained by pepsin digestion of the antibody. The Fc portion of the antibody is responsible for various effector functions such as ADCC, ADCP, and CDC, but does not play a role in antigen binding.

[0216] "Fv" with respect to an antibody refers to the smallest fragment of the antibody that carries the complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. "dsFv" refers to a disulfide bond-stabilized Fv fragment, in which the connection between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0217] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody formed by directly connecting the variable region of the light chain to the variable region of the heavy chain or by connecting them through a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)). "scFv dimer" refers to a single chain containing two heavy chain variable regions and two light chain variable regions with linkers. In certain embodiments, the "scFv dimer" is a bivalent bifunctional antibody or a bivalent ScFv (BsFv), the bivalent bifunctional antibody or BsFv comprising (connected by a peptide linker) V H -V L wherein the V H -V L partially dimerizes with another V H -V L such that the V H of one part coordinates with the V L of the other part and forms two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody, the bispecific bifunctional antibody comprising V L1 -V H2 (connected by a peptide linker) associated with V H1 -V L2 (also connected by a peptide linker) such that V H1 coordinates with V L1 and V H2 coordinates with V L2 , and each coordination pair has a different antigen specificity.

[0218] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0219] "Camelized single-domain antibody", "heavy-chain antibody", "nanobody" or "HCAb" refers to an antibody that comprises two V H domains and does not comprise a light chain (Riechmann L. and Muyldermans S., Journal of Immunological Methods. Dec 10; 231(1-2):25-38 (1999); Muyldermans S., Journal of Biotechnology. Jun; 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; US Patent No. 6,005,079). Heavy-chain antibodies were originally obtained from Camelidae (camels, dromedaries and llamas). Although lacking a light chain, camelized antibodies have a proven antigen-binding profile (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428):446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. Apr; 54(1):39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1):93-101 (2003)). The variable domain of the heavy-chain antibody (VHH domain) represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., The FASEB Journal. Nov; 21(13):3490-8. Epub Jun 15, 2007 (2007)). "Bifunctional antibody" comprises small antibody fragments having two antigen-binding sites, wherein the fragment comprises a V L domain linked to a V H domain in a single polypeptide chain (V H -V L or V L -V H)(See, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA Jul. 15; 90(14): 6444-8 (1993); EP404097; WO93 / 11161). Because the linker is too short, the two domains on the same chain cannot pair, thus forcing the domains to pair with the complementary domains on the other chain, thereby generating two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes).

[0220] "Domain antibody" refers to an antibody fragment containing only the variable region of the heavy chain or the variable region of the light chain. In certain embodiments, two or more V H domains are covalently linked by a peptide linker to produce a bivalent or multivalent domain antibody. The two V H domains of the bivalent domain antibody can target the same or different antigens.

[0221] In certain embodiments, "(dsFv) 2 " comprises three peptide chains linked by a peptide linker and bound to two V L portions by disulfide bridges: two V H portions.

[0222] In certain embodiments, "bispecific ds bifunctional antibody" comprises V H1 bound to V L1 by a disulfide bridge between V L1 -V H2 (linked by a peptide linker) and bound to V H1 -V L2 (also linked by a peptide linker).

[0223] In certain embodiments, "bispecific dsFv" or "dsFv-dsFv'" comprises three peptide chains: V H1 -V H2 portions, wherein the heavy chains are bound by a peptide linker (e.g., a long flexible linker) and paired with the V L1 and V L2 portions by disulfide bridges, respectively. Each heavy chain and light chain paired by a disulfide bond has a different antigen specificity.

[0224] As used herein, the term "humanized" means that an antibody or antigen-binding fragment comprises CDRs derived from a non-human animal, FR regions derived from a human, and, when applicable, constant regions derived from a human. In certain embodiments, the amino acid residues of the variable region framework of a humanized CLDN18.2 antibody are substituted for sequence optimization. In certain embodiments, the variable region framework sequence of a humanized CLDN18.2 antibody chain is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the corresponding human variable region framework sequence.

[0225] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy and / or light chain derived from one species and the remaining portion of the heavy and / or light chain derived from a different species. In illustrative examples, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human species (such as derived from a mouse).

[0226] The term "germline sequence" refers to a nucleic acid sequence encoding a variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by germline immunoglobulin variable region sequences. A germline sequence can also refer to a variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. A germline sequence can be only framework regions, only complementarity-determining regions, framework regions and complementarity-determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that include a variable region. Sequence identity can be determined using the methods described herein, such as by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. A germline nucleic or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a reference variable region nucleic or amino acid sequence. Germline sequences can be determined, for example, through the publicly available International ImMunoGeneTics database (IMGT) and V-base.

[0227] As used herein, an "anti-CLDN18.2 antibody" or an "antibody against CLDN18.2" refers to an antibody that can specifically bind to CLDN18.2 (e.g., human or non-human CLDN18.2) with sufficient affinity, for example, to provide diagnostic and / or therapeutic uses.

[0228] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0229] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein bind with ≤10 -6 M (e.g., ≤5×10 -7 M, ≤2×10 -7M, ≤10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M, ≤2×10 -9 M or ≤10 -9 The binding affinity (K D ) of M) specifically binds to human and / or non-human CLDN18.2. As used herein, K D refers to the ratio of the dissociation rate to the association rate (k off / k on ), and said ratio can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS methods, and flow cytometry (such as FACS) methods. In certain embodiments, the K D value can be appropriately determined by using flow cytometry methods. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein (see, e.g., Harlow and Lane, Using Antibodies, A Laboratory Manual (1998), for descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal that is at least twice the background signal and more typically at least 10-fold to 100-fold the background.

[0230] The "percent sequence identity (%)" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum correspondence. Alignment for determining the percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., Journal of Molecular Biology, 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tools or can appropriately customize the parameters according to the needs of the alignment, for example, by selecting a suitable algorithm. In certain embodiments, different residue positions may differ due to conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson, (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference.

[0231] As used herein, the terms "homologue sequence" and "homologous sequence" are used interchangeably and refer to a polynucleotide sequence (or its complementary strand) or an amino acid sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optionally aligned.

[0232] As used herein, the term "antibody-radionuclide conjugate" refers to an antibody or an antigen-binding fragment thereof conjugated to a radionuclide, such as a diagnostic radionuclide or a therapeutic radionuclide. Examples of diagnostic radionuclides suitable for conjugation to an antibody include, but are not limited to 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 l, 124 l, 125 l, 131 l, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194lr, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac. Examples of therapeutic radionuclides suitable for conjugation to antibodies include, but are not limited to 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb.

[0233] It should be noted that in the present disclosure, terms such as "comprises", "comprised", "comprising", "contains" or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0234] The terms "determining / determine", "measuring", and "detecting / detect" may be used interchangeably and refer to quantitative and semi-quantitative determinations.

[0235] As used herein, "likelihood" and "likely" with respect to an object's response to treatment are a measure of how likely a treatment response will occur in the object. It may be used interchangeably with "probability". Likelihood refers to a probability that is greater than speculation but less than certainty. Thus, if a reasonable person using common sense, training, or experience concludes that a treatment response is likely in a given situation, then the treatment response is likely. In one embodiment, the terms "likelihood" and "likely" represent the percentage of the odds of a treatment response occurring. In some embodiments, a cancer object identified as "likely to respond" refers to a cancer object having a greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% chance of responding to a CLDN18.2-targeted therapy.

[0236] The terms "response" or "responsiveness" as used in the context of an object's treatment response to a therapy may be used interchangeably and refer to a beneficial response of the object to the treatment, rather than an adverse response, i.e., an adverse event. In an object with a tumor, a beneficial response can be expressed in terms of a number of clinical parameters, including complete response (e.g., disappearance of the detectable tumor) or partial response (e.g., reduction in tumor size and / or reduction in the number of cancer cells). A decrease in tumor growth rate, thereby prolonging overall survival, enhancing the anti-tumor immune response, may cause tumor regression or rejection; alleviating one or more tumor-related symptoms to some extent; prolonging survival after treatment; and / or reducing the mortality rate at a specific time point after treatment. Continued increase in tumor size and / or number of cancer cells (in the absence of any decrease in growth rate that is beneficial for overall survival) and / or tumor metastasis indicates a lack of a beneficial response to the treatment and thus decreased responsiveness.

[0237] As used herein, "cancer" refers to the general term for a variety of malignant cell tumors, which are characterized by unregulated growth, lack of differentiation, and the potential or ability to invade local tissues and metastasize. These neoplastic malignancies affect every tissue and organ of the body with varying degrees of prevalence. Cancer involves the presence of cells with the typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. Typically, cancer cells will form tumors, but such cells can exist alone or can circulate in the bloodstream as independent cells such as leukemia cells. The terms cancer and tumor are used interchangeably herein. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, hematological, or solid, and all stages and grades of cancer, including pre-metastatic and post-metastatic cancers.

[0238] As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In many embodiments, the subject is a human. A subject can be a patient, which refers to a human who presents to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". A subject can have or be susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0239] Unless otherwise specified, the term "treating" or "treatment" of a CLDN18.2-related disease as used herein means partially or completely reversing, alleviating, inhibiting the progression of, or preventing the progression of a subject's CLDN18.2-related disease.

[0240] As used herein, the term "prognose" or "prognosing" refers to the prediction or anticipation of the future course or outcome of a disease or condition.

[0241] The term "CLDN18.2" refers to a Claudin-18 splice variant 2 derived from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.2 is human CLDN18.2. Exemplary sequences of human CLDN18.2 include the human CLDN18.2 protein (NCBI Reference Sequence No. NP_001002026.1, or SEQ ID NO:30). Exemplary sequences of CLDN18.2 include the mouse CLDN18.2 protein (NCBI Reference Sequence No. NP_001181852.1), the cynomolgus macaque (Macaca fascicularis) CLDN18.2 protein (NCBI Reference Sequence No. XP_015300615.1). CLDN18.2 is expressed in cancer cells. In one embodiment, the CLDN18.2 is expressed on the surface of cancer cells.

[0242] As used herein, the term "about" in reference to a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself. For example, a description of "about X" includes a description of "X". A numerical range includes the numbers defining the range. In general, the term "about" means an indicated value of a variable and all values of the variable within the experimental error of the indicated value (e.g., within 95% confidence interval of the mean) or within 10% of the indicated value, whichever is greater. In the context of using the term "about" in reference to a period of time (years, months, weeks, days, etc.), the term "about" means the period of time plus or minus the next subordinate period (e.g., about 1 year means 11 - 13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6 - 8 days; etc.), or within 10% of the indicated value, whichever is greater.

[0243] CLDN18.2 as a Biomarker

[0244] Claudin-18 (CLDN18) molecules (GenBank accession numbers: splice variant 1 (CLDN18A1 or CLDN18.1): NP_057453, NM_016369 and splice variant 2 (CLDN18A2 or CLDN18.2): NM_001002026, NP_001002026) are integral transmembrane proteins with a molecular weight of approximately 27.9 / 27.72 kD. The CLDN18 protein is located within the tight junctions of epithelia and endothelia, which organize a network of interconnected strands of intramembranous particles between adjacent cells. CLDN18 and occludin are the most important transmembrane protein components in tight junctions. Due to the strong intercellular adhesion properties of these tight junction proteins, the tight junction proteins form the main barrier to prevent and control paracellular transport of solutes, and also limit the lateral diffusion of membrane lipids and proteins to maintain cell polarity.

[0245] CLDN18 exhibits several different conformations that can be selectively resolved by antibodies (see Sahin U, Koslowski M, Dhaene K et al. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development [J]. Clinical Cancer Research, 2008, 14(23): 7624-7634). CLDN18-conformation-1 has all four hydrophobic regions as transmembrane domains (TMs) and forms two extracellular loops (loop 1 surrounded by hydrophobic regions 1 and 2; loop 2 surrounded by hydrophobic regions 3 and 4), as described for the vast majority of CLDN family members. The second conformation (CLDN18-conformation-2) means that, as described for PMP22, the second and third hydrophobic domains do not fully span the plasma membrane, such that the portion between the first and fourth transmembrane domains (loop D3) is extracellular. The third conformation (CLDN18-conformation-3) shows a large extracellular domain in which two internal hydrophobic regions are surrounded by the first and fourth hydrophobic regions. Due to the classical N-glycosylation site in loop D3, the CLDN-18 topological variants CLDN18 topology-2 and CLDN18 topology-3 have additional extracellular N-glycosylation sites.

[0246] CLDN18 has two different splice variants that exist in mice and humans. The splice variants CLDN18.1 and CLDN18.2 differ in the first 21 amino acids at the N-terminus including the first TM and loop 1, while the protein sequences in the C-terminus are the same (see Niimi T, Nagashima K, Ward J M et al. Claudin-18, a novel downstream target gene for the T / EBP / NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing [J]. Molecular and Cellular Biology, 2001, 21(21): 7380-7390).

[0247] CLDN18.1 is selectively expressed on normal lung and gastric epithelial cells, while CLDN18.2 is expressed only on gastric cells. Most importantly, CLDN18.2 expression is restricted to the differentiated short-lived cells of the gastric epithelium but not the gastric stem cell area. Using sensitive RT-PCR, the variant was not detected in any other normal human organ. However, the said CLDN18.2 is highly expressed in several cancer types, including gastric tumors, esophageal tumors, pancreatic tumors, and lung tumors, as well as human cancer cell lines (see Matsuda Y, Semba S, Ueda J, et al. Gastric and intestinal claudin expression at the invasive front of gastric carcinoma [J]. Cancer science, 2007, 98(7): 1014-1019.).

[0248] Since CLDN18.2 is highly specific for normal tissues and is expressed in multiple cancers, CLDN18.2 has become a potential target for epithelial tumors. In clinical trials, anti-CLDN18.2 antibodies have shown antitumor effects in patients with advanced gastric adenocarcinoma (7-10). The FAST study showed that the therapy of CLDN18.2 antibody drug IMAB362 (dose 800 mg / m2) combined with chemotherapy (EOX: epirubicin + oxaliplatin + capecitabine) could significantly prolong the survival of patients with advanced gastric and gastroesophageal junction tumors (PFS: 5.7 months vs. 7.9 months; OS: 8.7 months vs. 12.5 months) (11). And the curative effect of the group with strong CLDN18.2 expression was more significant (IHC: CLDN18.2 expression intensity ≥2+ and the proportion of expressing cells ≥70%) (PFS: 6.1 months vs. 9.1 months; OS: 9.3 months vs. 16.6 months). Therefore, in this new targeted therapy, a comprehensive assessment of the CLDN18.2 expression of patients is crucial.

[0249] Without wishing to be bound by any theory, it is believed that the molecular and functional characteristics of CLDN18.2 render it a highly attractive target for antibody-based cancer diagnostics and therapies. These include (i) the absence of CLDN18 in most toxicity-related normal tissues; (ii) the restricted expression of CLDN18.2 variants to dispensable cell populations, as differentiated gastric cells that can be replenished by the target-negative stem cells of the stomach; (iii) potential glycosylation differences between normal and neoplastic cells; and (iv) the presence of different conformational topologies.

[0250] It has been found that the molecular weight of the CLDN18 protein differs between tumors and adjacent normal tissues. A higher molecular weight CLDN18 protein was observed in healthy tissues, and the CLDN18 protein could be reduced to the same molecular weight as that observed in tumors by treating normal tissue lysates with the deglycosylating compound PNGase F. This indicates that CLDN18 is less N-glycosylated in tumors compared to its counterpart in normal tissues. The classical N-glycosylation motif is located in amino acid residue 116 within the loop D3 domain of the CLDN18 molecule. The molecular weight difference and the inferred structural difference may represent altered epitopes for antibody binding.

[0251] In addition, CLDN18, as a tight junction protein, can also contribute to good diagnostic specificity and a good therapeutic window. Since tumor cells express CLDN but generally do not form classical tight junctions through homotypic and heterotypic associations of CLDN as seen in normal epithelial tissues, the tumor cells may have a substantial amount of free CLDN, which is suitable for extracellular antibody binding and immunotherapy. The binding epitopes of CLDN in healthy epithelia may be shielded within the tight junctions, preventing access for antibody binding.

[0252] Anti - CLDN18.2 Antibody - Radionuclide Conjugate

[0253] In one aspect, the present disclosure provides an anti-CLDN18.2 antibody-radionuclide conjugate, which comprises an anti-CLDN18.2 antibody or an antigen-binding fragment thereof conjugated to a radionuclide.

[0254] A radionuclide is an agent characterized by an unstable atomic nucleus that is capable of undergoing radioactive decay. Radionuclides that can be used in the present disclosure include gamma emitters, positron emitters, Auger electron emitters, X-ray emitters, and fluorescent emitters. In certain embodiments, the radionuclide is diagnostic.

[0255] Diagnostic radionuclides can be used as imaging agents. Imaging agents can indicate the location of radionuclides and their adherents in the cells or tissues of an animal or human subject or in cells or tissues under in vitro conditions. In certain embodiments, the radionuclide is a radionuclide that can be detected externally in a non-invasive manner after being administered in vivo. Radionuclides (such as imaging agents) for diagnostic use preferably have relatively low cytotoxicity but decay with emissions suitable for imaging. Radionuclides having diagnostic use can include, for example 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 lr, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi,223 Ra and 225 Ac. Paramagnetic ions that can be used as diagnostic agents according to embodiments of the present disclosure include, but are not limited to, ions of transition metals and lanthanide metals (e.g., metals with atomic numbers 6 to 9, 21 - 29, 42, 43, 44, or 57 - 71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

[0256] In certain embodiments, the radionuclide is a radioactive metal ion, a γ - emitting radioactive halogen, and a positron - emitting radioactive non - metal. In certain embodiments, the radionuclide can be detected by positron emission tomography (PET) or single - photon emission computed tomography (SPECT). For example, radionuclides that decay with γ - emission are suitable for planar and single - photon computed emission tomography (SPECT) imaging, while radionuclides that decay with positron (β) emission and annihilation photons are suitable for positron emission tomography (PET). Diagnostic radionuclide labels that can be detected by imaging techniques such as PET or SPECT include, for example, but are not limited to 64 Cu, 67 Cu, 89 Zr, 124 I, 86 Y, 90 Y, 111 In, 123 / 124 / 131 I, 177 Lu, 11 C, 14 C, 41 Ca, 67 Ga, 68 Ga, 13 N, 15 O, 44 Sc, 18 F, 99m Tc, 90m Tc, etc.

[0257] In some embodiments, the radionuclide is non - metallic. In some embodiments, the radionuclide includes radioactive halogens. In some embodiments, the radionuclide is 124 I or 123 I or 131 I. In some embodiments, the anti - CLDN18.2 antibody - radionuclide conjugates provided herein include the anti - CLDN18.2 antibody or antigen - binding fragment thereof provided herein conjugated to 124 I or 123 I or 131 I.

[0258] Methods for preparing radioiodinated antibodies are known in the art, see, for example, Grassi, J. et al. (1987). Radioiodination and Other Labeling Techniques. Handbook of Experimental Pharmacology, 91 - 141, Eclelman, W.C. et al., Cancer Research, 40, 3036 - 3042, 1998, which are hereby incorporated by reference in their entirety.

[0259] In some embodiments, 124 I or 123 I or 131 I is labeled to at least one phenylhydroxy group of the antibody or its antigen - binding fragment.

[0260] In certain embodiments, in the presence of a radioactive iodine such as Na 124 I, the antibody or its antigen - binding fragment is directly iodinated by electrophilic substitution of tyrosine residues. In certain embodiments, the antibody or its antigen - binding fragment is indirectly iodinated by covalent attachment of a pre - labeled compound or a compound capable of post - labeling (e.g., Bolton and Hunter reagent for peptides).

[0261] In some embodiments, in the presence of an oxidizing agent, 124 I or 123 I or 131 I is labeled to the antibody or its antigen - binding fragment. Any suitable oxidizing agent can be used, including chemical oxidizing agents and enzymatic oxidizing agents. Exemplary oxidizing agents include, but are not limited to, N - bromosuccinimide, chloramine T, chlorine gas, or lactoperoxidase.

[0262] In some embodiments, 124 I or 123 I or 131 I is labeled to the antibody or its antigen - binding fragment by reaction with N - bromosuccinimide (NBS).

[0263] Methods for preparing radionuclide-conjugated antibodies and antigen-binding fragments are known in the art. Generally, methods for metal radionuclides and non-metal radionuclides are different. Conjugation of metal radionuclides usually requires a chelating agent for conjugation, which may not be necessary for non-metal radionuclides. Methods for conjugating metal radionuclides to antibodies are known in the art, for example, through a suitable chelating agent (see, for example, WO94 / 11026; Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., editors, published by Wiley-Interscience, New York, N.Y. (1991)).

[0264] As used herein, the term "chelating agent" refers to a chemical structure capable of binding a metal with two or more bonds. A chelating agent can have one or more chelating groups that can bind to a metal ion. For example, a chelating agent can include at least one heteroatom suitable for coordinating with a metal ion and isolating the metal ion from an aqueous solution.

[0265] In some embodiments, the chelating agent includes three or more atoms for chelation, each atom selected from the group consisting of nitrogen, sulfur, oxygen, and phosphorus.

[0266] Examples of chelating agents that can be used according to the present disclosure include, but are not limited to, DFO (deferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), TRITA (1,4,7,10-tetraazacyclotridecane-N,N',N",N"'-tetraacetic acid); TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid); and HETA (1,5,9,13-tetraazacyclohexadecane-N,N',N",N"'-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid), ΝΕΤΑ ({4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazacyclononan-1-yl}-acetic acid), TACN-TM (N,N',N", tris(2-mercaptoethyl)1,4,7-triazacyclononane), TRAP (1,4,7-triazacyclononane-1,4,7-tri[methyl(2-carboxyethyl)phosphinic acid]), CP256,PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadec-1(15),11,13-triene-3,6,9,-triacetic acid), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and derivatives thereof.

[0267] In some embodiments, the anti-CLDN18.2 radionuclide conjugates provided herein include a metal radionuclide conjugated to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof provided herein through a chelator. Those skilled in the art can select a suitable chelator for the radionuclide based on the knowledge known in the art, such as described by Price E.W. et al., Chem. Soc. Rev., 2014, 43, 260-290.

[0268] In some embodiments, the metal radionuclide can be 64 Cu, 67 Cu, 89 Zr, 86 Y, 90 Y, 111 In, 177 Lu, 67 Ga, 44 Sc or 99m Tc. In some embodiments, the radionuclide is 177 Lu, 64 Cu, 67 Cu, or 89 Zr. In some embodiments, 177 Lu, 64 Cu, 67 Cu or 89 Zr is labeled to the antibody or its antigen-binding fragment through a chelator.

[0269] In some embodiments, the chelator further includes a reactive functional group for conjugating with an antibody. The chelate can be directly linked to an antibody or a peptide, for example, as disclosed in U.S. Patent No. 4,824,659, which is incorporated herein by reference in its entirety. Such reactive groups enable the chelator to react with functional groups in one or more amino acid residues of the antibody, such as free cysteine, lysine, etc. Such amino acid residues (i.e., conjugation sites) to be reacted with the chelator (directly or indirectly) can be, for example, natural or engineered and can be present, for example, on the heavy or light chain of the antibody. Cysteine conjugation sites can be obtained by insertion, mutation, or reduction of antibody disulfide bonds. Methods for preparing cysteine-engineered antibodies have been disclosed, for example, in WO2011 / 056983. Site-specific conjugation methods can also be used to direct the conjugation reaction to specific sites on the antibody, achieve the desired stoichiometry, and / or achieve the desired ratio of chelator to antibody. Such conjugation methods are known in the art, such as cysteine engineering and enzymatic and chemoenzymatic methods, including but not limited to Q295 conjugation, glutamine conjugation, and transglutaminase-mediated conjugation, and the methods described in J. Clin. Immunol., 36:100 (2016), which is incorporated herein by reference in its entirety. Suitable reactive functional groups are generally capable of effectively and readily coupling to the anti-CLDN18.2 antibody or its antigen-binding fragment. Reactive functional groups that are reactive with lysine and cysteine sites include electrophilic groups known in the art. In certain embodiments, when the desired conjugation site is lysine, the reactive moiety is an isothiocyanate, such as para-isothiocyanatobenzyl or a reactive ester. In certain embodiments, when the desired conjugation site is cysteine, the reactive moiety is a maleimide.

[0270] Examples of reactive groups include maleimide, aminobenzyl, N-hydroxysuccinimide ester, etc.

[0271] In certain embodiments, the chelator is conjugated to an antibody or antigen-binding fragment thereof provided herein through a bifunctional linker reagent. Examples of such bifunctional linkers include, but are not limited to, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), methyltetrazine-amine, p-isothiocyanobenzene, bis-diazo derivatives (such as bis-(p-diazobenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene), BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPRH, SBAP, SIA, SIAB, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSG (succinimidyl-(4-vinylsulfone)benzoate). Those linker reagents are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A., see pages 467-498 of the 2003-2004 Applications Handbook and Catalog).

[0272] In some embodiments, the radionuclide is 64 Cu or 67 Cu, and the chelator includes TETA, NOTA, NODA, or NODGA. In some embodiments, the radionuclide is 89 Zr, and the chelator includes DFO. When the chelator is DFO, suitable bifunctional linker reagents include, but are not limited to, isothiocyanobenzyl, n-hydroxysuccinimidyl ester, 2,3,5,6-tetrafluorophenol ester, n-succinimidyl-S-acetylthioacetate, and those described in BioMed Research International, Volume 2014, Article ID 203601, which is incorporated herein by reference in its entirety. In certain embodiments, the chelator that includes a reactive functional group reactive with the conjugation site is p-isothiocyanobenzyl-deferoxamine (p-NCS-Bz-DFO):

[0273]

[0274] In some embodiments, the radionuclide is 177 Lu, and the chelator comprises DOTA. When the chelator is DOTA, a suitable bifunctional linker reagent may be methyltetrazine-amine or absent. In certain embodiments, the chelator conjugated with the bifunctional linker reagent is methyltetrazine-DOTA:

[0275]

[0276] In some embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein may be further conjugated with a nanoparticle. For example, the nanoparticle may be used as a drug carrier in a therapeutic application, and when the drug carrier is conjugated with a CLDN18.2-specific antibody or fragment of the present invention, a chemotherapeutic agent, a radiotherapeutic agent, a toxin, or any other cytotoxic agent or anti-cancer agent known in the art is delivered to cancer cells overexpressing CLDN18.2 on the cell surface.

[0277] In some embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein may be further conjugated with a drug (e.g., at the ε-amino group of a lysine residue), and the carrier may incorporate additional therapeutic or diagnostic agents.

[0278] i. Antibody Sequence

[0279] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein comprises a CLDN18.2 antibody or an antigen-binding fragment thereof comprising heavy chain HCDR1, HCDR2, and HCDR3 and / or light chain LCDR1, LCDR2, and LCDR3 sequences, wherein

[0280] the HCDR1 sequence comprises GYNMN (SEQ ID NO:1) or a homologous sequence having at least 80% sequence identity therewith;

[0281] the HCDR2 sequence comprises X 1 IDPYYX 2 X 3 TX 4 YNQKFX 5 G (SEQ ID NO:32) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity therewith;

[0282] the HCDR3 sequence comprises X 6 X 7 X 8GNAFDY (SEQ ID NO:33) or a homologous sequence having at least 80% sequence identity thereto;

[0283] The LCDR1 sequence includes KSSQX 9 LX 10 NX 11 GNX 12 KNYLT (SEQ ID NO:34) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto;

[0284] The LCDR2 sequence includes WASTRX 13 S (SEQ ID NO:35) or a homologous sequence having at least 80% sequence identity thereto; and

[0285] The LCDR3 sequence includes QNDYSX 15 PX 16 T (SEQ ID NO:36) or a homologous sequence having at least 80% sequence identity thereto;

[0286] wherein X 1 is N or Y or H, X 2 is G or V, X 3 is A or G or T, X 4 is R or T or S, X 5 is K or R, X 6 is S or M, X 7 is Y or F, X 8 is Y or H, X 9 is S or N, X 10 is L or F, X 11 is S or N, X 12 is Q or L, X 13 is E or K, X 15 is F or Y and X 16 is F or L.

[0287] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein includes an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region includes:

[0288] a) HCDR1 includes a sequence selected from SEQ ID NO:1;

[0289] b) HCDR2 includes a sequence selected from the following: SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:19, and SEQ ID NO:22; and

[0290] c) The HCDR3 comprises a sequence selected from SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:21, and / or

[0291] a light chain variable region, the light chain variable region comprising:

[0292] d) The LCDR1 comprises the sequences of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:20;

[0293] e) The LCDR2 comprises the sequences of SEQ ID NO:4 and SEQ ID NO:16; and

[0294] f) The LCDR3 comprises a sequence selected from SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:18.

[0295] In certain embodiments, the heavy chain variable region is selected from the group consisting of:

[0296] a) a heavy chain variable region comprising: HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:3, and HCDR3 comprising the sequence of SEQ ID NO:5;

[0297] b) a heavy chain variable region comprising: HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:7, and HCDR3 comprising the sequence of SEQ ID NO:5;

[0298] c) a heavy chain variable region comprising: HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:9, and HCDR3 comprising the sequence of SEQ ID NO:11;

[0299] d) a heavy chain variable region comprising: HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:19, and HCDR3 comprising the sequence of SEQ ID NO:21; and

[0300] e) a heavy chain variable region comprising: HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:22, and HCDR3 comprising the sequence of SEQ ID NO:5.

[0301] In certain embodiments, the light chain variable region is selected from the group consisting of:

[0302] a) a light chain variable region comprising: LCDR1 comprising the sequence of SEQ ID NO:2, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:6;

[0303] b) a light chain variable region comprising: LCDR1 comprising the sequence of SEQ ID NO:2, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:8;

[0304] c) a light chain variable region comprising: LCDR1 comprising the sequence of SEQ ID NO:10, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:6;

[0305] d) a light chain variable region comprising: LCDR1 comprising the sequence of SEQ ID NO:14, LCDR2 comprising the sequence of SEQ ID NO:16, and LCDR3 comprising the sequence of SEQ ID NO:18; and

[0306] e) a light chain variable region comprising: LCDR1 comprising the sequence of SEQ ID NO:20, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:6.

[0307] In certain embodiments, in the anti-CLDN18.2 radionuclide conjugates provided herein:

[0308] a) the heavy chain variable region comprises HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:3, and HCDR3 comprising the sequence of SEQ ID NO:5; and the light chain variable region comprises LCDR1 comprising the sequence of SEQ ID NO:2, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:6 (referred to as antibody "7C12");

[0309] b) the heavy chain variable region comprises HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:7, and HCDR3 comprising the sequence of SEQ ID NO:5; and the light chain variable region comprises LCDR1 comprising the sequence of SEQ ID NO:2, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:8 (referred to as antibody "11F12");

[0310] c) The heavy chain variable region comprises HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:9, and HCDR3 comprising the sequence of SEQ ID NO:11; and the light chain variable region comprises LCDR1 comprising the sequence of SEQ ID NO:10, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:6 (referred to as antibody "22G6");

[0311] d) The heavy chain variable region comprises HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:19, and HCDR3 comprising the sequence of SEQ ID NO:21; and the light chain variable region comprises LCDR1 comprising the sequence of SEQ ID NO:14, LCDR2 comprising the sequence of SEQ ID NO:16, and LCDR3 comprising the sequence of SEQ ID NO:18 (referred to as antibody "18B10"); or

[0312] e) The heavy chain variable region comprises HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:22, and HCDR3 comprising the sequence of SEQ ID NO:5; and the light chain variable region comprises LCDR1 comprising the sequence of SEQ ID NO:20, LCDR2 comprising the sequence of SEQ ID NO:4, and LCDR3 comprising the sequence of SEQ ID NO:6 (referred to as antibody "12E9").

[0313] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein comprises a CLDN18.2 antibody or an antigen-binding fragment thereof comprising heavy chain HCDR1, HCDR2, and HCDR3 and / or light chain LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 are identical to the HCDR1, HCDR2, and HCDR3 of a heavy chain variable region comprising a sequence selected from SEQ ID NO:37, 39, 41, 45, or 47, and the LCDR1, LCDR2, and LCDR3 are identical to the LCDR1, LCDR2, and LCDR3 of a light chain variable region comprising a sequence selected from SEQ ID NO:38, 40, 42, 46, or 48.

[0314] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of the CLDN18.2 antibodies 7C12, 11F12, 26G6, 18B10, and 12E9.

[0315] Table 1. Sequences of the CDR regions of CLDN18.2 antibodies

[0316]

[0317] Table 2. Sequences of VH / VL of murine / chimeric antibodies

[0318]

[0319]

[0320] In certain embodiments, the CLDN18.2 antibody conjugates provided herein include suitable framework region (FR) sequences, provided that the antibody and its antigen-binding fragments can specifically bind to CLDN18.2. The CDR sequences provided in Table 1 were obtained from murine antibodies, but the sequences can be transplanted into any suitable FR sequences of any suitable species such as murine, human, rat, rabbit, etc. using suitable methods known in the art such as recombinant techniques.

[0321] In certain embodiments, the anti-CLDN18.2 radionuclide conjugates provided herein are humanized. Humanized antibodies or antigen-binding fragments are desirable in that they reduce the immunogenicity in humans. Humanized antibodies are chimeric in their variable regions in that non-human CDR sequences are transplanted into human or substantially human FR sequences. Humanization of an antibody or antigen-binding fragment can generally be carried out by replacing the corresponding human CDR genes in human immunoglobulin genes with non-human (such as murine) CDR genes (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0322] Suitable human heavy and light chain variable domains can be selected using methods known in the art for this purpose. In illustrative examples, a "best fit" method can be used, where a non-human (e.g., rodent) antibody variable domain sequence is screened against a database of known human variable domain germline sequences or BLASTed, and the human sequence closest to the non-human query sequence is identified and used as the human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al., (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). Alternatively, frameworks derived from consensus sequences of all human antibodies can be used to graft non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0323] In certain embodiments, in addition to the non-human CDR sequences, the humanized antibody conjugates provided herein are composed of substantially all human sequences. In some embodiments, the variable region FRs and the constant region (if present) are entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be derived from different human immunoglobulin genes, e.g., the FR sequences are from one human antibody and the constant region is from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment includes human heavy and / or light chain FR1-4.

[0324] In some embodiments, the FR regions derived from humans can include the same amino acid sequences as the human immunoglobulins from which they are derived. In some embodiments, one or more amino acid residues of the human FRs are replaced with the corresponding residues from the parental non-human antibody. In certain embodiments, this may be desirable to make the humanized antibody or its fragment very close to the non-human parental antibody structure, thereby reducing or avoiding immunogenicity and / or improving or retaining binding activity or binding affinity.

[0325] In certain embodiments, the humanized antibody conjugates provided herein include no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each human FR sequence of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all of the FRs of the heavy or light chain variable domains. In some embodiments, such changes in amino acid residues can be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. In certain embodiments, the one or more amino acid residue mutations, for example, revert to the corresponding residues found in the non-human parental antibody (e.g., murine framework region) from which the CDR sequences are derived. One of ordinary skill in the art can select suitable mutation positions according to principles known in the art. For example, mutation positions can be selected where: 1) the residues in the germline sequence framework are rare (e.g., less than 20% or less than 10% in the human variable region sequence); 2) the position is adjacent to one or more of the three CDRs in the primary sequence of the germline chain, since the position may interact with residues in the CDRs; or 3) the position is close to the CDRs in the three-dimensional model and thus has a high probability of interacting with the amino acids in the CDRs. The residues at the selected positions can be mutated back to the corresponding residues in the parental antibody, or mutated back to corresponding residues that are neither in the germline sequence nor in the parental antibody, but rather to typical residues of the human sequence, i.e., residues that occur more frequently at the position in known human sequences belonging to the same subgroup as the germline sequence (see U.S. Patent No. 5,693,762).

[0326] In certain embodiments, the humanized light and heavy chains of the present disclosure are substantially non-immunogenic in humans and retain an affinity that is substantially the same as or even higher than the affinity of the parental antibody for CLDN18.2.

[0327] In certain embodiments, with or without back mutations, the humanized antibody conjugates provided herein include one or more light chain FR sequences of the germline framework sequence VK / 4-1 and / or one or more heavy chain FR sequences of the germline framework sequence VH / 1-46. If desired, back mutations can be introduced into the germline framework sequences. In certain embodiments, in the heavy chain framework sequence VH / 1-46, the humanized antibody 18B10 can contain one or more back mutations selected from the group consisting of: R71I, T73K, T28S, M69L, R38K, and M48I, all based on Kabat numbering. In the light chain framework sequence VK / 4-1, the humanized antibody 18B10 can contain one or more back mutations selected from the group consisting of: S63T and I21M, all based on Kabat numbering.

[0328] In certain embodiments, in the anti-CLDN18.2 antibody conjugates provided herein, the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:45, and SEQ ID NO:47, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity but retaining specific binding affinity for CLDN18.2 (specifically human CLDN18.2).

[0329] In certain embodiments, in the anti-CLDN18.2 antibody conjugates provided herein, the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a light chain variable region, and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity but retaining specific binding affinity for CLDN18.2 (specifically human CLDN18.2).

[0330] In certain embodiments, the anti-CLDN18.2 radionuclide conjugates provided herein comprise:

[0331] A heavy chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO:25, and the light chain variable region comprising the sequence of SEQ ID NO:26;

[0332] A heavy chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO:27, and the light chain variable region comprising the sequence of SEQ ID NO:28;

[0333] A heavy chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO:29, and the light chain variable region comprising the sequence of SEQ ID NO:26 or 28;

[0334] A heavy chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO:37, and the light chain variable region comprising the sequence of SEQ ID NO:38;

[0335] A heavy chain variable region, said heavy chain variable region comprising the sequence of SEQ ID NO:39, and said light chain variable region comprising the sequence of SEQ ID NO:40;

[0336] A heavy chain variable region, said heavy chain variable region comprising the sequence of SEQ ID NO:41, and said light chain variable region comprising the sequence of SEQ ID NO:42;

[0337] A heavy chain variable region, said heavy chain variable region comprising the sequence of SEQ ID NO:45, and said light chain variable region comprising the sequence of SEQ ID NO:46; or

[0338] A heavy chain variable region, said heavy chain variable region comprising the sequence of SEQ ID NO:47, and said light chain variable region comprising the sequence of SEQ ID NO:48.

[0339] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein further comprises one or more of heavy chain HFR1, HFR2, HFR3, and HFR4 and / or one or more of light chain LFR1, LFR2, LFR3, and LFR4, wherein:

[0340] Said HFR1 comprises QVQLVQSGAEVKKPGASVKVSCKASGYX 17 FT (SEQ ID NO:54) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto;

[0341] Said HFR2 comprises WVX 18 QAPGQGLEWX 19 G (SEQ ID NO:55) or a homologous sequence having at least 80% (or at least 90%) sequence identity thereto;

[0342] Said HFR3 sequence comprises RVTX 20 TIDKSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO:56) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto;

[0343] Said HFR4 comprises WGQGTTVTVSS (SEQ ID NO:57) or a homologous sequence having at least 80% sequence identity thereto;

[0344] Said LFR1 comprises DIVMTQSPDSLAVSLGERATX 21NC (SEQ ID NO:58) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto;

[0345] Said LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO:59) or a homologous sequence having at least 80% (or at least 85%, 90%) sequence identity thereto;

[0346] Said LFR3 comprises GVPDRFX 22 GSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:60) or a homologous sequence having at least 80% (or at least 85%, 90%, 95%) sequence identity thereto; and

[0347] Said LFR4 comprises FGGGTKVEIK (SEQ ID NO:61) or a homologous sequence having at least 80% (or at least 90%) sequence identity thereto;

[0348] Wherein X 17 is T or S, X 18 is R or K, X 19 is M or I, X 20 is M or L, X 21 is I or M, and X 22 is S or T.

[0349] In certain embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NO:62 and 63, HFR2 comprises a sequence selected from the group consisting of SEQ ID NO:64 and 65, HFR3 comprises a sequence selected from the group consisting of SEQ ID NO:66 and 67, HFR4 comprises the sequence of SEQ ID NO:57, LFR1 comprises the sequences consisting of the group of SEQ ID NO:68 and 69, LFR2 comprises the sequence of SEQ ID NO:59, LFR3 comprises a sequence selected from the group consisting of SEQ ID NO:70 and 71, and LFR4 comprises the sequence of SEQ ID NO:61.

[0350] Table 3-1. Framework (FR) sequences of humanized CLDN18.2 antibody 18B10

[0351]

[0352] Table 3-2 shows the sequences of the variable regions of humanized 18B10 antibody.

[0353] Table 3-2. Sequences of humanized 18B10

[0354]

[0355]

[0356] In certain embodiments, the anti-CLDN18.2 radionuclide conjugates provided herein comprise 124 I- or 123 I- or 131 I or 89 Zr or 177 Lu-labeled 18B10, such as 124 I or 89 Zr or 177 Lu-labeled humanized 18B10 (e.g., as listed in Table 3-2). In certain embodiments, the 124 I or 89 Zr or 177 Lu-labeled humanized 18B10 radionuclide conjugate comprises: a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:19, and HCDR3 comprising the sequence of SEQ ID NO:21; and the light chain variable region comprises LCDR1 comprising the sequence of SEQ ID NO:14, LCDR2 comprising the sequence of SEQ ID NO:16, and LCDR3 comprising the sequence of SEQ ID NO:18.

[0357] In certain embodiments, the 124 I or 89 Zr or 177 Lu-labeled humanized 18B10 radionuclide conjugate comprises:

[0358] a heavy chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO:25, and the light chain variable region comprising the sequence of SEQ ID NO:26 (“Hu18B10HaLa”);

[0359] a heavy chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO:27, and the light chain variable region comprising the sequence of SEQ ID NO:28 (“Hu18B10HbLb”); or

[0360] a heavy chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO:29, and the light chain variable region comprising the sequence of SEQ ID NO:26 or 28.

[0361] In certain embodiments, the humanized anti-CLDN18.2 radionuclide conjugate provided herein can include a heavy chain variable region fused to a constant region of human IgG1 isotype and a light chain variable region fused to a constant region of human kappa chain.

[0362] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein further includes an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG. In some embodiments, the immunoglobulin constant region includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region includes a CH1 region, a hinge region, and / or a CH2-CH3 region. In certain embodiments, the heavy chain constant region includes an Fc region. In certain embodiments, the light chain constant region includes Cκ or Cλ.

[0363] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein includes a full-length antibody (e.g., humanized 18B10) conjugated to a radionuclide provided herein. In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein includes a scFv-Fc antibody derived from the humanized 18B10 provided herein (e.g., Hu18B10HaLa) conjugated to a radionuclide provided herein.

[0364] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate is conjugated to 89 Zr (e.g., via a chelator including DFO), and such CLDN18.2 radionuclide conjugate does not bind to FcγR. For example, the CLDN18.2 89 Zr conjugate lacks an Fc domain or includes an engineered Fc domain that lacks binding to FcγR. In certain embodiments, the anti-CLDN18.2 radionuclide conjugate is conjugated to 177 Lu (e.g., via a chelator including DOTA).

[0365] In certain embodiments, the anti-CLDN18.2 radionuclide conjugate provided herein further includes a constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the anti-CLDN18.2 antibody and its antigen-binding fragment provided herein include a constant region of IgG1 isotype. In certain embodiments, the constant region of human IgG1 includes SEQ ID NO:49 or a homologous sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0366] Anti-CLDN18.2 antibodies and antigen-binding fragments thereof have been disclosed in the published PCT application WO2021 / 032157, which is incorporated herein by reference in its entirety. These anti-CLDN18.2 antibodies and antigen-binding fragments thereof have been shown to have strong binding affinity and specificity for human CLDN18.2 and have been shown to bind to a unique epitope on human CLDN18.2. All binding characteristics and experimental data of these anti-CLDN18.2 antibodies as disclosed in WO2021 / 032157 are incorporated herein by reference.

[0367] In certain embodiments, the EC50 value of the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein for binding to human CLDN18.2 (or mouse CLDN18.2) expressing cells is no more than 70 μg / ml (or no more than 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12 or 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 μg / ml), as measured by flow cytometry. In certain embodiments, the EC50 value of the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein for binding to human CLDN18.2 (or mouse CLDN18.2) expressing cells is no more than 500 nM (or no more than 250, 200, 100, 50, 45, 40, 35, 30, 25, 20, 15, 12 or 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nM), as measured by flow cytometry. In certain embodiments, the EC50 value of the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein for binding to human CLDN18.2 (or mouse CLDN18.2) expressing cells is no more than 1.0 nM (e.g., no more than 0.8 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM or no more than 0.3 nM), as measured by ELISA. In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein specifically bind to human CLDN18.2 expressing cells with an EC50 value that is no more than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 1% or 0.1% of the EC50 value of IMAB362, as measured by flow cytometry. In certain embodiments, the EC50 is determined using the NUGC4 cell line, KATOIII cell line, SNU-601 cell line, SNU-620 cell line or equivalent cells having a human CLDN18.2 protein expression level equivalent to or no more than that of the NUGC4 cell line, KATOIII cell line, SNU-601 cell line or SNU-620 cell line, e.g., a human CLDN18.2 low-expression cell line or a human CLDN18.2 medium-expression cell line. In certain embodiments, the EC50 is determined using a human CLDN18.2 high-expression cell line.

[0368] In certain embodiments, the EC50 values of the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein for binding to a human CLDN18.2 high-expression cell line or a human CLDN18.2 medium-expression cell line do not exceed 5, 4, 3, or 2 μg / ml.

[0369] In certain embodiments, the EC50 values of the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein for binding to NUGC4 cells do not exceed 70 μg / ml (or do not exceed 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / ml), as measured by flow cytometry.

[0370] In certain embodiments, the anti-CLDN18.2 radionuclide conjugates provided herein do not bind to CLDN18.1 (e.g., human CLDN18.1 or mouse CLDN18.1).

[0371] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 antibody conjugates provided herein are capable of specifically binding to mouse CLDN18.2 (e.g., cells expressing mouse CLDN18.2) with an EC50 value of no more than 1.5 μg / ml, as measured by flow cytometry. In certain embodiments, the anti-CLDN18.2 radionuclide conjugates provided herein bind to mouse CLDN18.2 with an EC50 of 0.1 μg / ml - 1.5 μg / ml (e.g., 0.1 μg / ml - 1.2 μg / ml, 0.2 μg / ml - 1 μg / ml, 0.5 μg / ml - 1 μg / ml, 0.6 μg / ml - 1 μg / ml, 0.6 μg / ml - 0.8 μg / ml, or 0.67 μg / ml), as measured by flow cytometry.

[0372] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein bind to an epitope comprising at least one or more (e.g., one, two, three, or more) amino acid residues at positions D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79, and R80 of human CLDN18.2, wherein the human CLDN18.2 has the amino acid sequence of SEQ ID NO:30.

[0373] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can include specific amino acids that directly contact the antibody, sugar side chains, phosphoryl groups, or sulfonyl groups. One of ordinary skill in the art will recognize that, without undue experimentation, it can be determined whether an antibody binds to the same or overlapping or adjacent epitopes as an antibody of the present disclosure (e.g., a hybridoma / chimeric or humanized antibody 7C12, 11F12, 26G6, 18B10, and any of its chimeric and humanized variants provided herein) by determining whether the two compete for binding to a CLDN18.2 antigen polypeptide.

[0374] As used herein, the term "compete for binding with..." with respect to two antigen-binding proteins (e.g., antibodies) means that one antigen-binding protein blocks or reduces the binding of another antigen-binding protein to an antigen (e.g., human / mouse CLDN18.2), as determined by a competitive binding assay. Competitive binding assays are well known in the art and include, for example, direct or indirect radioimmunoassay (RIA), direct or indirect enzyme immunoassay (EIA), and sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253). Generally, such assays involve the use of a purified antigen bound to a solid surface or cells bearing the antigen, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Generally, the test antibody is present in excess. If two antibodies compete for binding to CLDN18.2, then the two antibodies bind to the same or overlapping epitopes, or to adjacent epitopes that are sufficiently close to the epitope bound by the other antibody so as to produce steric hindrance. Generally, when the competing antibody is present in excess, the competing antibody inhibits (e.g., reduces) the specific binding of the test antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90% or more.

[0375] In certain embodiments, the epitope or amino acid residues in the epitope to which the antibody binds can be determined by mutating specific residues (i.e., CLDN18.2) in the antigen. If the antibody binds to a mutant CLDN18.2 having an amino acid residue mutated to, for example, alanine at a level significantly reduced relative to its binding to wild-type CLDN18.2, this indicates that the mutated residue is directly involved in the binding of the antibody to the CLDN18.2 antigen or is very close to the antibody when it binds to the antigen. Such mutated residues are considered to be within the epitope, and the antibody is considered to specifically bind to the epitope comprising the residue. As used herein, a significantly reduced binding level means that the binding affinity (e.g., EC50, Kd, or binding capacity) between the antibody and the mutant CLDN18.2 is reduced by greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to the binding between the antibody and wild-type CLDN18.2. Such binding measurements can be performed using any suitable method known in the art and disclosed herein, such as, but not limited to, KinExA assays and flow cytometry.

[0376] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein exhibit significantly lower binding to mutant CLDN18.2, wherein the residue in wild-type CLDN18.2 is replaced with alanine, and the residue is selected from the group consisting of D28, W30, V43, N45, Y46, L49, W50, R51, R55, E56, F60, E62, Y66, L72, L76, V79, and R80 of human CLDN18.2 (SEQ ID NO:30). In certain embodiments, the residue is E56. In certain embodiments, the residue is selected from the group consisting of W30, L49, W50, R55, and E56. In certain embodiments, the residue is selected from the group consisting of T41, N45, Y46, R51, F60, E62, and R80. In certain embodiments, the residue is selected from the group consisting of D28, V43, N45, Y46, Y66, L72, L76, and V79.

[0377] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein exhibit at least an 80%, 90%, 95%, or 99% or more reduction in binding to mutant CLDN18.2 comprising E56A of human CLDN18.2 relative to the binding between the antibody and wild-type CLDN18.2.

[0378] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein exhibit at least a 50%, 60%, 70%, 80%, or 90% reduction in binding to mutant CLDN18.2 relative to the binding between the antibody and wild-type CLDN18.2, wherein the mutant CLDN18.2 comprises one or more mutant residues selected from the group consisting of W30A, L49A, W50A, R55A, and E56A of human CLDN18.2.

[0379] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein exhibit at least a 30%, 35%, 40%, 45%, or 50% reduction in binding to mutant CLDN18.2 relative to the binding between the antibody and wild-type CLDN18.2, wherein the mutant CLDN18.2 comprises one or more mutant residues selected from the group consisting of D28, V43, N45, Y46, Y66, L72, L76, and V79 of human CLDN18.2.

[0380] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein exhibit at least a 10%, 15%, 20%, 25%, or 30% reduction in binding to mutant CLDN18.2 relative to the binding between the antibody and wild-type CLDN18.2, wherein the mutant CLDN18.2 comprises one or more mutant residues selected from the group consisting of T41A, N45A, Y46A, R51A, F60A, E62A, and R80A of human CLDN18.2.

[0381] In certain embodiments, the anti-CLDN18.2 antibodies, antigen-binding fragments thereof, and anti-CLDN18.2 radionuclide conjugates provided herein do not bind to A42 and / or N45.

[0382] ii. Antibody Variants

[0383] The anti-CLDN18.2 antibodies and antigen-binding fragments thereof in the anti-CLDN18.2 antibody conjugates provided herein also encompass various types of variants of the antibody sequences provided herein.

[0384] In certain embodiments, the variant comprises one, two, or three CDR sequences as provided in Table 1, one or more FR sequences, a heavy chain variable region sequence or a light chain variable region sequence provided herein, and / or one or more modifications or substitutions in a constant region (e.g., the Fc region). Such antibody variants retain the specific binding affinity of their parental antibody for CLDN18.2 but have desired properties conferred by the modification or substitution. For example, the antibody variant can have improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamidation, reduced or increased effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility with conjugation (e.g., one or more introduced cysteine residues), etc.

[0385] a). Affinity Variants

[0386] The affinity variant retains the specific binding affinity of the parental antibody for CLDN18.2, or even has a higher CLDN18.2 specific binding affinity than the parental antibody. Various methods known in the art can be used to achieve this purpose. For example, a library of antibody variants (such as Fab or scFv variants) can be generated, expressed using phage display technology, and then screened for binding affinity to human CLDN18.2. As another example, computer software can be used for virtual simulation of the binding of the antibody to human CLDN18.2 and to identify the amino acid residues on the antibody that form the binding interface. Such residues can be avoided from being substituted to prevent a decrease in binding affinity, or targeted for substitution to achieve stronger binding.

[0387] b). Glycosylation Variants

[0388] The anti-CLDN18.2 antibodies and antigen-binding fragments in the anti-CLDN18.2 antibody conjugates provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment. As used herein, the term "glycosylation" refers to the enzymatic process of attaching a glycan such as fucose, xylose, mannose, or GlcNAc phosphoserine glycan to a protein, lipid, or other organic molecule. Depending on the carbon to which the glycan is linked, glycosylation can be classified into five categories, including: N-linked glycosylation, O-linked glycosylation, phosphoglycosylation, C-linked glycosylation, and glycosylphosphatidylinositolization.

[0389] c). Cysteine Engineered Variants

[0390] The anti-CLDN18.2 antibodies and antigen-binding fragments in the anti-CLDN18.2 antibody conjugates provided herein also encompass cysteine-engineered variants, which include one or more introduced free cysteine amino acid residues.

[0391] d). Fc Variants

[0392] The anti-CLDN18.2 antibodies and antigen-binding fragments in the anti-CLDN18.2 antibody conjugates provided herein also encompass Fc variants, which include one or more amino acid residue modifications or substitutions at their Fc regions and / or hinge regions.

[0393] Humanized variants of the 18B10 heavy and light chains are linked to the human IgG1 heavy chain constant region and the κ light chain constant region as follows:

[0394] Human IgG1 heavy chain constant region (SEQ ID NO:49):

[0395] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0396] Human κ light chain constant region (SEQ ID NO:50):

[0397] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0398] In certain embodiments, the constant region of the antibody or its antigen-binding fragment provided herein includes one or more amino acid residue substitutions relative to SEQ ID NO:49 (i.e., the wild-type sequence), and the one or more amino acid residue substitutions are selected from the group consisting of: L235V, F243L, R292P, Y300L, P396L, or any combination thereof.

[0399] iii. Antigen - Binding Fragments

[0400] The anti-CLDN18.2 antibody conjugates provided herein may also include anti-CLDN18.2 antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-CLDN18.2 antibodies provided herein, including, for example, the exemplary antibodies whose CDR sequences are shown in Table 1 and their different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants, etc.).

[0401] In certain embodiments, the anti-CLDN18.2 antigen-binding fragments provided herein are bispecific antibodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bispecific antibodies (ds bispecific antibodies), single-chain antibody molecules (scFv), scFv-Fc antibodies, scFv dimers (bivalent bispecific antibodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0402] A variety of techniques can be used to generate such antigen-binding fragments. Illustrative methods include enzymatic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science 229:81 (1985)), recombinant expression in host cells such as Escherichia coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form an F(ab')2 fragment (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for generating antibody fragments will be apparent to the skilled practitioner.

[0403] In certain embodiments, the antigen-binding fragment is an scFv. The production of scFv is described, for example, in WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. The scFv can be fused to an effector protein at the amino or carboxyl terminus to provide a fusion protein (see, for example, Antibody Engineering, edited by Borrebaeck).

[0404] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof in the anti-CLDN18.2 antibody conjugates provided herein are bivalent, tetravalent, hexavalent, or multivalent. As used herein, the term "valence" refers to the presence of a specified number of antigen-binding sites in a given molecule. Thus, the terms "bivalent", "tetravalent", and "hexavalent" indicate the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule. Any molecule with a valence greater than bivalent is considered multivalent, encompassing, for example, trivalent, tetravalent, hexavalent, etc.

[0405] A bivalent molecule can be monospecific if both binding sites bind specifically to the same antigen or the same epitope. In certain embodiments, this provides stronger binding to the antigen or epitope than the monovalent counterpart. Similarly, a multivalent molecule can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valence of the binding site and the second valence of the binding site are structurally the same (i.e., having the same sequence) or structurally different (i.e., having different sequences but the same specificity).

[0406] A bivalent can also be bispecific if the two binding sites have specificities for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific when two binding sites are monospecific for a first antigen (or epitope) and a third binding site is specific for a second antigen (or epitope).

[0407] Methods of Using Anti - CLDN18.2 Radionuclide Conjugates

[0408] On the other hand, the present disclosure provides methods of using such anti-CLDN18.2 radionuclide conjugates provided herein (specifically, those conjugated to diagnostic radionuclides, and preferably 124 I-18B10) for detecting or visualizing CLDN18.2 protein, for diagnosing a subject with a CLDN18.2-related disease, for identifying a subject in need who may respond to a CLDN18.2-targeted therapy, for monitoring the progression of a CLDN18.2-related disease in a subject during a monitoring period, and for monitoring the therapeutic efficacy of a subject with a CLDN18.2-related disease who has been treated with a therapy for a certain treatment period, etc. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0409] The anti-CLDN18.2 antibody-radionuclide conjugates provided herein can be used in vivo to provide information regarding: (i) the presence or expression level of the CLDN18.2 protein; (ii) the location and / or distribution of CLDN18.2; and (iii) changes in the expression level or location / distribution of CLDN18.2. Compared to currently available diagnostic methods for CLDN18.2 such as immunohistochemistry (IHC), the antibody-radionuclide conjugates provided herein have advantages in non-invasive treatment, quantifiability, whole-body assessment, and repeated administration and assessment at multiple time points.

[0410] As used herein, "CLDN18.2-related disease" refers to any disease or condition caused by, exacerbated by, or otherwise associated with an increase or decrease in the expression or activity of CLDN18.2. In certain embodiments, the CLDN18.2-related disease is a CLDN18.2-positive tumor or a CLDN18.2-positive non-cancerous lesion (e.g., a gastric lesion).

[0411] In certain embodiments, the CLDN18.2-related condition is cancer. In certain embodiments, the CLDN18.2-related condition is a cancer that expresses CLDN18.2.

[0412] In certain embodiments, the cancer is selected from the following: gastric cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, esophageal adenocarcinoma, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, and adenocarcinoma.

[0413] Other examples of cancers include, but are not limited to, non-small cell lung cancer (squamous / non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal breast cancer, ductal carcinoma, and lobular breast cancer), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, myeloma, mycosis fungoides, Merkel cell cancer, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancies, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, chronic myeloid (granulocytic) leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumors, EBV-positive and -negative PTLD, and diffuse large B-cell lymphoma (DLBCL), plasmacytic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia, primary CNS lymphoma, spinal axis tumors, brainstem glioma, astrocytoma, medulloblastoma, craniopharyngioma, epididymal tumors, pineal tumors, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0414] In certain embodiments, the cancer is a cancer that expresses CLDN18.2. As used herein, "cancer that expresses CLDN18.2" refers to any cancer or tumor involving cancer cells that express CLDN18.2 and that do not substantially form classical tight junctions found in normal epithelial tissue. Such CLDN18.2-expressing cells are susceptible to binding by extracellular antibodies and can thus be detected as CLDN18.2 positive. Examples of cancers that express CLDN18.2 include gastric cancer, esophageal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer such as non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), ovarian cancer, colon cancer, colorectal cancer, gastrointestinal stromal tumor (GIST), gastrointestinal carcinoid tumor, rectal cancer, anal cancer, cholangiocarcinoma, small intestine cancer, appendiceal cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), liver cancer, head and neck cancer, and gallbladder cancer and metastases thereof, e.g., gastric cancer metastases such as Krukenberg tumor, peritoneal metastases, and lymph node metastases.

[0415] In certain embodiments, the cancer that expresses CLDN18.2 can be an adenocarcinoma, e.g., advanced adenocarcinoma. In certain embodiments, the cancer is selected from gastric adenocarcinoma, esophageal adenocarcinoma, pancreatic ductal adenocarcinoma, cholangiocarcinoma adenocarcinoma, lung adenocarcinoma, and ovarian adenocarcinoma. In certain embodiments, the cancer that expresses CLDN18.2 includes gastric cancer, esophageal cancer, particularly lower esophageal cancer, esophagogastric junction cancer, and gastroesophageal cancer.

[0416] In certain embodiments, the cancer is gastric cancer, ovarian cancer, pancreatic cancer, cholangiocarcinoma, colorectal cancer, lung cancer, or esophageal adenocarcinoma.

[0417] In certain embodiments, CLDN18.2-related diseases involve non-cancerous lesions such as gastric lesions. In certain embodiments, the CLDN18.2-related disease is gastric ulcer.

[0418] The anti-CLDN18.2 antibody-radionuclide conjugates provided herein can be used in conjunction with in vivo nuclear imaging modalities to visualize CLDN18.2 protein within the topography of an object's body. In addition to diagnosing conditions or diseases associated with CLDN18.2 expression (e.g., cancer), the anti-CLDN18.2 antibody-radionuclide conjugates provided herein can also be used to diagnose, predict responsiveness to CLDN18.2-targeted therapies, monitor disease progression, monitor treatment efficacy, etc. in an object according to the methods provided herein.

[0419] In certain embodiments, the object is a human. In certain embodiments, the object is a non-human animal. In certain embodiments, the object has or is suspected of having a CLDN18.2-related disease. In certain embodiments, the object has been diagnosed with a CLDN18.2-related disease.

[0420] In certain embodiments, the subject has or is suspected of having cancer that expresses CLDN18.2. In certain embodiments, the subject has or is suspected of having a risk of cancer metastasis.

[0421] In certain embodiments, the subject has not received any CLDN18.2-targeted therapy. Without wishing to be bound by any theory, it is believed that a subject who has not received any CLDN18.2-targeted therapy may have higher CLDN18.2 expression than a subject treated with CLDN18.2-targeted therapy. Thus, a subject who has not received any CLDN18.2-targeted therapy may have a higher detection signal, such as in radionuclide imaging.

[0422] In certain embodiments, the subject has received CLDN18.2-targeted therapy.

[0423] Administration of Anti - CLDN18.2 Radionuclide Conjugates

[0424] The methods provided herein include administering to the subject a detectably effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate provided herein. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate includes an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0425] As described herein, the term "detectably effective amount" with respect to an anti-CLDN18.2 antibody-radionuclide conjugate refers to an amount sufficient to be taken up by CLDN18.2-expressing tissues in a subject and to produce a detectable signal in the subject to generate an acceptable image using a suitable device for radionuclide imaging such as positron emission tomography (PET), single photon emission computed tomography (SPECT). The term "detectable" with respect to radionuclide imaging means that a radionuclide signal originating from the imaging device can be detected and distinguished from the background signal generated by the imaging device. In certain embodiments, the detectable signal is significantly different from the background signal, such as at least about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30% or more different. In certain embodiments, the ratio of a particular signal to the background signal is at least 1.5, 2, 2.5, 3, 4, 5 or greater.

[0426] A detectably effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate can be administered in a single injection or alternatively in more than one injection and may vary depending on factors such as the susceptibility of the individual, the sex, age and weight of the individual, the specific response of the subject, etc. The detectably effective amount may also vary depending on the instrument and imaging method used. Optimization of these factors is well known in the art.

[0427] The anti-CLDN18.2 antibody-radionuclide conjugates provided herein can be administered to a subject by any suitable route known in the art, such as parenterally (e.g., subcutaneously, intraperitoneally, intravenously, including intravenous infusion, intramuscularly, or intradermally). Other routes of administration may also be useful, such as orally, topically, subcutaneously, peritoneal, intraarterially, inhaled, vaginal, rectal, nasal, intrathecally, or inhaled. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugates provided herein are administered via an intravenous catheter inserted into the contralateral ulnar vein.

[0428] The anti-CLDN18.2 antibody-radionuclide conjugates provided herein can be administered in a suitable amount of antibody protein, such as from about 0.1 mg to about 30 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about 2 mg of antibody protein. In certain embodiments, such an amount of antibody protein is administered intravenously, e.g., in a single injection or infusion.

[0429] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugates provided herein (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) can be administered to humans at a range of about 0.1 mg to 10 mg of anti-CLDN18.2 antibody-radionuclide conjugate per square meter of body surface area for a typical adult, e.g., as a single bolus injection, but lower or higher doses may also be administered depending on the circumstances.

[0430] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugates provided herein (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) can be administered to humans in the ranges of 0.01 mg to 10 mg, 0.05 mg to 10 mg, 0.1 mg to 5 mg, 0.5 mg to 5 mg, and 0.5 mg to 2 mg, e.g., as a single bolus injection.

[0431] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugates provided herein (e.g., 124 I-18B10 or 89 Zr-18B10 or177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) can be administered to a human at a dose of from 0.015 mg / kg body weight to 1 mg / kg body weight per day, such as from 0.015 mg / kg body weight to 0.5 mg / kg, such as per day, from 0.015 mg / kg body weight to 0.1 mg / kg body weight, such as per day, from 0.015 mg / kg body weight to 0.075 mg / kg body weight, such as per day, or from 0.015 mg / kg body weight to 0.05 mg / kg body weight, such as per day, for example as a single bolus injection.

[0432] The dosage regimen can be adjusted to provide a desired detectable amount so as to obtain a clear image of the tissue or cells that have taken up the anti-CLDN18.2 antibody-radionuclide conjugate provided herein (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106). In certain embodiments, the amount of the anti-CLDN18.2 antibody-radionuclide conjugate provided herein (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) required for imaging in a human subject will be determined by the prescribing physician, where the dose will generally vary depending on the emission of the radionuclide, to obtain an amount that effectively achieves the desired uptake of the anti-CLDN18.2 antibody-radionuclide conjugate (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) in the cells or tissues of a particular subject without being toxic to the subject.

[0433] In particular, full attention must be paid to the exposure dose of the subject. The anti-CLDN18.2 antibody-radionuclide conjugate at a saturating dose (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177Lu-DOTA-SF106) can be administered to a patient. For example, the amount of radioactivity of an anti-CLDN18.2 antibody-radionuclide conjugate to be administered to a subject can be in the range of 18.5 megabecquerels (MBq) to 370 MBq, 18.5 MBq to 350 MBq, 18.5 MBq to 300 MBq, 18.5 MBq to 250 MBq, 18.5 MBq to 200 MBq, 18.5 MBq to 150 MBq, 18.5 MBq to 125 MBq, or 18.5 MBq to 100 MBq. Alternatively, the dose can be measured in millicuries (mCi). In some embodiments, the 124 I or 89 Zr or 177 the amount of Lu imaging agent administered for an imaging study of a subject is 0.1 to 10 mCi, 0.2 to 10 mCi, 0.3 to 10 mCi, 0.4 to 10 mCi, 0.5 to 10 mCi, 0.5 to 7.5 mCi, 0.5 to 5 mCi, 0.5 to 2.5 mCi, or 0.5 to 2 mCi. In some embodiments, the detectable effective amount will be the amount of the compound sufficient to produce emissions in the range of 0.5 to 10 mCi, 0.5 to 7.5 mCi, 0.5 to 5 mCi, 0.5 to 2.5 mCi, or 0.5 to 2 mCi. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate is administered to a human subject in an amount of 0.5 to 10 mCi, 0.5 to 7.5 mCi, 0.5 to 5 mCi, 0.5 to 2.5 mCi, or 0.5 to 2 mCi.

[0434] In some embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate is in a composition having a specific activity of 3.0 - 6.0 GBq / μmol, such as 3.0 - 5.0 GBq / μmol, 3.5 - 5.0 GBq / μmol, 3.0 - 4.5 GBq / μmol, or 4.0 - 4.5 GBq / μmol.

[0435] In certain embodiments, 124 I or 89 Zr or 177 the Lu-labeled anti-CLDN18.2 antibody-radionuclide conjugate (such as 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) is administered as a composition that respectively contains 95 - 99% of 124 I or 89 Zr or 177Lu-labeled anti-CLDN18.2 antibody-radionuclide conjugates (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) and 1 - 5% of non-radiolabeled anti-CLDN18.2 antibody-radionuclide conjugates. In certain embodiments, the ratio is 98% of 124 I or 89 Zr or 177 Lu-labeled anti-CLDN18.2 antibody-radionuclide conjugates and 2% of non-radiolabeled anti-CLDN18.2 antibody-radionuclide conjugates. 124 The purity of I-labeled anti-CLDN18.2 antibody-radionuclide can be measured by radio thin layer chromatography (TLC) or radio high performance liquid chromatography (HPLC).

[0436] Detecting or Visualizing CLDN18.2 Protein

[0437] The methods provided herein further include performing radionuclide imaging on a subject to obtain an image.

[0438] Suitable methods of in vivo radionuclide imaging that can be used according to the methods described herein include, but are not limited to, positron emission tomography (PET) and single photon emission computed tomography (SPECT).

[0439] As used herein, the term "positron emission tomography (PET)" refers to a nuclear imaging technique used in the medical field to visualize target tissues or organs or activities. PET measures two annihilation photons that are continuously produced after a positron-emitting radionuclide-conjugated tracer molecule emits a positron. The radionuclide-conjugated tracer molecule is selected to label a specific function in the body at the biochemical level. PET provides molecular imaging of biological functions rather than anatomy. PET can examine a patient by generating images of many functions of the human body that cannot be obtained by other imaging techniques. After injecting a short-lived positron-emitting radioactive tracer into a subject, it can be distributed in the body according to the physiological pathways related to the stable counterpart. When the tracer is a targeting molecule specifically directed to the target of interest, the tracer allows visualization of the tissue or organ expressing such a target.

[0440] As used herein, the term "SPECT" refers to "single photon emission computed tomography", which is a nuclear medicine tomographic imaging technique using gamma rays. The SPECT is similar to conventional nuclear medicine planar imaging using a gamma camera and can provide true 3D information. This information is typically presented in the form of cross-sectional slices of a patient but can be freely reformatted or manipulated as needed. Imaging requires injection of a gamma-emitting radionuclide or its conjugate into an object.

[0441] PET may be accompanied by other scanning or imaging techniques for anatomical reference purposes. In some embodiments, radionuclide imaging is combined with CT, MRI, ultrasound. In some embodiments, radionuclide imaging is combined with low-dose or diagnostic CT scans.

[0442] As used herein, the term "CT" refers to "computed tomography", which is a non-invasive medical examination or procedure using specialized X-ray equipment to generate cross-sectional images of the body. These cross-sectional images are used for various diagnostic and therapeutic purposes. CT images of internal organs, bones, soft tissues, and blood vessels are clearer and more detailed than conventional X-ray images such as chest X-rays. Thus, CT is a valuable medical tool that can help diagnose diseases, trauma, or abnormalities, plan and guide interventional or therapeutic procedures, and monitor the effectiveness of therapies (such as cancer treatment). A PET / CT combined imager can be used to perform PET and CT in combination.

[0443] As used herein, the term "MRI" refers to "magnetic resonance imaging", which is a medical imaging procedure used to image internal structures of the body. An MRI scanner uses a strong magnetic field and radio waves (radiofrequency energy) to generate images. The signals in MR images mainly come from protons in fat and water molecules in the body. MRI is a non-invasive method for doctors to examine organs, tissues, and the skeletal system. The MRI produces high-resolution images of the interior of the body, which helps diagnose various problems, including abnormalities of the brain and spinal cord, heart and blood vessels, bones and joints, other internal organs, etc.

[0444] Ultrasonic imaging uses high-frequency sound waves to view the interior of the body. Since ultrasonic images are captured in real time, the ultrasonic images can also show the movement of internal organs of the body and the blood flowing through blood vessels. In an ultrasonic examination, a sensor (probe) is placed directly on the skin or inside a body opening. A thin layer of gel is applied to the skin so that ultrasonic waves are transmitted from the transducer through the gel into the body. The intensity (amplitude) of the sound signal and the time required for the wave to pass through the body provide the information needed to generate an image. Ultrasonic imaging is an ideal medical tool that can help visualize abdominal tissues and organs, evaluate bone fragility, view the heart, etc.

[0445] The object is scanned by a radionuclide imaging system to obtain an image. Generally, the object can be positioned in a PET camera, and an image can be obtained by scanning the object for an amount of time suitable for the particular radionuclide being used.

[0446] The object is exposed to radionuclide imaging at subsequent time points after administration of the anti-CLDN18.2 antibody-radionuclide conjugate.

[0447] In certain embodiments, radionuclide imaging is performed at least 2 hours after administration of the anti-CLDN18.2 antibody-radionuclide conjugate (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) to the object. In certain embodiments, radionuclide imaging is performed at a time point between 2 hours and 144 hours, between 12 hours and 144 hours, or preferably between 24 hours and 144 hours, between 24 hours and 120 hours, or between 24 hours and 96 hours after administration of the anti-CLDN18.2 antibody-radionuclide conjugate (e.g., 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106) to the object. In any of the above embodiments, radionuclide imaging is performed one, two, three, or more times at different time points between 2 hours and 144 hours, between 12 hours and 144 hours, or preferably between 24 hours and 144 hours, between 24 hours and 120 hours, or between 24 hours and 96 hours after administration of the anti-CLDN18.2 antibody-radionuclide conjugate to the object. For example, radionuclide imaging is performed one or more times at the 2nd hour, 24th hour, 48th hour, and / or 96th hour after administration of the anti-CLDN18.2 antibody-radionuclide conjugate to the object. After administration of the anti-CLDN18.2 antibody-radionuclide conjugate, the object may rest for 24 hours to 96 hours (e.g., 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours), and then radionuclide detection is performed.

[0448] In certain embodiments, radionuclide imaging is performed on the site of interest of the object. In some embodiments, the site of interest is a site that expresses or is suspected of expressing claudin 18.2.

[0449] In some embodiments, the site of interest has or is suspected of having a tumor. In some embodiments, the site of interest can be the whole body, torso, head, or limbs.

[0450] Determining or Visualizing the Presence of Claudin18.2 Protein

[0451] In certain embodiments, the methods provided herein further comprise determining or visualizing the presence of the CLDN18.2 protein in the site of interest of the subject according to the image, wherein the presence and / or location of the radionuclide conjugate above background indicates the presence and / or location of the CLDN18.2 protein.

[0452] The anti-CLDN18.2 antibody-radionuclide conjugate provided herein can bind to tissues and cells expressing CLDN18.2, thereby allowing detection or visualization of such CLDN18.2-expressing tissues and cells based on the bound radionuclide signal, or in other words, radionuclide uptake.

[0453] Radionuclide imaging (such as PET imaging) with the anti-CLDN18.2 antibody-radionuclide conjugate provided herein can be used for qualitative or quantitative detection of the CLDN18.2 protein.

[0454] To quantify radionuclide uptake, a clinician can visually identify one or more lesions of interest (such as tumor lesions or gastric lesions or suspected metastatic lesions) on a PET or CT scan and determine the region of interest (ROI) around these lesions. In these ROIs, the uptake of the anti-CLDN18.2 antibody-radionuclide conjugate (such as 124 I-18B10 or 89 Zr-18B10 or 177 Lu-18B10 or 124 I-SF106 or 177 Lu-DOTA-SF106, a radiotracer) can be corrected for body weight and injection dose and quantified as the standardized uptake value (SUV). SUV refers to the ratio of the concentration of the radionuclide in a certain volume of tissue (in microcuries per volume of injected reagent) to the concentration in the body when uniformly distributed (determined by a standard body phantom). The SUV has no unit. When the counting rate equals the counting rate of the uniformly distributed activity in the body phantom, the SUV in any tissue volume reaches 1.0. The results are usually normalized relative to body weight. To calculate the SUV, a circular region of interest can be drawn around the region of interest (such as the primary site of the tumor or metastatic lesion) in the transverse axis slice and adapted to a three-dimensional volume of interest (VOI).

[0455] In certain embodiments, the maximum standardized uptake value (SUVmax) of a suspected lesion is measured. In certain embodiments, the SUVmax can be the SUV with the highest value within the volume of interest.

[0456] In certain embodiments, the average standardized uptake value (SUVmean) of certain tissues or organs, such as normal tissues or organs, is measured.

[0457] The presence and / or location of radionuclide uptake above background indicates the presence and / or location of the CLDN18.2 protein.

[0458] Tomographic images are obtained by image reconstruction. To determine the distribution of the radiotracer, ROIs can be drawn on the reconstructed images, including but not limited to the stomach, peritoneum, lung, liver, heart, kidney, lymph nodes, ovary, bone, pancreas, intestine, skin, or other organs and tissues (such as cancerous tissues). The radiotracer uptake over time in these regions is used to generate time-activity curves (TACs) for the various dose paradigms being examined. The data can be expressed as radioactivity per unit time per unit volume (μci / cc / mCi injected dose).

[0459] There are two types of PET procedures, involving static tracer imaging or dynamic tracer imaging. Static tracer imaging involves obtaining a single time-point estimate of tracer uptake or static imaging that provides a spatial map of regional tracer concentration. Under static imaging, only the mean value (e.g., standardized uptake value, SUV) is measured. On the other hand, dynamic tracer imaging can provide more information about in vivo biology by depicting the temporal and spatial patterns of tracer uptake. See, for example, Muzi et al., Magn Reson Imaging. 2012 30(9):1203-1215. As 124 Anti-CLDN18.2 antibody-radionuclide conjugates such as I-18B10 can be used in either static tracer imaging or dynamic tracer imaging.

[0460] The methods provided herein have a wide range of uses in preclinical and clinical settings, including but not limited to detecting or visualizing the CLDN18.2 protein in a subject (e.g., directly visualizing the in vivo saturation of the CLDN18.2 protein) according to the methods provided herein; diagnosing conditions or diseases associated with CLDN18.2 expression (such as cancer); quantifying tissues expressing CLDN18.2 or diseased tissues; predicting responsiveness to CLDN18.2-targeted therapies; monitoring disease progression or tumor metastasis; or monitoring the therapeutic efficacy over time; monitoring drug resistance over time; or monitoring uptake in normal tissues to predict toxicity or patient-to-patient differences, etc.

[0461] Methods for Detection or Visualization

[0462] The present disclosure describes methods for detecting or visualizing CLDN18.2 protein at a site of interest of an object, the methods comprising:

[0463] a) administering to the object a detectably effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate; and

[0464] b) performing radionuclide imaging on the object at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain an image; and

[0465] c) determining or visualizing the presence of the claudin18.2 protein in the site of interest of the object based on the image,

[0466] wherein the presence and / or location of the radionuclide conjugate above background indicates the presence and / or location of the CLDN18.2 protein.

[0467] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0468] In certain embodiments, the object has not been treated with or received a CLDN18.2-targeted therapy.

[0469] In certain embodiments, the method further comprises administering to the object a therapeutically effective amount of a CLDN18.2-targeted therapy that has been identified as having the CLDN18.2 protein present in the site of interest.

[0470] In certain embodiments, CLDN18.2-targeted therapies include therapies that specifically target CLDN18.2 or cells that express CLDN18.2 (i.e., cells that aberrantly express CLDN18.2 in a manner that substantially does not form classical tight junctions found in normal epithelial tissue). The CLDN18.2-targeted therapies include therapies selected from the group consisting of: anti-CLDN18.2 antibody therapies, CLDN18.2-targeted compounds, CLDN18.2-targeted nucleic acid therapies, CLDN18.2-targeted peptides, CLDN18.2-targeted cell therapies, and CLDN18.2-targeted gene therapies. In certain embodiments, anti-CLDN18.2 antibody therapies include monoclonal anti-CLDN18.2 antibodies, multispecific antibodies that target CLDN18.2 and a second antigen, and / or antibody-drug conjugates (ADCs) that target CLDN18.2. In certain embodiments, anti-CLDN18.2 antibody therapies include anti-CLDN18.2 antibody conjugates conjugated to a therapeutic radionuclide, which include the anti-CLDN18.2 antibodies provided herein conjugated to a therapeutic radionuclide, such as 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194Ir, 198 Au, 199 Au, 199 Au and 211 Pb. In certain embodiments, the anti-CLDN18.2 antibody therapy comprises a therapeutically radiolabeled anti-CLDN18.2 antibody conjugate, which comprises an anti-CLDN18.2 antibody provided herein conjugated to 177 Lu.

[0471] In certain embodiments, the therapeutically radiolabeled nuclide is the same as the diagnostically radiolabeled nuclide. In certain embodiments, both the therapeutically radiolabeled nuclide and the diagnostically radiolabeled nuclide are 177 Lu. In certain embodiments, the anti-CLDN18.2 antibody-diagnostically radiolabeled nuclide conjugate is the same as the anti-CLDN18.2 antibody-therapeutically radiolabeled nuclide (e.g., the same radiolabeled nuclide, the same sequence of the anti-CLDN18.2 antibody or its antigen-binding fragment, and / or the same conjugation site). In certain embodiments, the therapeutically effective amount is at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% of the detectable effective amount.

[0472] Methods for Diagnosis

[0473] Methods for diagnosing a subject with a CLDN18.2-related disease are described herein; methods for identifying a subject in need who may respond to a CLDN18.2-targeted therapy; methods for monitoring the progression of a CLDN18.2-related disease in a subject during a monitoring period; methods for monitoring the therapeutic efficacy of a subject with a CLDN18.2-related disease who has been treated with a therapy for a certain treatment period; and kits for the above methods. In certain embodiments, the anti-CLDN18.2 antibody-radiolabeled nuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostically radiolabeled nuclide conjugate.

[0474] On the one hand, the present disclosure provides a method for diagnosing a subject with a CLDN18.2-related disease. In certain embodiments, the method comprises: administering to the subject an effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate provided herein; performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain an image; diagnosing the subject's CLDN18.2-related disease based on the presence or location of CLDN18.2 protein in the site of interest of the subject as determined from the image, wherein the presence and / or location of radionuclide uptake above background indicates the presence and / or location of the CLDN18.2 protein. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0475] The presence and / or location of radionuclide uptake above background can be determined qualitatively or quantitatively. As described in the previous section, radionuclide uptake can be quantified using SUVmax or SUVmean.

[0476] In certain embodiments, the level of radionuclide uptake at the site of interest is compared with a corresponding reference level to determine the difference from the reference level.

[0477] In certain embodiments, the reference level can be the level of radionuclide uptake in non-diseased tissue or normal tissue (e.g., muscle tissue). In some other embodiments, the reference level can be the level of radionuclide uptake in tissue of the same type but not diseased (and thus not known to express CLDN18.2). Such reference levels can be empirical or arbitrary.

[0478] In certain embodiments, if the difference is statistically significant, or if the difference reaches a predetermined threshold, the subject can be diagnosed as having a CLDN18.2-related disease.

[0479] In certain embodiments, the reference level can be the level of radionuclide uptake at a standard-confirmed lesion. A standard-confirmed lesion can be a lesion that has been confirmed to have CLDN18.2 expression by other methods such as immunohistochemistry (IHC) methods. In certain embodiments, the standard-confirmed lesion is tissue of the same type as the tissue of interest. By comparing with the reference level of the radionuclide uptake at the standard-confirmed lesion, the level (i.e., high, medium, or low) of CLDN18.2 protein at the site of interest in the subject can be estimated.

[0480] In some embodiments, the subject has not been treated with a CLDN18.2-targeted therapy or has not received the CLDN18.2-targeted therapy. Without wishing to be bound by any theory, it has surprisingly been found that, compared to subjects who have received a CLDN18.2-targeted therapy, subjects who have not received a CLDN18.2-targeted therapy show a much higher signal (e.g., SUV) in radionuclide imaging. Accordingly, the radionuclide imaging method provided herein can be particularly useful for diagnosing CLDN18.2-related diseases in subjects who have not been treated with a CLDN18.2-targeted therapy or have not received the CLDN18.2-targeted therapy.

[0481] In some embodiments, the method further comprises administering a therapeutically effective amount of a CLDN18.2-targeted therapy to the subject diagnosed with a CLDN18.2-related disease.

[0482] In some embodiments, a CLDN18.2-targeted therapy comprises a therapy that specifically targets CLDN18.2 or cells expressing CLDN18.2 (i.e., cells that aberrantly express CLDN18.2 in a manner that substantially does not form classical tight junctions found in normal epithelial tissue). The CLDN18.2-targeted therapy comprises a therapy selected from the group consisting of: anti-CLDN18.2 antibody therapy, CLDN18.2-targeted compounds, CLDN18.2-targeted nucleic acid therapy, CLDN18.2-targeted peptides, CLDN18.2-targeted cell therapy, and CLDN18.2-targeted gene therapy. In some embodiments, the anti-CLDN18.2 antibody therapy comprises a monoclonal anti-CLDN18.2 antibody, a multispecific antibody that targets CLDN18.2 and a second antigen, and / or an antibody-drug conjugate (ADC) that targets CLDN18.2. In some embodiments, the anti-CLDN18.2 antibody therapy comprises a therapeutically radiolabeled anti-CLDN18.2 antibody conjugate comprising an anti-CLDN18.2 antibody provided herein conjugated to a therapeutically radiolabeled, such as 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr,153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb. In certain embodiments, the anti-CLDN18.2 antibody therapy comprises a therapeutically radiolabeled anti-CLDN18.2 antibody conjugate comprising an anti-CLDN18.2 antibody provided herein conjugated to 177 Lu.

[0483] In certain embodiments, the therapeutic radionuclide is the same as the diagnostic radionuclide. In certain embodiments, both the therapeutic radionuclide and the diagnostic radionuclide are 177 Lu. In certain embodiments, the anti-CLDN18.2 antibody-diagnostic radionuclide conjugate is the same as the anti-CLDN18.2 antibody-therapeutic radionuclide conjugate (e.g., the radionuclide is the same, the sequence of the anti-CLDN18.2 antibody or its antigen-binding fragment is the same and / or the conjugation site is the same). In certain embodiments, the therapeutically effective amount is at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% of the detectable effective amount.

[0484] In certain embodiments, the anti-CLDN18.2 antibody-diagnostic radionuclide conjugate and the anti-CLDN18.2 antibody-radionuclide conjugate

[0485] Methods for Prognosis

[0486] On the other hand, the present disclosure provides a method for identifying a subject in need who may respond to a CLDN18.2-targeted therapy. In certain embodiments, the method comprises: administering to the subject an effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate; performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain an image; identifying that the subject may respond to the CLDN18.2-targeted therapy based on the presence and / or location of CLDN18.2 protein in the site of interest of the subject as determined according to the image, wherein the presence and / or location of the radionuclide conjugate above background indicates the presence and / or location of the CLDN18.2 protein. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0487] The presence and / or location of radionuclide uptake above background can be determined qualitatively or quantitatively. As described in the previous section, radionuclide uptake can be quantified using SUVmax or SUVmean.

[0488] In certain embodiments, the level of the radionuclide conjugate at the site of interest is compared with a corresponding reference level to determine the difference from the reference level. In certain embodiments, the method further comprises identifying that the subject may respond to the CLDN18.2-targeted therapy when the difference is statistically significant or when the difference reaches a predetermined threshold.

[0489] These identified subjects may be recommended for additional tests to confirm the conclusion.

[0490] In certain embodiments, the method further comprises administering a therapeutically effective amount of a CLDN18.2-targeted therapy to the subject identified as likely to respond.

[0491] In certain embodiments, when the subject is identified as unlikely to respond to a CLDN18.2-targeted therapy, the method further comprises recommending against treatment with a CLDN18.2-targeted therapy.

[0492] In certain embodiments, CLDN18.2 targeting therapies include therapies that specifically target CLDN18.2 or cells that express CLDN18.2 (i.e., cells that aberrantly express CLDN18.2 in a manner that substantially does not form classical tight junctions found in normal epithelial tissue). The CLDN18.2 targeting therapies include therapies selected from the group consisting of: anti-CLDN18.2 antibody therapies, CLDN18.2 targeting compounds, CLDN18.2 targeting nucleic acid therapies, CLDN18.2 targeting peptides, CLDN18.2 targeting cell therapies, and CLDN18.2 targeting gene therapies. In certain embodiments, anti-CLDN18.2 antibody therapies include monoclonal anti-CLDN18.2 antibodies, multispecific antibodies that target CLDN18.2 and a second antigen, and / or antibody-drug conjugates (ADCs) that target CLDN18.2. In certain embodiments, anti-CLDN18.2 antibody therapies include anti-CLDN18.2 antibody conjugates conjugated to a therapeutic radionuclide, which include the anti-CLDN18.2 antibodies provided herein conjugated to a therapeutic radionuclide, such as 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194Ir, 198 Au, 199 Au, 199 Au and 211 Pb. In certain embodiments, the anti-CLDN18.2 antibody therapy comprises a therapeutically radiolabeled anti-CLDN18.2 antibody conjugate comprising an anti-CLDN18.2 antibody provided herein conjugated to 177 Lu. In certain embodiments, the therapeutically radiolabeled nuclide is the same as the diagnostically radiolabeled nuclide. In certain embodiments, both the therapeutically radiolabeled nuclide and the diagnostically radiolabeled nuclide are 177 Lu. In certain embodiments, the anti-CLDN18.2 antibody-diagnostically radiolabeled nuclide conjugate is the same as the anti-CLDN18.2 antibody-therapeutically radiolabeled nuclide. In certain embodiments, the therapeutically effective amount is at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% of the detectable effective amount.

[0493] In certain embodiments, the CLDN18.2-targeted cell therapy comprises chimeric antigen receptor (CAR) T cells, TCR T cells or CAR NK cells that target CLDN18.2. A chimeric antigen receptor (CAR) is an engineered chimeric receptor that combines an antigen-binding domain (such as that of an antibody) with one or more signaling domains for immune cell activation. Immune cells such as T cells and natural killer (NK) cells can be genetically engineered to express a CAR. T cells expressing a CAR are referred to as CAR-T cells. A CAR can mediate antigen-specific cellular immune activity in T cells, enabling CAR-T cells to eliminate cells (such as tumor cells) that express the targeted antigen (such as CLDN18.2).

[0494] The CLDN18.2-targeted therapy can specifically bind to CLDN18.2 and exert a therapeutic effect on cells expressing CLDN18.2 such as cancer cells. In certain embodiments, the CLDN18.2-targeted therapy can specifically kill cells expressing CLDN18.2.

[0495] Methods for Monitoring Disease Progression

[0496] On the other hand, the present disclosure provides a method for monitoring the progression of CLDN18.2-related diseases in a subject during a monitoring period. In certain embodiments, the method comprises: administering to the subject a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate after the monitoring period; performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain a post-monitoring image; and comparing the post-monitoring image with a pre-monitoring image to determine a change in the level of CLDN18.2 protein during the monitoring period based on the images, wherein the level of the radionuclide conjugate above background indicates the level of the CLDN18.2 protein, and wherein the change indicates the presence or absence of disease progression. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0497] In certain embodiments, the pre-monitoring image is obtained from the subject before the monitoring period by: administering an effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate, and subsequently performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain the pre-monitoring image.

[0498] In certain embodiments, an increase in CLDN18.2 level during the monitoring period indicates disease progression, and the absence of such an increase in CLDN18.2 level during the monitoring period indicates the absence of disease progression.

[0499] In certain embodiments, the subject has not been treated with a CLDN18.2-targeted therapy or has not received the CLDN18.2-targeted therapy. In certain embodiments, the subject has been treated with a CLDN18.2-targeted therapy or has received the CLDN18.2-targeted therapy.

[0500] In certain embodiments, the disease is a tumor.

[0501] In certain embodiments, the progression is metastasis of the tumor. In certain embodiments, the spread of CLDN18.2 expression to sites where CLDN18.2 expression was previously undetectable indicates the presence of metastasis.

[0502] In certain embodiments, the level of the CLDN18.2 protein comprises the amount, distribution, and / or location of the CLDN18.2 protein.

[0503] In certain embodiments, the subject is at risk of metastasis.

[0504] Methods for Monitoring Therapeutic Efficacy

[0505] On the other hand, the present disclosure provides a method for monitoring the therapeutic efficacy of a subject having a CLDN18.2-related disease and who has been treated with a therapy for a certain treatment period. In certain embodiments, the method comprises:

[0506] a) administering to the subject a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate after the treatment period;

[0507] b) performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain a post-treatment image; and

[0508] c) comparing the post-treatment image with a pre-treatment image to determine a change in the level of CLDN18.2 protein during the treatment period based on the images, wherein the level of the radionuclide conjugate above background indicates the level of the CLDN18.2 protein,

[0509] wherein the change indicates the presence or absence of the therapeutic efficacy.

[0510] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

[0511] In certain embodiments, the pre-treatment image is obtained from the subject prior to the treatment period by: administering an effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate, and subsequently performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain the pre-treatment image.

[0512] In certain embodiments, an increase in the CLDN18.2 level during the treatment period indicates the absence or poor therapeutic efficacy, and / or wherein the absence of an increase in the CLDN18.2 level during the treatment period indicates the presence or positive therapeutic efficacy.

[0513] In certain embodiments, the therapy can be any therapeutic agent useful for treating CLND18.2-related diseases. In certain embodiments, the CLDN18.2-related disease is cancer. In certain embodiments, the CLDN18.2-related disease is gastric cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0514] For example, the therapy can be an anti-cancer drug or a drug for treating gastric diseases such as gastric ulcers.

[0515] Examples of anti-cancer drugs include, but are not limited to, chemotherapeutic agents, anti-cancer drugs, radiotherapy, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormonal therapeutic agents, or cytokines.

[0516] As used herein, the term "immunotherapy" refers to types that stimulate the immune system to combat diseases such as cancer or strengthen the immune system in general. Immunotherapy includes passive immunotherapy, which is carried out by delivering agents with established tumor immunoreactivity (such as effector cells), can directly or indirectly mediate anti-tumor effects and does not necessarily rely on an intact host immune system (such as antibody therapy or CAR-T cell therapy). Immunotherapy can further include active immunotherapy, in which the treatment relies on in vivo stimulation of the endogenous host immune system to respond to diseased cells by administering immune response modulators.

[0517] Examples of immunotherapy include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0518] Checkpoint modulators can interfere with the ability of cancer cells to avoid immune system attacks and help the immune system mount a stronger response against tumors. Immune checkpoint molecules can mediate co-stimulatory signals to enhance the immune response or can mediate co-inhibitory signals to suppress the immune response. Examples of checkpoint modulators include, but are not limited to, PD-1, PD-L1, PD-L2, CLTA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, and CD83.

[0519] Adoptive cell transfer, which is a treatment that attempts to enhance the natural ability of T cells to fight cancer. In this treatment, T cells are taken from the patient and expanded and activated in vitro. In certain embodiments, the T cells are modified to be CAR-T cells in vitro. The most anti-cancer active T cells or CAR-T cells are cultured in large batches in vitro for 2 to 8 weeks. During this time period, the patient will receive treatments such as chemotherapy and radiotherapy to reduce the body's immunity. After these treatments, the in vitro cultured T cells or CAR-T cells will be re-administered to the patient. In certain embodiments, the immunotherapy is CAR-T therapy.

[0520] Cytokine therapy can also be used to enhance the presentation of tumor antigens to the immune system. Two main cytokines used in the treatment of cancer are interferons and interleukins. Examples of cytokine therapy include, but are not limited to, interferons such as interferon-α, -β, and -γ; colony-stimulating factors such as macrophage-CSF, granulocyte macrophage CSF, and granulocyte-CSF; insulin growth factor (IGF-1); vascular endothelial growth factor (VEGF); transforming growth factor (TGF); fibroblast growth factor (FGF); interleukins such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12; tumor necrosis factors such as TNF-α and TNF-β or any combination thereof.

[0521] Oncolytic viruses are genetically modified viruses that can kill cancer cells. Oncolytic viruses can specifically infect tumor cells, leading to lysis of the tumor cells and subsequent release of a large amount of tumor antigens, which trigger the immune system to target and eliminate cancer cells with such tumor antigens. Examples of oncolytic viruses include, but are not limited to, talimogenelaherparepvec.

[0522] Therapeutic vaccines combat cancer by enhancing the immune system's response to cancer cells. Therapeutic vaccines can include non-pathogenic microorganisms (such as Mycobacterium bovis bacillus Calmette-Guérin, BCG), genetically modified viruses targeting tumor cells, or one or more immunogenic components. For example, BCG can be directly inserted into the bladder using a catheter and can elicit an immune response against bladder cancer cells.

[0523] Anti-angiogenic agents can prevent the growth of blood vessels that support tumor growth. Some anti-angiogenic agents among anti-angiogenic agents target VEGF or its receptor VEGFR. Examples of anti-angiogenic agents include, but are not limited to, Axitinib, Bevacizumab, Cabozantinib, Everolimus, Lenalidomide, Lenvatinib mesylate, Pazopanib, Ramucirumab, Regorafenib, Sorafenib, Sunitinib, Thalidomide, Vandetanib, and Ziv-aflibercept.

[0524] "Targeted therapy" is a type of therapy that acts on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not in normal cells or are more abundant in cancer cells, or target molecules in the cancer microenvironment that contribute to cancer growth and survival. Targeted therapy targets the therapeutic agent to the tumor, thereby sparing normal tissues from the effects of the therapeutic agent.

[0525] Targeted therapies can target, for example, tyrosine kinase receptors and nuclear receptors. Examples of such receptors include erbB1 (EGFR or HER1), erbB2 (HER2), erbB3, erbB4, FGFR, platelet-derived growth factor receptor (PDGFR), and insulin-like growth factor-1 receptor (IGF-1R), estrogen receptor (ER), nuclear receptor (NR), and PR.

[0526] Targeted therapies can target molecules in the tyrosine kinase or nuclear receptor signaling cascades, such as Erk and PI3K / Akt, AP-2α, AP-2β, AP-2γ, mitogen-activated protein kinase (MAPK), PTEN, p53, p19ARF, Rb, Apaf-1, CD-95 / Fas, TRAIL-R1 / R2, caspase-8, forkhead, box 03A, MDM2, IAP, NF-kB, Myc, P13K, Ras, FLIP, heregulin (HRG) (also known as gp30), Bcl-2, Bcl-xL, Bax, Bak, Bad, Bok, Bik, Blk, Hrk, BNIP3, BimL, Bid, and EGL-1.

[0527] Targeted therapies can also target tumor-associated ligands, such as estrogen, estradiol (E2), progesterone, estrone, androgen, glucocorticoid, prolactin, thyroid hormone, insulin, P70 S6 kinase protein (PS6), survivin, fibroblast growth factor (FGF), EGF, neuregulin (NDF), transforming growth factor alpha (TGF-α), IL-1A, TGF-β, IGF-1, IGF-II, IGFBP, IGFBP protease, and IL-10.

[0528] In certain embodiments, the therapy can be a CLDN18.2 targeted therapy as provided herein. In certain embodiments, the CLDN18.2 targeted therapy includes a therapy selected from the group consisting of: anti-CLDN18.2 antibody therapy, CLDN18.2 targeted compounds, CLDN18.2 targeted nucleic acid therapy, CLDN18.2 targeted peptides, CLDN18.2 targeted cell therapy, and CLDN18.2 targeted gene therapy.

[0529] In certain embodiments, the method further comprises increasing the dose of the therapy or stopping the therapy when a poor therapeutic efficacy is determined.

[0530] In certain embodiments, the method further comprises advising the subject to continue the therapy when a positive therapeutic efficacy is determined.

[0531] In certain embodiments, the therapy comprises a CLDN18.2-targeted therapy.

[0532] In certain embodiments, the method further comprises advising the subject to switch to a therapy other than the CLDN18.2-targeted therapy when the CLDN18.2 level after treatment is lower than a corresponding reference level. In certain embodiments, the method further comprises advising the subject to switch to a therapy other than the CLDN18.2-targeted therapy when the CLDN18.2 level after treatment is reduced by at least 40% (or at least 50%, 60%, 70%, 80%, 90% or 95%) relative to the CLDN18.2 level before treatment.

[0533] In certain embodiments, the therapy is not a CLDN18.2-targeted therapy.

[0534] In certain embodiments, the site of interest has or is suspected of having a tumor or gastric lesion. In certain embodiments, the site of interest is the whole body. In certain embodiments, the tumor includes metastases therefrom.

[0535] In certain embodiments, the subject is at risk of metastasis.

[0536] Pharmaceutical Compositions

[0537] The present disclosure further provides a pharmaceutical composition comprising the anti-CLDN18.2 antibody conjugate provided herein and one or more pharmaceutically acceptable carriers. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate has at least one of the following properties: a) having a radiochemical purity of at least 90% (such as 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%); b) having a radiolabeling rate of at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%); and c) being able to specifically bind to CLDN18.2 with a Kd value of no more than 15 nM (such as no more than 14 nM, no more than 13 nM, no more than 12 nM, no more than 10 nM, no more than 8 nM, no more than 6 nM, no more than 4 nM or no more than 4 nM); and d) being able to specifically bind to CLDN18.2 with an EC50 value of no more than 1.0 nM (such as no more than 0.8 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM or no more than 0.3 nM), as measured by ELISA.

[0538] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate comprises a therapeutic radionuclide selected from the group consisting of: 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb or a diagnostic radionuclide selected from the group consisting of: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac.

[0539] In certain embodiments, the therapeutic radionuclide is 177 Lu or 124 I. In certain embodiments, the diagnostic radionuclide is 124 I, 89 Zr or 177 Lu. In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate further comprises a chelator. In certain embodiments, the chelator is DFO or DOTA. In certain embodiments, the chelator is DFO and the radionuclide is 89 Zr. In certain embodiments, the chelator is DOTA and the radionuclide is 177 Lu. In certain embodiments, when the radionuclide is 89 Zr, the chelator conjugated to the bifunctional linker reagent is p-isothiocyanatobenzyl-desferrioxamine (p-NCS-Bz-DFO):

[0540]

[0541] In certain embodiments, when the radionuclide is 177 Lu, the anti-CLDN18.2 antibody-radionuclide conjugate comprises a compound moiety of formula (II):

[0542]

[0543] In certain embodiments, the composition comprises 89 Zr and a compound of formula (I):

[0544] wherein A is the anti-CLDN18.2 antibody or an antigen-binding fragment thereof provided herein, and k is 1 - 40 (such as 3 - 5, 5 - 35, 10 - 30, 15 - 25 or 20).

[0545] In certain embodiments, the pharmaceutical compositions provided herein are suitable for parenteral administration, such as suitable for bolus, intravenous or intratumoral injection. In certain embodiments, the pharmaceutical composition is in a unit dose injectable form.

[0546] Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquids, gels or solid phase carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, polyvalent chelating or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art or various combinations thereof.

[0547] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifying agents or stabilizers such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, mercaptoacetic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene and / or propyl gallate. As disclosed herein, including one or more antioxidants such as methionine in compositions comprising an antibody or antigen-binding fragment and conjugate as provided herein reduces oxidation of the antibody or antigen-binding fragment. This reduction in oxidation prevents or reduces loss of binding affinity, thereby enhancing antibody stability and maximizing shelf life. Accordingly, in certain embodiments, there are provided compositions comprising one or more antibody conjugates as disclosed herein and one or more antioxidants such as methionine.

[0548] For further illustration, pharmaceutically acceptable carriers may include, for example: aqueous vehicles such as sodium chloride injection, Ringer's injection, isosmotic dextrose injection, sterile water injection or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil; antimicrobial agents at bacteriostatic or fungistatic concentrations; isosmotic agents such as sodium chloride or dextrose; buffering agents such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (TWEEN-80); polyvalent chelating or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid or lactic acid. The antimicrobial agents used as carriers can be added to the pharmaceutical composition in multi-dose containers, and the antimicrobial agents include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0549] In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions or solid forms suitable for producing liquid solutions, suspensions or emulsions. Injectable preparations may include sterile and / or pyrogen-free solutions ready for injection, sterile dry soluble products ready to be combined with a solvent before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle before use, and sterile and / or pyrogen-free emulsions. The solutions can be aqueous or non-aqueous.

[0550] In certain embodiments, unit dose parenteral formulations are packaged in ampoules, vials or syringes with needles. As is known and practiced in the art, all formulations for parenteral administration should be sterile and pyrogen-free.

[0551] In certain embodiments, the anti-CLDN18.2 antibody-radionuclide conjugate is formulated as a parenteral composition in a unit dosage form for ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically discrete unit suitable as a single dosage for a subject to be administered the anti-CLDN18.2 antibody-radionuclide conjugate. The specifications of the unit dosage form described herein are governed by and directly depend on: (a) the unique characteristics of the anti-CLDN18.2 antibody in the antibody-radionuclide conjugate; (b) the tissue or cells to be targeted; (c) the limitations inherent in the imaging technique used.

[0552] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the antibody or its antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may include excipients that may improve the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Excipients that may be used include, but are not limited to, water, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, and in one embodiment, the pH is about neutral. The solution is then sterile filtered and subsequently lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of the anti-CLDN18.2 antibody conjugate or its composition. Overfilling the vials by a small amount (e.g., about 10%) in an amount greater than that required for a given dose or set of doses is acceptable to facilitate accurate sample removal and accurate dosing. The lyophilized powder may be stored under appropriate conditions (such as at about 4°C to room temperature).

[0553] Reconstitution of the lyophilized powder with water for injection provides a formulation for use upon parenteral administration. In one embodiment, for reconstitution, sterile and / or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder. The exact amount depends on the selected therapy being administered and can be determined empirically.

[0554] Kits

[0555] On the other hand, the present disclosure further provides one or more reagents that can be used in any of the methods described herein. The reagents may include the anti-CLDN18.2 antibody-radionuclide conjugate.

[0556] On the other hand, the present disclosure provides a kit for the methods described above. Generally, the kit contains reagents that can be used in any of the methods provided herein in a carrier or a compartmentalized container. The carrier can be a container or support in the form of, for example, a bag, box, tube, rack, and is optionally compartmentalized.

[0557] In certain embodiments, the kit may further include a standard negative control and / or a standard positive control.

[0558] Additionally, the kit may include instructional materials containing guidelines (i.e., protocols) for practicing the methods provided herein. While the instructional materials typically include written or printed materials, the instructional materials are not limited thereto.

[0559] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials, and methods described below fall, in whole or in part, within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but are only used to illustrate specific embodiments that fall within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive faculty and without departing from the scope of the invention. It should be understood that many variations can be made in the procedures described herein while still remaining within the bounds of the invention. The intention of the inventors is that such variations are all included within the scope of the invention.

[0560] Examples

[0561] Although the present disclosure has been specifically shown and described with reference to specific embodiments, some of which are preferred embodiments, those skilled in the art should understand that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

[0562] Example 1

[0563] Example 1.1 Background

[0564] Claudin 18.2 (CLDN18.2) is specifically expressed in differentiated gastric mucosal epithelial cells but not in the gastric stem cell region, and the loose structure of the cancer cell stromal space makes it possible for CLDN18.2 to be exposed to protein macromolecular drugs, which supports its reliability as a target for the detection and clinical significance of epithelial tumors, especially digestive system tumors. However, there is currently no predictive technology for accurately mapping the expression of CLDN18.2 in patients systemically.

[0565] Preclinical experiments of murine antibody 5C9 have shown that the novel CLDN18.2-targeted probe can be used as a supplement to clinical routine to help localize tumors and guide the surgery of CLDN18.2-positive tumors in multiple ways (14). Previous studies have also shown that 124 I-labeled antibody 124 I-18B10 has good radiochemical properties and stability and specifically accumulates in CLDN18.2-positive tumors (15). Recently, the interim results of a phase 1 trial using Claudin18.2-specific CAR T cells in patients with gastrointestinal cancers were reported (12), and the interim results showed promising efficacy in patients with CLDN18.2-positive digestive system cancers who had been over-pretreated.

[0566] Therefore, an open-label, single-center, single-group, first-in-human phase 0 trial was sponsored to study the safety, whole-body distribution / dosimetry, and CLDN18.2 targeting ability of patients, and to explore the main relationship between it and patient prognosis before and after anti-CLDN18.2 therapy.

[0567] Here, the present invention reports the prognosis of non-pre-specified interim results from this ongoing trial (NCT04883970). A detectable CLDN18.2-targeted radioactive probe with high specificity and sensitivity was established ( 124 I-18B10), which was conducted in the first-in-human studies of 124 I-18B10 PET / CT and PET / MR in patients with gastric cancer, pancreatic cancer, or cholangiocarcinoma. 124 Using PET functional imaging, I-18B10 was shown to be safe and feasible for indicating CLDN18.2-overexpressing tumor lesions.

[0568] The primary objective of the study was to evaluate 124 the safety of the I-18B10 tracer and the feasibility of mapping whole-body CLDN18.2 expression using PET functional imaging.

[0569] The second objective of the study was to detect lesions using 18F-FDG and the novel 124 I-18B10 PET molecular imaging technique.

[0570] 124 The I-18B10 PET imaging study was conducted in cancer patients who had received or would receive CLDN18.2-targeted therapy. It was hypothesized that patients with strong CLDN18.2 expression might obtain more clinical benefits.

[0571] Example 1.2 Pre - clinical and Patients

[0572] Manually synthesize using Hu18B10-Ha and Hu18B10-La (i.e., SEQ ID NO:25 and SEQ ID NO:26) 124 I-18B10 probe, and perform preclinical experiments including binding affinity and specific targeting ability after testing in in vitro and in vivo model cells and / or mice.

[0573] Patients with pathologically confirmed digestive system tumors were enrolled in an ongoing, open-label, single-group, first-in-human (FiH) phase 0 trial (NCT04883970). Perform within one week 124 I-18B10 PET / CT or PET / MR and 18 F-FDG PET.

[0574] In the lesions 124 I-18B10 and 18 The uptake of F-FDG was calculated as the maximum standardized uptake value (SUVmax), and compared with CLDN immunohistochemistry of tumor lesions, and the uptake of normal organs was calculated as the SUV mean. Tumor response was evaluated according to (i) RECIST v1.1.

[0575] Example 1.3 Results

[0576] 124 I-18B10 was successfully constructed with a radiochemical yield exceeding 95%. The preclinical experimental results showed the described 124 I-18B10 had high stability in saline and maintained high affinity (Kd = 4.11 nM) in cells overexpressing CLDN18.2 and specific uptake in tumors of model mice.

[0577] Seventeen patients were enrolled, including 12 patients with gastric cancer, 4 patients with pancreatic cancer, and 1 patient with cholangiocarcinoma (phase 0). The optimal time point for PET scan was selected as 48h - 96h. As indicated by the semi-quantification of PET, 124 I-18B10 showed high uptake in the spleen and liver, with mean SUV means of 2.52 ± 1.38 and 1.85 ± 0.65 respectively, and showed slight uptake in the bone marrow, lung, stomach, and pancreas, with mean SUV means of 0.83 ± 0.41, 0.48 ± 0.20, 1.2 ± 0.80, and 0.77 ± 0.12 respectively.

[0578] A total of 65 lesions from 17 patients were analyzed. The tracer uptake range of tumor lesions with SUVmax was 0.4 to 19.5. Compared with the lesions treated with CLDN18.2-targeted therapy, the 124The uptake of I-18B10 was significantly higher (SUVmax at 24 h: 6.00 ± 7.38 vs. 2.37 vs. 1.43, P = 0.042). For two gastric cancer patients, after regional 124 I-18B10 PET / MR, metastatic lymph nodes showed high signal and high tracer uptake on the T2WI sequence.

[0579] Example 1.4 Conclusions

[0580] 1. 124 I-18B10 was successfully prepared and maintained high binding affinity and CLDN18.2 target specificity in preclinical studies.

[0581] 2. 124 I-18B10 is a first-in-human CLDN18.2 PET tracer that maintains safety through acceptable dosimetry and clearly indicates most CLDN18.2 overexpressing lesions.

[0582] 3. Tumor lesions showed different levels of tracer uptake.

[0583] Example 2 Study Design

[0584] The objective of this invention was to evaluate the biodistribution, safety, and dosimetry of a single-dose injection 124 of I-18B10 in patients for PET / CT or PET / MR imaging.

[0585] Study the 124 uptake of I-18B10 in tumor lesions and its relationship with patient prognosis. The clinical study was approved by the Ethics Committee of Peking University Cancer Hospital (2021KT57) and registered on ClinicalTrials.gov (NCT04883970).

[0586] (1) The primary objective of the study was to map systemic CLDN18.2 expression.

[0587] (2) The second objective was to detect lesions using 18 F-FDG and the novel 124 I-18B10 PET molecular imaging technique.

[0588] (3) 124 The I-18B10 PET imaging study was conducted in cancer patients who had received or would receive CLDN18.2-targeted therapy.

[0589] Example 3124 Construction and Preclinical Study of I-18B10

[0590] Example 3.1 124 Production, Quality Control and In Vitro Experiments of I-18B10

[0591] Example 3.1.1 124 Production and Quality Control of I-18B10

[0592] The CLDN18.2 antibody used in the study was provided by Suzhou Transcenta Therapeutics co., LTD (Suzhou, China). 124 I was produced and purified by the cyclotron team of the Department of Nuclear Medicine, Peking University Cancer Hospital. 124 The radiolabeling and quality control methods of I-18B10 are as follows.

[0593] Prepared by NBS reaction 124 I-18B10: Add 0.8 mL of 0.1 M pH 7.2 PB buffer, 0.1 mL (29.2 mg / mL) Hu18B10HaLa monoclonal antibody solution (prepared with H 2 O) and 36 μg of NBS to 1.0 mL of 59.2 KBq / μL Na 124 I solution in sequence, incubate at 37 °C for 60 seconds, then add 0.1 mL of 10% human serum albumin to terminate the reaction; purify the final reaction solution through a PD-10 column to obtain the target product.

[0594] Before use, first equilibrate the PD-10 column with 0.01 M pH 7.4 PBS solution, add 5 mL of PBS each time, dry the column at gravity flow rate, repeat 5 times; then purify the target product with 0.01 M pH 7.4 PBS solution.

[0595] Radioactive TLC and radioactive HPLC are used to determine the radiolabeling rate and radiochemical purity. Radioactive TLC detection: Add 2 μL of free Na 124 I with a radioactivity of 37 - 74 kBq (1 - 2 μCi) and purified 124 I-18B10 to 20 μL of saturated EDTA. Drop 2 μL of the above solution 1 cm from the bottom of No. 1 Whatman filter paper, and place it in a physiological saline developing system. After complete development, take out the filter paper and dry it for radioactive TLC detection. Radioactive HPLC detection: Add 2 μL of free Na 124 I with a radioactivity of 37 - 74 kBq (1 - 2 μCi) and purified 124I-18B10 was diluted to 50 μL in 0.01 M PBS at pH 7.4 for radio-HPLC analysis. Analysis conditions: Agilent Bio SEC-3 gel filtration / size exclusion chromatography column, flow rate 1 mL / min, mobile phase 0.01 M PBS at pH 7.4.

[0596] Synthesized 124 The radiochemical purity and yield of I-18B10 exceeded 95%. Details regarding 124 quality control of I-18B10 can be seen Figure 9 . It was demonstrated 124 the stability of I-18B10 in PBS within 48 h ( Figure 7A ).

[0597] Example 3.1.2 124 Cell uptake and saturation binding studies of I-18B10

[0598] Cell uptake experiments were performed by adding 124 I-18B10 (20 μL, 37 kBq, 1.37×10−7 M) as the experimental group and negative control, respectively, into wells (n = 4) containing MKN45CLDN18.2+ and MKN45 cells. And 124I-IgG (immunoglobulin G) was added as a control in MKN45CLDN18.2+. The mixtures were incubated in a 37 °C 5% CO2 incubator for 10, 30, 60, and 120 minutes, collected in 2 mL tubes, centrifuged at 2000 rpm for 3 minutes, and washed twice with 1 mL of cold PBS (pH 7.4, 0.01 M) (the supernatant was removed after each centrifugation). For the blocking control, 10 μL of PBS (pH 7.4, 0.01 M) containing 60 μg of 18B10 was added to wells (n = 4) containing MKN45CLDN18.2+ cells, and the mixture was incubated in a 37 °C 5% CO2 incubator for 2 h, followed by the steps described above.

[0599] Saturation binding experiments were performed to determine 124 the binding affinity between I-18B10 and CLDN18.2. 124 I-18B10 was added to wells (n = 4) containing MKN45CLDN18.2+ cells with six concentration gradients (0 kBq, 0.037 kBq, 0.37 kBq, 3.7 kBq, 37 kBq, and 370 kBq), 50 μL per well. After incubation in a 37 °C 5% CO 2 incubator for 4 h, the mixtures were processed as described above. The value of the binding constant Kd was calculated according to the fitted curve of the pooled data.

[0600] The radioactivity of the cells and the total added radioactivity were counted using a γ counter. The percentage of cell uptake was calculated as cell count / total added count. The final results of four independent parallel experiments were expressed as mean ± standard deviation (SD).

[0601] The results of the cell uptake experiment showed that the uptake of 124 I-18B10 in MKN45CLDN18.2+ cells increased over time (from 20.08 ± 0.83% at 10 minutes to 23.51 ± 0.47% at 120 minutes). As a comparison, a significant decrease was observed in the MKN45 group (from 3.31 ± 0.54% at 10 minutes to 8.69 ± 0.35% at 120 minutes). At each selected time point, the uptake of positive cells was significantly higher than that of negative cells or blocked controls (p < 0.0001)( Figure 7B ).

[0602] The binding constant (Kd = 4.11 nM) of 124 I-18B10 to the CLDN18.2 receptor was also determined using MKN45CLDN18.2+ cells by cell saturation binding assay( Figure 7C ). These results demonstrated 124 the targeted and specific binding of

[0603] Example 3.2 124 I-18B10 microPET / CT functional imaging and IHC analysis

[0604] Example 3.2.1 Model Mouse Construction and Biodistribution

[0605] All animal experiments were conducted in accordance with the guidelines established by the Peking University Cancer Hospital Animal Care and Use Committee.

[0606] Gastric Ulcer Mouse Model:

[0607] Fifteen female KM mice (18 - 22 g) were fasted for 24 hours. Ten of them were given absolute ethanol by gavage at 0.15 ml / mouse, and the other five served as blank controls.

[0608] PDX (Patient - Derived Xenograft) Mouse Model:

[0609] A PDX mouse model was established by subcutaneously transplanting biopsied human gastric cancer tissue into the flanks of 6-week-old female NOD / SCID mice (Vital River, China). When the tumor volume reached 150 mm3 When the above conditions are met, the mice are randomly divided into groups (5 - 6 mice per group) for further experiments. The tumor volume and mouse body weight are measured every 3 days. The tumor growth inhibition rate (TGI%) is calculated using the formula: tumor volume (length × width²) / 2.

[0610] 124 Biodistribution study of I-18B10:

[0611] Female BALB / c nude mice (20 g, 4 - week - old), purchased from Vital River Laboratories (China), were intravenously injected via the tail vein with 200 μL 124 of I - 18B10 (0.37 - 0.74 MBq). The mice were sacrificed after isoflurane anesthesia at 2, 24, 60 h and 120 h after injection. The major organs, including heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, brain and blood, were collected, weighed and the radioactivity was measured using a gamma counter. As a standard, ten samples of 1% of the injected dose were taken out and measured. The results were expressed as percentage of injected dose per gram (%ID / g).

[0612] The biodistribution study in normal mice (n = 4) showed a decrease over time in all tissues, indicating that the probe has no specificity in normal organs ( Figure 7D ). Among all the tissues tested, blood had the highest radioactivity and decreased rapidly (from 22.45 ± 6.24 %ID / g at 2 h to 8.89 ± 0.18 %ID / g at 120 h).

[0613] Example 3.2.2 MicroPET / CT Imaging and IHC

[0614] Small animal PET imaging was performed using a micro - PET / CT (Super Nova PET / CT, PINGSENG, Shanghai, China).

[0615] At 0, 4, 24, 48 and 96 h after gavage, micro - PET / CT imaging of gastric ulcers and normal (n = 5 per group) was carried out. At each imaging time point, the mice in the parallel groups were sacrificed by cervical dislocation, the whole stomach was removed, and the gastric ulcer was observed macroscopically after removing the contents. The samples were sectioned for IHC and HE staining to detect the changes in CLDN18.2 expression and the degree of gastric injury before and after gavage. 124 When the tumor volume was estimated to be 400 - 900 mm

[0616] ³, the mice were used for small animal PET imaging. Under isoflurane inhalation, CLDN18.2 - positive or negative PDX mice (n = 4 per group) were intravenously injected with 200 μL of 5.55 MBq 3 124 ​I-18B10 for microPET imaging. Scans were performed at 2, 60, and 120 h after administration. And 5.55 MBq was injected. 124 I-IgG was used as a control (n = 4), while 124 I-18B10 was injected into CLDN18.2 low-expression PDX mice as a negative control. And the counts per second values of the region of interest (ROI) on the tumor were collected.

[0617] MicroPET Scanning of Gastric Ulcer Model:

[0618] Since no specific uptake was found in normal stomachs with high CLDN18.2 expression (both in mice and humans) in previous imaging, an acute gastric ulcer model was constructed using ethanol gavage to verify whether gastric ulcer lesions could uptake more 124 I-18B10 tracer.

[0619] Imaging showed high gastric uptake within 4 h after gavage (2.53 ± 0.27 at 4 h and 2.98 ± 0.24 at 24 h), and as the gastric injury gradually healed, the uptake gradually decreased ( Figure 8A ). No death was seen in the gavage mice, and eating and defecation were normal.

[0620] After autopsy, the gastric body was found to show local redness, punctate bleeding of varying degrees, and congestion of the gastric body ( Figure 8B ).

[0621] IHC data showed that the expression level of CLDN18.2 in the gastric mucosa was the same before and after injury (normal stomach ++ vs. 0 h ++, 4 h ++, 24 h ++, 48 h ++, 96 h ++). In addition, according to the HE results, it could be seen that except at 4 h after gavage, there was no obvious injury to the gastric tissue at all time points ( Figure 8C ).

[0622] It could be seen that the gradual recovery of acute gastric ulcer after 24 hours had no effect on the continuous accumulation of antibody probes in the stomach. And as Figure 8D shown, more critically, at longer time points (1.81 ± 0.20 at 48 h vs. 0.74 ± 0.05, and 1.01 ± 0.10 at 96 h vs. 0.46 ± 0.07), higher uptake was observed in the gastric tissue approaching healing in the gastric ulcer model compared to normal mice (p < 0.05). This simulation experiment seems to provide partial support for the hypothesis that the spatial structure of gastric mucosal cells limits their uptake of mAb probes.

[0623] MicroPET Scanning of PDX Model:

[0624] At 2, 60, and 120 h after radioactive tracer injection, the in vivo distribution and metabolic characteristics of 124 I-18B10 were evaluated in real-time and non-invasively by small animal PET / CT imaging ( Figure 1 ). PET data were quantified by analyzing the ROI to demonstrate the dynamic changes of the tracer in tumors at different post-injection time points ( Figure 2A ).

[0625] 124 I-18B10 began to accumulate in positive tumors at 2 h and then showed a stable trend until 120 h. The tumor uptake values of the negative control were lower than those of the experimental group at each time point (at 2 h 0.85 ± 0.10 vs. 1.22 ± 0.21, at 60 h 0.64 ± 0.10 vs. 1.32 ± 0.12, at 120 h 0.45 ± 0.06 vs. 1.02 ± 0.05, p < 0.05), and the same was true for the blank control (at 2 h 0.53 ± 0.07, at 60 h 0.56 ± 0.08, at 120 h 0.55 ± 0.10).

[0626] To verify 124 the in vivo specificity of I-18B10, the ratios of tumor / muscle probe uptake were calculated and compared among the three experimental groups. It was found that the tumor / muscle ratio in the positive group increased over time (from 4.52 ± 0.02 at 2 h to 5.37 ± 0.02 at 120 h), while that in the negative control was opposite (from 3.70 ± 0.02 at 2 h to 2.14 ± 0.01 at 120 h), and the blank control showed no obvious change trend over time (3.12 ± 0.01 at 2 h, 3.11 ± 0.01 at 60 h, 3.06 ± 0.01 at 120 h).

[0627] In addition, paraffin-embedded tissue sections of mice were subjected to IHC staining ( Figure 2B ). CLDN18.2 was widely stained in tumors of the positive group (++), while no positive signal was observed in the negative group, which demonstrated the usefulness of these two xenograft models in evaluating the in vivo binding specificity of the radioactive tracer 124 I-18B10. Notably, CLDN18.2 also showed high expression in the heart (++) and stomach (+++ / ++) of both groups.

[0628] According to Figure 2C the maximum intensity projection (MIP) images shown, although 124 I-18B10 showed accumulation in the heart and blood pool at the early time point (2 h), which was considered normal, PET imaging specifically and clearly depicted the PDX tumors from 2 h. The uptake in the other two control groups was similar to that in the experimental group, except that due to124 The non-specificity of I-IgG or low uptake in the tumor area due to negative CLDN18.2 expression in the negative PDX model. These results are consistent with the tumor uptake quantification by analyzing the ROI.

[0629] Example 4 First-in-human Clinical Translation Study

[0630] Example 4.1 Patient Eligibility Criteria and Characteristics

[0631] Patients with gastric cancer or pancreatic cancer or cholangiocarcinoma confirmed by pathology were eligible by using the Eastern Cooperative Oncology Group performance (status ≤ 2). Other inclusion criteria included age greater than 18 years; life expectancy of at least 3 months; and sufficient liver and kidney function. Written informed consent was obtained from all patients.

[0632] From May 2021 to April 2022, 17 patients with pathological results were included in the analysis, namely, 6 males and 11 females, with a median age of 51 (29 - 65) years. Figure 10 Patient characteristics are summarized in

[0633] Example 4.2 18 F-FDG PET / CT and 124 I-18B10 PET / CT and / or PET / MR

[0634] All patients received 124 I-18B10 PET / CT imaging within 1 week before and after 124 I-18B10 PET / CT and 18F-FDG PET / CT ( Figure 1 ).

[0635] For 124 I-18B10 PET imaging, 124 Lugol's potassium iodide (ten drops each time, three times a day) was taken 3 days before and 7 days after the administration of I-18B10 to block the patient's thyroid gland. At 2 h, 24 h, 72 h, and 96 h after the administration of 124 I-18B10, 124 I-18B10 PET / CT scans were obtained using a Siemens Biograph mCT Flow 64 scanner (Erlangen, Germany).

[0636] I-18B10 PET / MR was performed using an integrated TOF PET / MR system (uPMR 790 PET / MR, United Imaging, Shanghai, China). 124 I-18B10 PET / MR.

[0637] The images were read by two experienced nuclear medicine physicians familiar with the patient's medical history. The maximum standardized uptake value (SUVmax) of primary and metastatic lesions of the tumor was measured, and the SUV mean of the patient's major organs was measured simultaneously.

[0638] Example 4.3 Statistical Analysis

[0639] Descriptive statistics included median, mean, and standard deviation (SD). Independent samples t-tests were used to analyze between-group comparisons. Spearman correlation analysis was used to analyze the relationship between the two groups. Statistical analysis was performed using SPSS 20.0. P < 0.05 was considered significant.

[0640] Example 4.4 124 I-18B10 Whole Body CLDN18.2 Expression Mapping

[0641] The CLDN18.2 expression level in the tumor tissue of one patient was zero, while the CLDN18.2 expression levels in the tumor tissues of the other 16 patients were all positive. Among the enrolled patients, 10 patients had received CLDN18.2-targeted therapy before 124 I-18B10 PET imaging, while 7 patients had not received CLDN18.2-targeted therapy. The first patient in this trial received two 124 I-18B10 PET scans before and after receiving CLDN18.2-targeted therapy. Figure 10 Patient characteristics are shown.

[0642] In the patient 124 The biodistribution of I-18B10 was derived from the images of the first five patients ( Figure 3A , B). The patient's thyroid was fully blocked. The tracer concentrated in the blood after injection and decreased over time, while the tracer concentration in the spleen and liver increased over time. Among all organs, the spleen had the highest activity at 24, 48, and 72 h. There was also tracer uptake in the kidneys, while the uptake in the brain, lungs, and bones was low. No obvious tracer uptake was seen in the normal gastric wall.

[0643] Example 4.5 In tumor lesions 124 Functional PET imaging of I-18B10

[0644] A total of 65 tumor lesions from 17 patients were analyzed, among which 25 lesions were not treated with CLDN18.2-targeted therapy before imaging, and 40 lesions had been treated with CLDN18.2-targeted therapy. The tracer uptake range of tumor lesions was 0.4 to 19.5 SUV max. There was no difference in tumor uptake among patients with gastric cancer, pancreatic cancer, and cholangiocarcinoma (SUV max at 24 h: 2.24 ± 1.51 vs. 2.42 ± 1.18 vs. 10.00 ± 10.66, P > 0.05; at 48 h: 2.14 ± 1.71 vs. 2.95 ± 0.87 vs. 6.86 ± 6.63, P > 0.05). The uptake in lesions not treated with CLDN18.2-targeted therapy was significantly higher than that in lesions treated with CLDN18.2-targeted therapy (SUV max at 24 h: 6.00 ± 7.38 vs. 2.37 vs. 1.43, P = 0.042)( Figure 3D ).

[0645] There were significant differences in the uptake of 124 I-18B10 in tumors at different lesion sites (P < 0.0001)( Figure 3C ). Ovarian metastases showed the highest uptake, with an average SUV max of 23.65 ± 2.05, while lung metastases showed the lowest uptake, with an SUV max of 1.5.

[0646] Among seven patients who did not receive CLDN18.2-targeted therapy, a 57-year-old female patient (Patient 4) had moderately differentiated adenocarcinoma of cholangiogenic origin and liver, peritoneal, bone metastases, and suspected right ovarian metastases. The patient's primary lesion had been resected, and the CLDN18.2 expression level was 3 + 30% by pathological examination. 124 I-18B10 PET / CT showed increased uptake of 124 I-18B10 in some liver and bone metastases, with SUV max of 3.2 and 4.4 respectively, and two soft tissue masses with abnormally high uptake were observed in both ovaries, with SUV max of 12.5 and 17.6 on the left and right respectively( Figure 4 ).

[0647] After injection of 124 I-18B10, two patients with gastric cancer underwent regional 124 I-18B10 PET / MR at 96 h. A 36-year-old female patient (Patient 8) with gastric cancer and lymph node metastases and suspected left ovarian metastases had previously received CLDN18.2-targeted therapy, and the CLDN18.2 expression level was 3 +, 90%( Figure 5 Small figure D). 124I-18B10 PET / MR showed high uptake in the lymph nodes beside the left iliac vessels, with a maximum SUV of 6.6 ( Figure 5 small figures A-C) and slight uptake in the left ovarian metastatic tumor, with a maximum SUV of 1.3, which showed high signal on the T2WI sequence. Another patient was a 43-year-old female with gastric cancer and lymph node metastases, and had also received CLDN18.2-targeted therapy previously. The CLDN18.2 expression level was also 3+, 90%. Two retroperitoneal metastatic lymph nodes showed high signal on the T2WI sequence and high uptake on 124 I-18B10 PET / MR, with maximum SUVs of 2.6 and 2.5 respectively ( Figure 5 small figures E-G).

[0648] Example 4.6 124 I-18B10 PET uptake and response to therapy

[0649] A gastric cancer patient who had failed two lines of treatment including capecitabine, oxaliplatin, and paclitaxel underwent 124 I-18B10 PET / CT and 18F-FDG PET / CT. On the pre-treatment 124 I-18B10 PET, several peritoneal metastases showed 124 high I-18B10 uptake, with maximum SUVs of 3.1, 3.2, and 4.2. Then the patient received an infusion of CT041, CLDN18.2-targeted CAR T cell therapy, and 4 months later underwent 124 I-18B10 PET / CT and 18F-FDG PET / CT again, on which the previously highly-uptaken peritoneal metastases did not show obvious uptake. And without disease progression, this patient survived for up to 40 weeks.

[0650] Example 4.7 124 I-18B10 Human Dosimetry Assessment

[0651] Volumes of interest (VOIs) were drawn for the major organs including the brain, lungs, heart, liver, spleen, kidneys, muscles, and bones, and the activity concentrations of the major organs were measured using PET / CT images through the IntelliSpace Portal workstation (Philips, Netherlands). The values of standard male / female organ masses were taken from the OLINDA / EXM software (Ver2.2, HERMES Medical Solutions, Inc, Canada) to estimate human dosimetry.

[0652] Throughout the examination, the patient did not report any issues related to 124Adverse events related to I-18B10. The OLINDA dosimetry estimates showed that the three organs receiving the highest absorbed dose were the spleen, kidney, and liver, with mean values of 1.20, 0.717, and 0.616 mGy / MBq ( Figure 11 ), respectively. The mean effective dose was 0.213 mSv / MBq (0.788 rem / mCi).

[0653] Discussion of Example 5

[0654] Targeted radionuclide labeling technology can achieve real-time diagnosis and treatment through in vivo imaging, thus comprehensively reflecting the spatial distribution and expression intensity of all focal targets. The targeted radionuclide labeling technology provides a non-invasive and effective molecular imaging method for precision medicine, which can achieve patient screening, monitoring expression heterogeneity, predicting efficacy, and evaluating efficacy.

[0655] Targeting CLDN18.2 124 The first-in-human study of I-18B10 PET / CT and PET / MR in cancer patients was confirmed. The results showed that 124 I-18B10 PET is a safe and invasive imaging for detecting CLDN18.2 in patients receiving CLDN18.2-targeted therapy.

[0656] 124 The biodistribution of I-18B10 in humans was mainly concentrated in the liver, spleen, and kidney, and gradually decreased over time. The uptake in the brain, muscle, and other tissues was very low, which was consistent with 124 the distribution characteristics of I-labeled monoclonal antibodies in humans (16). Previous studies have shown that CLDN18.2 can also be highly expressed in normal gastric wall tissues (5), but no obvious uptake was observed in the normal gastric wall tissues of the enrolled patients. This may be because CLDN18.2 is usually limited to the inside of tight junctions, and monoclonal antibodies in normal mucosal cells are basically inaccessible (17). There are two possible mechanisms to explain this phenomenon, which have not been confirmed and require further study. One possible mechanism is that CLDN18.2 on the apical membrane is farther from the microvessels and more difficult to reach than CLDN18.2 on the basolateral membrane, while another possible mechanism is that IgG antibodies are difficult to bind to targets surrounded by several tight junction components (11).

[0657] To prove whether the low uptake observed in normal gastric tissues was due to the above speculation, a gastric ulcer model was simply constructed, attempting to change the external environment and spatial relationship of normal gastric mucosal cells, and interpreting the negative imaging of the stomach from the perspective of the structure-activity relationship of monoclonal antibody probes. The high uptake observed in mice with unhealed / healed gastric ulcers indicated that the binding of the mAb probe to the gastric mucosa was negatively correlated with inflammation, but more human results were still needed for verification.

[0658] Several clinical trials have shown remarkable progress in CLDN18.2-targeted therapies for CLDN18.2-positive patients. However, these studies have also demonstrated the limitations of CLDN18.2 testing and used different cut-off values (18). There is a great need to develop unified detection methods to reduce the impact of using different detection methods and different positive thresholds in trials. PET imaging using radiolabeled antibodies is a non-invasive molecular imaging method that can avoid the effects of sampling errors and tumor heterogeneity (19). So far, several studies have shown that PET imaging may have the potential to screen patients who may benefit from PD-1 / L1 blockade immunotherapy (20 - 22). Therefore, PET imaging targeting CLDN18.2 may also become an effective method for studying CLDN18.2-targeted therapies.

[0659] Included 124 Patients who had or had not received CLDN18.2-targeted therapy before I-18B10 PET imaging. Compared with the lesions treated with CLDN18.2-targeted therapy, the uptake in the lesions not treated with CLDN18.2-targeted therapy was significantly higher. Tumor uptake was not related to the CLDN18.2 expression level, either in any patient group or in all patients. In patients who had not received CLDN18.2-targeted therapy, the expression level of CLDN18.2 in a cholangiocarcinoma patient was only 3 + 30%. But in 124 I-18B10 PET, there was significantly high uptake in multiple bone metastases, liver metastases, and ovarian metastases, especially abnormal concentrations in ovarian metastases, indicating that the expression of CLDN18.2 in different lesions of the same patient may have significant heterogeneity.

[0660] After receiving CLDN18.2-targeted therapy, tumor cells with high CLDN18.2 expression in the patient's tumor lesions may induce apoptosis through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) caused by anti-18B10-18.2 antibody (23, 24). Therefore, although the CLDN18.2 expression level was high in previous pathological tests, the lesion uptake was negative on CLDN18.2 PET. The negative uptake of the lesion on CLDN18.2 PET indicates that there are no tumor cells with high CLDN18.2 expression in the lesion, and the effect of CLDN18.2-targeted therapy may not meet expectations, suggesting that the treatment plan should be changed. After receiving CLDN18.2-targeted therapy, the patient's tumor lesion still showed significantly high uptake in CLDN18.2 PET, indicating that there are still tumor cells with high CLDN18.2 expression in the lesion, and CLDN18.2-targeted therapy may still have obvious efficacy.

[0661] A 30-year-old female gastric cancer patient who had undergone gastrectomy and resection of ovarian metastases (with ovarian and peritoneal metastases) (Patient 1) had two 124 I-18B10 PETs before and after receiving CLDN18.2-targeted therapy. The patient's CLDN18.2 expression level was 90%, 3+. On the 124 pre-treatment 124 I-18B10 PET, several peritoneal metastases showed Figure 6 high I-18B10 uptake, with maximum SUV values of 3.1, 3.2, and 4.2 ( Figure 6 small panels A-C). Nearly 4 months after receiving CLDN18.2-targeted therapy, the patient had another 124 I-18B10 PET, on which the previously highly-uptaken peritoneal metastases did not show obvious uptake ( Figure 6 small panels D-E). Therefore, 124 I-18B10 PET can dynamically monitor the expression level of CLDN18.2 in tumor lesions and may play an important role in guiding CLDN18.2-targeted treatment strategies.

[0662] In summary, the present invention shows that 124 I-18B10 PET imaging is safe, has acceptable dosimetry, and shows good human biodistribution. Tumor lesions show different levels of tracer uptake, and the uptake may be related to treatment response.

[0663] References for the background art, the specific embodiments of the present invention, and Examples 1-5 can be found in Table 7.

[0664] Example 6

[0665] Example 6.1 Abstract

[0666] In this study, 18B10 labeled with the solid target radionuclide zirconium-89 (< 89 89Zr) was constructed to detect the expression in the human gastric cancer BGC823 CLDN18.2 cell line. 89 89Zr]Zr-DFO-18B10 showed high radiochemical purity (RCP, >99%) and specific activity (24.15 ± 1.34 GBq / μmol), and was stable in 5% HSA and PBS (radiochemical purity >85% at 96 h). The 50% maximum effective concentration (EC 50 50) values of 18B10 and DFO-18B10 were as high as 0.413 ± 0.055 nM and 0.361 ± 0.058 nM, respectively (P > 0.05). Two days after (p.i.) injection, the uptake of the radiotracer in CLDN18.2-positive tumors was significantly higher than that in CLDN18.2-negative tumors (1.11 ± 0.02 vs. 0.49 ± 0.03, P = 0.0016). The BGC823 CLDN18.2 mouse model showed a high T / M value at p.i. 96 h, in which 89 89Zr]Zr-DFO-18B10 was much higher than that of 89 89Zr]Zr-DFO-18B10 in other imaging groups. The immunohistochemical results showed that BGC823 CLDN18.2 tumors were highly positive (+++) for CLDN18.2, while tumors in the BGC823 group did not express CLDN18.2 (-). The results of the ex vivo biodistribution study showed that the distribution in BGC823 CLDN18.2 tumor-bearing mice (2.05 ± 0.16% ID / g) was higher than that in BGC823 mice (0.69 ± 0.02% ID / g) and the blocking group (0.72 ± 0.02% ID / g). The dosimetry estimation study showed that 89 the effective dose of 89Zr]Zr-DFO-18B10 was 0.0705 mSv / MBq, within the acceptable dose range for nuclear medicine research. In summary, these results indicate that the GMP generated by this immunological PET probe can detect tumors overexpressing CLDN18.2.

[0667] Example 6.2 Introduction

[0668] According to the cancer epidemiology report released in 2022, lung cancer is the leading cause of cancer death, followed by digestive tract tumors (such as gastric cancer, colorectal cancer, liver cancer, esophageal cancer, etc.). In China, gastrointestinal cancers account for 45% of cancer-related deaths, probably because most gastrointestinal cancers are diagnosed at an advanced stage and patients often have a poor prognosis [1-3]. In addition to traditional chemotherapy and immunotherapy, there has been little progress in new chemotherapy and targeted therapies for gastrointestinal tumors [4-7]. Among the 70 new first-line agents approved for cancer treatment, only 5 drugs are approved for advanced gastrointestinal cancer, and according to data from the past five years, the survival rate remains low [8]. Therefore, strategies to improve the survival rate of patients with advanced gastrointestinal cancer remain an unmet medical need.

[0669] CLDN18.2 is a tight junction protein belonging to the CLDN protein family (CLDN), which is involved in the formation of intercellular adhesion structures and controls cell polarity and intercellular material exchange [9-11]. The expression of CLDN18.2 is strictly limited to normal gastric mucosal cells but is overexpressed during the proliferation, division, and metastasis of tumor cells, making it an emerging therapeutic target for digestive tract tumor therapies [12,13]. Zolbetuximab (IMAB362) is the first antibody targeting CLDN18.2, which kills tumor cells through antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and is combined with first-line epirubicin, oxaliplatin, and capecitabine (EOX) to provide longer progression-free and overall survival

[14] . 18B10 is a monoclonal antibody against CLDN18.2 developed globally after IMAB362. Compared with IMAB362, 18B10 has higher affinity and stronger NK cell-mediated ADCC tumor-killing activity. In a phase I clinical study of 18B10 (NCT04396821) combined with capecitabine and oxaliplatin (CAPOX) as a first-line agent for advanced gastric / gastroesophageal junction adenocarcinoma, 73.3% achieved partial response and 26.7% achieved disease stability

[15] . A phase I study of CLDN18.2 CAR-T therapy (NCT03874897) conducted by Shen et al. showed that after receiving CLDN18.2 CAR-T infusion, the overall response rate (ORR) and disease control rate (DCR) reached 48.6% and 73.0%, respectively

[16] . Interestingly, both clinical studies showed that the CLDN18.2 expression level is related to drug efficacy, indicating that patients with high CLDN18.2 expression in tumors have more clinical benefits. Therefore, patient selection based on the CLDN18.2 expression level is crucial for CLDN18.2-targeted therapies. Currently, the main method for detecting CLDN18.2 protein is immunohistochemistry (IHC), and other methods include molecular beacons and RT-PCR

[17] . IHC is invasive and requires endoscopic biopsy, and the sampling sites and numbers are limited. Due to the heterogeneous nature of tumors, it is impossible to comprehensively and dynamically reflect the distribution and expression level of CLDN18.2 in patients in real time. Molecular imaging can be used as a non-invasive diagnostic tool to detect the expression and distribution of CLDN18.2 in lesions using the radioactive signals emitted by radioactive tracers, thereby helping clinical screening of patients with potential benefits, evaluating the efficacy of CLDN18.2-targeted therapies, and guiding the precise diagnosis and treatment of tumors. A recent study showed that including SUV maximum, MTV, and TLG of 18F-FDG PET / CT parameters cannot predict the CLDN18.2 expression status in diffuse gastric cancer

[18] . Hu et al. developed three antibodies with different molecular weights for PET / CT imaging (anti-CLDN18.2 VHH, anti-CLDN18.2 VHH-ABD, and anti-CLDN18.2 VHH-Fc), and identified 89 Zr]-anti-CLDN18.2 VHH-ABD as the most suitable imaging agent (high tumor uptake and low liver uptake) in preclinical studies

[19] . However, in subsequent clinical studies, 89 Zr]-VHH-Fc was found to be more specific and persistent than 89 Zr]-anti-CLDN18.2 VHH-ABD, and was also considered a molecular imaging tracer with potential cancer diagnostic value because it contains CLDN18.2

[20] . Recently, CLDN18.2-specific murine mAb 5C9 was explored by DNA immunization and labeled with 124 I, Cy5.5, and FD1080. These research results support targeted therapy for CLDN18.2-positive tumors by using immuno-PET imaging and near-infrared fluorescence II imaging to localize tumors and guide surgery for in-situ CLDN18.2-positive tumors

[21] .

[0670] Due to the superior targeting specificity and high sensitivity of molecular imaging technology, the 18B10 antibody meeting GMP requirements was used to construct an immuno-PET molecular probe 89 Zr]Zr-DFO-18B10. The goal of this study was to evaluate 89 Zr]Zr-DFO-18B10's ability to characterize CLDN18.2 expression.

[0671] Heavy chain variable region of IMAB362 (SEQ ID NO:72)

[0672] QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS

[0673] Light chain variable region of IMAB362 (SEQ ID NO:73)

[0674] DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYW ASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK

[0675] Example 6.3 Materials and Methods

[0676] 1) Materials

[0677] All reagents were obtained from Sigma - Aldrich. p - NCS - Bz - DFO was purchased from Macrocyclics. The GMP - grade CLDN18.2 antibody 18B10 (Hu18B10HaLa) was kindly provided by Transcenta Holding Ltd. (Suzhou, China). The radionuclide 89 89Zr was produced and purified by the cyclotron team in the Department of Nuclear Medicine, Peking University Cancer Hospital.

[0678] 2) Radio - labeling of 18B10 with 89Zr

[0679] For 89 Zr labeling, 0.25 M 2 - [4 - (2 - hydroxyethyl) - 1 - piperazinyl] ethanesulfonic acid (HEPES) and 1 M Na 2 2CO 3 buffer was used to neutralize 89 89Zr - oxalic acid to pH 7.0, and then it was mixed with the previously described DFO - 18B10 at 37 °C for 60 minutes. The reaction mixture was purified through a PD - 10 column (2.5 ml, 0.01 M Ph 7.4 PBS).

[0680] 3) 89 Small animal PET imaging of [89Zr]Zr - DFO - 18B10

[0681] Normal KM mice and BGC823 CLDN18.2 / BGC823 model nude mice were injected via the tail vein with 7.4 MBq 89 89Zr]Zr - DFO - 18B10 (n = 3). Then 10 - minute static PET scans were acquired at each time point (p.i. 2, 24, 48, and 72 h). As a non - specific control group, BGC823 CLDN18.2 mice (n = 3) were fasted for 6 h in advance and then injected via the tail vein with 7.4 MBq of 18 18F - fluorodeoxyglucose (FDG). At 18Mice were anesthetized with 2% isoflurane before and during F-FDG PET imaging. A small animal PET / CT scanner (Super Nova PET / CT, Pingseng Healthcare, China) was used to reconstruct PET images by Avatar 3, and the standardized uptake value (SUV) of the ROI was calculated by drawing ROIs on these organs.

[0682] 4) Ex vivo biodistribution.

[0683] KM mice were intravenously injected with 0.74 MBq 89 [

[18] Zr]Zr-DFO-18B10 via the tail vein and then sacrificed at 2, 24, 48, 72, and 144 h p.i. (n = 4). Tissues including blood, heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, and brain were dissected. The radioactivity of the tissues was measured using a gamma counter. The radioactivity of each organ was calculated as the percentage of the injected dose per gram (%ID / g). For the ex vivo biodistribution of the tumor model, female nude mice bearing BGC823 CLDN18.2 and BGC823 tumor xenografts were injected with 0.74 MBq 89 [

[18] Zr]Zr-DFO-18B10 via the tail vein to evaluate 89 [

[18] Zr]Zr-DFO-18B10 distribution in major organs and tumors (n = 4 per group). Mice (n = 4) were sacrificed and dissected at 48 h p.i., and tumors, kidneys, blood, and other major organs were collected and weighed. Blocking studies were also performed in BGC823 CLDN18.2 mice by co-injecting 0.74 MBq 89 [

[18] Zr]Zr-DFO-18B10 and an excess of cold 18B10 (1 mg). Blocked mice were sacrificed and dissected at 48 h p.i. Then the organ biodistribution of 89 [

[18] Zr]Zr-DFO-18B10 was determined.

[0684] 5) Dosimetry estimation

[0685] For human radiation dosimetry, animal biodistribution data were obtained by standard methods of organ dissection. Human organ radiation dosimetry data were extrapolated from the biodistribution data of 89 [

[18] Zr]Zr-DFO-18B10 in KM mice using OLINDA / EXM 2.0 software (Vanderbilt University, America).

[0686] 6) Statistical analysis

[0687] Quantitative data are presented as mean ± standard deviation (SD), where all error bars represent SD. The means were compared using Student's t test, and a P value of less than 0.05 was considered to indicate statistical significance.

[0688] 7) Conjugation and Identification of DFO - 18B10

[0689] The methods for modification and radiolabeling of 18B10 have been reported in previous studies. Specifically, after exchanging the solvent with 0.01 M PBS, 18B10 (3.3 nmol) was dissolved in NaHCO 3 buffer (0.1 M, pH 9.5). The pH was adjusted to 9.0 with 0.1 M Na 2 CO 3 solution. Then, 60 nmol of p-NCS-Bz-DFO dissolved in DMSO was added to the above solution, where the molar ratio of p-NCS-Bz-DFO to 18B10 was 20:1. After mixing and reacting at 37 °C for 1 h, the crude product was further purified by a PD-10 column (2.5 ml, 0.01 M PBS) and stored at -80 °C in a stabilizer.

[0690] The mass spectra of 18B10 and DFO-18B10 were measured by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker Dalton, Germany). For non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 10 μg of antibody samples were diluted with 0.01 M PBS and ×5 non-reducing sample buffer without dithiothreitol and then separated by electrophoresis on a 6% sodium dodecyl sulfate PAGE gel. The gel was stained with 0.5% Coomassie blue.

[0691] 8) Evaluation of CLDN18.2 Binding Affinity

[0692] The binding efficacy between 18B10 and DFO-18B10 and the full-length human CLDN18.2 protein-VLP (CL2-H52P7) was determined using enzyme-linked immunosorbent assay (ELISA). First, 100 μL of CL2-H52P7 solution (2 μg / mL) was added to each well of a 96-well polystyrene StripwellTM microplate (Corning Costar, CLS2481-100EA) and incubated overnight at 4 °C. Then, the antigen solution was discarded, and the protein was washed five times with PBST (0.01 M pH 7.4 PBS and 0.2% Tween-20). After that, 5% milk powder (diluted with PBS) was added to the microplate and incubated at 37 °C for 2 h to block other non-specific sites. Then, after discarding the 5% milk powder, it was washed five times with PBST. After washing the plate, diluted samples (IgG, 18B10, and DFO-18B10) were added to the microplate (100 μL / well) at seven concentration gradients of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 10, 50, 100, 500, and 1000 nM, and the membrane was covered at 15 - 25 °C for 2 h. The plate was washed again, and a secondary antibody was added: Goat anti-mouse IgG4 Fc (HRP) was diluted 1:3000 with secondary antibody diluent (100 μL / well) in the ELISA plate and incubated at 15 - 25 °C for 1 h. To develop color after washing, a chromogenic solution (100 μL / well) was added, and the plate was covered with a film and developed in the dark at 15 - 25 °C for 20 minutes. To terminate the reaction, a termination solution (50 μL / well) was added. During detection, the optical density (OD) value of each well was read at a detection wavelength of 450 nm using an ELISA reader. EC 50 values were also used to evaluate the affinity of anti-CLDN18.2 antibodies for CLDN18.2.

[0693] 9) 89 Preparation of Zr]Zr-DFO-IgG

[0694] The preparation of DFO-IgG was the same as that of DFO-18B10. For 89 Zr labeling, 0.25 M 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) and 1 M Na 2 CO 3 buffer was used to neutralize 89 Zr-oxalate to pH 7.0, and then it was mixed with DFO-IgG at 37 °C for 60 minutes. The reaction mixture was purified through a PD-10 column (2.5 ml, 0.01 M Ph 7.4 PBS).

[0695] 10) 89 Quality control of Zr]Zr-DFO-18B10

[0696] ​89 The quality control of the Zr]Zr-DFO-18B10 tracer is carried out in accordance with the "Quality Control Guidelines for Positron Radiopharmaceuticals".

[0697] (1) pH: Take 1 drop of the radiopharmaceutical, drop it on a precision pH test paper, and compare it with the standard color sample, that is, the pH value of the solution.

[0698] (2) Ethanol content: Ethanol is not added during the preparation of the tracer preparation and does not need to be inspected.

[0699] (3) Endotoxin: Take this product and inspect it according to General Chapter 1143 of the Chinese Pharmacopoeia (2020). This product should contain less than 15 EU / mL of endotoxin.

[0700] (4) Sterility: Take this product and inspect it according to General Chapter 1101 of the Chinese Pharmacopoeia (2020).

[0701] (5) Specific activity: It refers to the activity per unit mass of an element or its compound of a radionuclide.

[0702] 11) Radiochemical Purity and In Vitro Stability

[0703] The radiochemical yield and radiochemical purity are measured by radio thin layer chromatography (radio TLC) with a standard protocol. The products before and after purification 89 Zr]Zr-DFO-18B10 (2 μL) is dropped at 1 cm from the lower end of the TLC-SG test strip, and then the TLC-SG test strip is placed in an eluent of 0.5 M sodium citrate buffer (pH = 5.0). When the eluent has developed to 10 cm from the lower end, the eluent is taken out and dried, radio TLC analysis is carried out, and the Rf value is calculated. The Rf values of free Zr-89 and 89 Zr]Zr-DFO-18B10 are 0.9 - 1.0 and 0 - 0.1 respectively. An in vitro stability study of 89 Zr]Zr-DFO-18B10 incubated with 0.01 M PBS or 5% HSA at room temperature (RT) is carried out. Radio TLC is carried out at different incubation times (0, 2, 12, 24, 48, and 96 h).

[0704] 12) Cell Lines and Tumor - Bearing Models

[0705] The human gastric cancer cell line BGC823 was obtained from Peking University Cancer Hospital and Institute (Beijing, China). BGC823 was generated by transfection with full-length CLDN18.2 CLDN18.2Cell line. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotics from Invitrogen. All animal experiments were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Animal Care and Ethics Committee of Peking University Cancer Hospital. For PET / CT imaging and in vitro biodistribution experiments, BGC823 and BGC823 xenografts were established in female BALB / c nu / nu mice at 4 to 6 weeks of age. The mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). CLDN18.2 1×10 6 BGC823 / BGC823 cells suspended in 100 μL PBS were subcutaneously injected into the right axilla of the mice. CLDN18.2 Tumors grew for 3 weeks and reached an average volume of 100 mm 3 .

[0706] 13) Evaluation of CLDN18.2 Expression

[0707] As previously described, Western blotting was performed. After washing three times with cold PBS, 500 μL of RIPA lysis buffer was added to each cell culture dish and incubated on ice for 10 minutes. The cell lysates were centrifuged at 15,000 rpm for 5 minutes at 4 °C, and the supernatants were collected and stored on ice. Equal amounts of supernatant (20 μg) were separated to extract proteins by SDS-PAGE (250 mA, 90 minutes) and transferred to a polyvinylidene difluoride (PVDF) membrane. The blot was incubated with the following primary antibody: rabbit anti-human CLDN18.2 (ab213480, 1:500; Abcam). The blot was then incubated with an HRP-conjugated goat anti-rabbit secondary antibody (1:10,000). Proteins were detected using Clarity Western ECL substrate. Immunoblots were imaged using an Alliance Micro Q9 chemiluminescence imaging system (Alliance Micro Q9, UVITEC, Britain). Cells (2×10 5) and washed twice with cold PBS, stained with 2 μg / mL CLDN18.2 antibody (1D5, Peking University Cancer Hospital) for 1 h at room temperature, then stained with Alexa Fluor 488-conjugated antibody for 30 minutes, and then subjected to flow cytometry using a BD FACS Aria. Flow cytometry analysis was performed on a Beckman Coulter Cytomics FC500MPL.

[0708] 14) Cell Experiments.

[0709] The BGC823 / BGC823 CLDN18.2 cells were cultured in RPMI-1640 medium (2.0×10 5 cells / mL), and added to 24-well plates (1.0 mL per well) and cultured overnight. The 89 Zr]Zr-DFO-18B10 (20 μL, 37 kBq, 6.87×10 -12 M) was added to the wells containing adherent BGC823 / BGC823 CLDN18.2 cells (n = 4). The mixture was incubated in a 5% CO 2 incubator at 37 °C for 2, 10, 30, 60, and 120 minutes. After incubation, the medium was removed and the cells were washed twice with cold PBS (0.01 M). Inhibition was carried out in the presence of excess unlabeled 18B10 (50 μg) for 60 and 120 minutes. Then the cells were collected after digestion with 1 M NaOH and counted in a γ counter. The percentage of the added dose per 2.0×10 5 cells (% AD / 2.0×10 5 cells) was calculated based on the counts.

[0710] Example 6.4: Immunohistochemical Studies

[0711] The paraffin sections were dewaxed with xylene. After rehydration in distilled water, the antigen was retrieved by heating in EDTA (pH 9.0) for 10 minutes. The endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide at room temperature for 15 minutes. Nonspecific binding was blocked with goat blocking serum at room temperature for 1 h. The anti-CLDN18.2 rabbit monoclonal antibody (Abcam, ab222512) diluted 1:500 was added, and the slides were incubated overnight at 4 °C. After washing three times, the slides were incubated with Envision (DAKO) at room temperature for 45 minutes. Diaminobenzidine was used as the chromogen. The sections were counterstained with hematoxylin, dehydrated, and mounted. The immunohistochemical slides were evaluated using a Leica AT2 microscope.

[0712] Example 6.5 Results and Discussion

[0713] 1) Conjugated molecular characteristics

[0714] The molecular weight of the CLDN18.2 antibody 18B10 is approximately 148 kDa, and it is further accurately determined to be 148,723 Da( Figure 14A ). DFO-18B10 is chelated with an approximate bis-DFO chelator with a molecular weight of 150320 Da( Figure 14B ). SDS-PAGE shows that both 18B10 and DFO-18B10 have a band of approximately 150 kDa and no other bands( Figure 14C ), indicating excellent conjugation quality as no antibody aggregates or antibody fragments are detected. ELISA results show that the EC 50 value of DFO-18B10 binding to CLDN18.2 has no significant difference from the EC 50 value of 18B10 (0.413 nM ± 0.055 nM vs. 0.361 ± 0.058 nM, P>0.05, Figure 14D ). The binding assay shows that both 18B10 and DFO-18B10 can form strong binding with CLDN18.2, and the conjugation of the chelator DFO has no effect on the affinity of 18B10 for CLDN18.2.

[0715] 2) Radiosynthesis, quality control, and in vitro stability

[0716] Figure 15 Panel A shows the 89 synthesis process of 89 Zr]Zr-DFO-18B10. 89 Zr]Zr-DFO-18B10 was prepared manually, with a radiochemical labeling yield of 74.64% ± 4.4% (n = 3, non-decay corrected). Figure 15 Panel B). 89 The in vitro stability of Figure 15 Zr]Zr-DFO-18B10 in 0.01 M PBS or 5% HSA was demonstrated by an RCP exceeding 85% after 96 h at RT(

[0717] Panel C). The excellent in vitro stability also indicates that the 18B10 structural modification and labeling method are feasible. The quality control results are shown in Table 4. 89 Quality control of

[0718]

[0719] 3) In vitro CLDN18.2 expression of cell lines.

[0720] Western blot results confirmed that the expression of CLDN18.2 in BGC823 CLDN18.2 cells was significantly different from the expression of CLDN18.2 in BGC823 cells ( Figure 16 Figure A). The relative expressions of CLDN18.2 in BGC823 CLDN18.2 and BGC823 cell lines were 1.37 ± 0.24 and 0.23 ± 0.01, respectively (P = 0.0013, Figure 16 Figure B). Flow cytometry experiments showed that 86.2% of the cells in the BGC823 CLDN18.2 group were stained positive with anti-CLDN18.2 antibody (1D5) ( Figure 16 Figure C). Then, the differences in CLDN18.2 expression between the human gastric cancer cell line BGC823 and BGC823 CLDN18.2 measured by Western blot and flow cytometry were verified. The results of the cell uptake experiment showed that the uptake of CLDN18.2 Zr]Zr-DFO-18B10 in BGC823 89 cells increased in a time-dependent manner (7.33 ± 0.84% at 10 minutes, 7.97% ± 0.56% at 30 minutes, 11.47% ± 0.32% at 60 minutes, 13.37% ± 2.04% at 120 minutes), while no significant changes were observed in the BGC823 group (4.21% ± 0.21% at 10 minutes, 3.77% ± 0.53% at 30 minutes, 4.57% ± 0.36% at 60 minutes, 5.54% ± 0.21% at 120 minutes). At each selected time point, the uptake of BGC823 CLDN18.2 cells (CLDN18.2 positive) was significantly higher than that of BGC823 cells (CLDN18.2 negative) (p < 0.0004). Meanwhile, excessive unlabeled 18B10 significantly blocked the 89 uptake of Figure 16 Zr]Zr-DFO-18B10 (11.47% ± 0.32% at 60 minutes vs. 3.24% ± 0.36%, 13.37% ± 2.04% at 120 minutes vs. 5.64% ± 0.21%) ( CLDN18.2 Figure D). In the cell uptake experiment, the uptake of 89 Zr]Zr-DFO-18B10 by BGC823 89Specificity of [Zr]Zr-DFO-18B10 for CLDN18.2

[0721] 4) Dosimetry estimation

[0722] 89 The biodistribution study of [Zr]Zr-DFO-18B10 demonstrated good pharmacokinetics with a relatively long half-life in vivo (Supporting Information Figure 20A ). The human organ radiation dosimetry is shown in Table 5. The highest dose was received by the liver (0.360 mSv / MBq), followed by the gallbladder wall (0.155 mSv / MBq). The effective dose was 0.0705 mSv / MBq. When a patient was injected with 74 MBq of 89 [Zr]Zr-DFO-18B10 for imaging, the effective radiation dose was less than 5.217 mSv, which is acceptable in conventional nuclear medicine studies. Due to a single intravenous injection of 89 [Zr]Zr-DFO-18B10, the estimated human radiation burden was comparable to that of other 89 Zr-labeled monoclonal antibodies [22 - 24] and was suitable for clinical studies.

[0723] Table 5. Estimation of average absorbed radiation dose

[0724]

[0725] 5) Small animal PET / CT imaging and IHC analysis

[0726] Small animal PET / CT imaging of KM mice at different time points (2, 24, 48, 72, and 120 h) after injection of 89 [Zr]Zr-DFO-18B10 showed high uptake in the heart, liver, and spleen (Supporting Information Figure 20B ). Supporting Information Figure 20C showed the mean standardized uptake value (SUV mean) of some organs measured by the region of interest (ROI). After 2 h, the SUV mean in the heart was 2.57 ± 0.02, the SUV mean in the liver was 2.27 ± 0.01, and the SUV mean in the spleen was 1.86 ± 0.01. The ratio of heart to muscle (H / M) was 20.30 ± 0.91. After 120 h, the SUV means of the heart, liver, and spleen were 0.49 ± 0.01, 1.36 ± 0.02, and 1.21 ± 0.01, respectively, and almost no special uptake was observed in the stomach. The images were consistent with the biodistribution results.

[0727] At 2, 24, 48, 72 h, and 96 h post-injection (p.i.) of the radiotracer, [Zr]Zr-DFO-18B10 was evaluated in real-time and non-invasively by small animal PET / CT imaging 89 ​In vivo distribution and metabolic characteristics of [Zr]Zr-DFO-18B10. Meanwhile, the following three control groups were set up, and the control groups were blocked with excessive 18B10, negative CLDN18.2 expression in BGC823 cells, and 89 non-specific targeting of [Zr]Zr-DFO-IgG (7.4 MBq). By outlining the ROI of the immunological PET images, the average SUV data of BGC823 CLDN18.2 or BGC823 mouse organs were collected ( Figure 17A 、 17B 、17C, 17D and 17E). 89 There was still obvious uptake at the tumor site in the [Zr]Zr-DFO-18B10 group at 96 h p.i. In the 89 BGC823 CLDN18.2 model with [Zr]Zr-DFO-18B10, the average SUV continued to increase within 48 h p.i. and reached the maximum uptake value of 1.09 ± 0.03 at 48 h. In addition, until 96 h p.i., the average SUV of the BGC823 CLDN18.2 model was significantly different from the average SUV of the BGC823 model and the blocking group (1.03 ± 0.03, 0.41 ± 0.05, 0.51 ± 0.07 respectively, P < 0.0002). Using 89 [Zr]Zr-DFO-IgG as a negative control probe, the results showed that in the BGC823 CLDN18.2 model mice, except that the tumor uptake value was slightly higher than that of 89 [Zr]Zr-DFO-18B10 at 2 h after injection (0.51 ± 0.01 vs. 0.37 ± 0.02), at all other time points (24 h, 48 h, 72 h and 96 h) 89 the tumor uptake value of [Zr]-DFO-IgG was significantly lower than that of 89 [Zr]Zr-DFO-18B10 (0.55 ± 0.04 vs. 0.96 ± 0.12, 0.53 ± 0.02 vs. 1.10 ± 0.12, 0.54 ± 0.04 vs. 1.06 ± 0.06 and 0.47 ± 0.01 vs. 1.03 ± 0.01) (Figure 21). Over time, compared with other imaging groups, the uptake of [Zr]Zr-DFO-18B10 in the BGC823 CLDN18.2 model 89 was mainly concentrated in the tumor, and the uptake values in the heart, liver and other organs decreased significantly.

[0728] To compare with the gold standard probe 18 18F-fluorodeoxyglucose (FDG), BGC823 CLDN18.2Tumor-bearing mice with 18F-FDG, and images were collected at 1 h p.i.( Figure 18A ). The results showed that in CLDN18.2-positive mice 18 the uptake of F-FDG was similar to the background uptake. At 48 h p.i., the tumor accumulation of CLDN18.2 Zr]Zr-DFO-18B10 in BGC823 89 mice was approximately 4.15-fold that of the blocked group, 2.27-fold that of the BGC823 group, and 2.05-fold that of the 89 Zr]Zr-DFO-IgG group (mean SUV values were 1.11 ± 0.02, 0.27 ± 0.01, 0.49 ± 0.03, 0.54 ± 0.06)( Figure 18B ). The tumor / heart (T / H) ratio and tumor / muscle (T / M) ratio at each time point after injection of 89 Zr]Zr-DFO-18B10 were significantly higher than the T / H ratio and T / M ratio of other control groups( Figure 18C-18D ), and at 96 h p.i., the T / H ratio and T / M ratio reached their maximum values of 2.37 ± 0.04 and 14.95 ± 1.63, respectively.

[0729] When comparing the BGC823 CLDN18.2 model with other groups, the T / NT value of 89 Zr]Zr-DFO-18B10 was significantly different at 48 h p.i. Compared with previous studies, 18B10 is a humanized antibody with better immunoreactivity to the CLDN18.2 receptor. Secondly, patients need to receive iodine to block the thyroid before and during 124 I imaging, which greatly reduces patient compliance

[21] . Labeling with 89 Zr seems to be more reliable and more available. However, 89 Zr]Zr-DFO-18B10 also showed significantly high background in the liver and spleen, which may be the result of non-specific binding and hepatobiliary clearance. This is very similar to the previous 89 Zr-labeled antibody studies [25, 26]. From the imaging perspective, this not only causes problems in tumor localization in the liver and spleen regions, but may also cause false positive results when "tumor CLDN18.2 expression" occurs, and further leads to incorrect selection of candidate patients for this therapy. Although the interaction between FcγR expressed on immune effector cells and the antibody Fc region can trigger antibody-mediated therapeutic responses, they may not be beneficial in the context of molecular imaging. According to the study, there are three preliminary solutions to reduce non-specific uptake in the liver and spleen [27, 28]. First, use fragments such as Fab, F(ab) 2Using antibody fragments such as these to replace intact antibodies in the preparation of probes not only avoids the interaction of the Fc region with the immune system, but also gives the probe faster pharmacokinetic characteristics. Secondly, another strategy is expected to genetically engineer the Fc region of IgG to eliminate the binding of the Fc region to FcγR on immune cells while maintaining its ability to bind FcRn. Thirdly, a simpler and more modular approach may lie in manipulating the glycans of the Fc region. Additionally, considering the properties of radionuclides, 89 Zr is a radioactive metal ion that is first linked to an antibody through a suitable chelator (lysine groups are commonly used), and then indirectly labels the antibody by non-covalently chelating the radioactive metal ion. Once the antibody is internalized into tumor cells, the antibody undergoes catabolism through lysosomal degradation. The catabolites of the radioactive metal ion chelate remain trapped (residual) inside the cells, causing the radioactive metal (and the PET signal) to accumulate over time in the target tumor tissue and metabolic organs. However, iodine is usually directly labeled on the antibody through a simple and widely used procedure, and most iodine-containing catabolites are non-polar molecules that are rapidly lost from the liver and spleen

[29] . Based on this property of the radionuclide iodine, related studies on 124 I-labeled 18B10 have also been carried out, which may be more suitable for future clinical translation.

[0730] Related studies on 18 F-FDG PET / CT imaging were also carried out as a reference. In the BGC823 CLDN18.2 model, the mean tumor SUV of 89 Zr]Zr-DFO-18B10 at the tumor site was higher than that of 18F-FDG (1.10 ± 0.12 vs. 0.40 ± 0.02), and 89 the T / M value of 18 Zr]Zr-DFO-18B10 was also much higher than that of

[0731] F-FDG (10.23 ± 1.30 vs. 1.80 ± 0.22). CLDN18.2 IHC results showed that CLDN18.2 was highly and uniformly expressed in BGC823 Figure 18E tumors, and BGC823 xenograft tumors were negative for CLDN18.2 ( CLDN18.2 ). The stomachs of BGC823 CLDN18.2 and BGC823 tumor-bearing mice showed a basically positive expression of CLDN18.2. The liver and spleen tissues of both types of tumor-bearing mice did not express CLDN18.2. The IHC results showed that BGC823 89The Zr]Zr-DFO-18B10 probe has the ability to specifically target CLDN18.2. Additionally, strong positive expression of CLDN18.2(+++) was also observed in the gastric mucosa of all mice, but neither PET / CT imaging nor biodistribution showed any significant uptake and retention of the probe in the stomach, probably because the expression of CLDN18.2(+++) is limited to the gastric mucosa in vivo and the monoclonal antibody has difficulty accessing the hidden CLDN18.2 binding epitope in the gastric mucosa

[29] ( Figure 22 ).

[0732] 6) Ex vivo biodistribution

[0733] Figure 19 presents the biodistribution of CLDN18.2 Zr]Zr-DFO-18B10 in BGC823 89 and BGC823 tumor-bearing mice. At 48 h p.i., the liver showed relatively high uptake in all three groups (8.39 ± 0.59% ID / g in the BGC823 CLDN18.2 group, 9.28 ± 0.19% ID / g in the BGC823 group, and 20.96 ± 0.88% ID / g in the blocking group). The uptake value in the spleen was second only to that in the liver (3.54 ± 0.26% ID / g in the BGC823 CLDN18.2 group, 2.08 ± 0.29% ID / g in the BGC823 group, and 1.93 ± 0.24% ID / g in the blocking group). The tumor uptake in BGC823 CLDN18.2 tumor-bearing mice (2.05 ± 0.16% ID / g) was higher than the tumor uptake in BGC823 mice (0.69 ± 0.02% ID / g) and the blocking group (0.72 ± 0.02% ID / g).( Figure 19A ) The tumor / liver (T / L) ratio and tumor / brain (T / B) ratio of BGC823 CLDN18.2 tumors were significantly higher than the T / L ratio and T / B ratio of the other two control groups. (T / L: 0.075 ± 0.001 for BGC823 vs. 0.25 ± 0.003 for the BGC823 CLDN18.2 group vs. 0.035 ± 0.002 for the blocking group; T / B: 16.03 ± 1.66 for BGC823 vs. 40.35 ± 3.68 for the BGC823 CLDN18.2 group vs. 3.01 ± 0.53 for the blocking group, Figure 19B , 19D ). There was no significant difference in the tumor / stomach (T / S) ratio among the three groups (2.00 ± 0.13 for BGC823 vs. 2.04 ± 0.43 for BGC823 CLDN18.2 vs. 1.47 ± 0.50 for the blocking group,Figure 19C ). Consistent with the PET / CT results, the in vitro biodistribution data at 48 h p.i. showed that 89 Zr]Zr-DFO-18B10 accumulated in the liver and spleen, and the liver uptake in the blocking group was significantly higher than that in the other two groups, probably because the tumor uptake was blocked, leading to the probe directly entering the liver through the bloodstream for metabolism. The differences in tumor uptake values among the three groups also reflected 89 Zr]Zr-DFO-18B10's excellent specificity for CLDN18.2-positive tumors.

[0734] Example 6.6 Conclusion

[0735] GMP-grade Zr-labeled anti-CLDN18.2 recombinant humanized antibody 18B10 was successfully prepared. 89 Zr]Zr-DFO-18B10 showed good specificity and rapid tumor accumulation at the cellular level and remained positive from 24 to 96 h. The 89 Zr]Zr-DFO-18B10 provided a promising molecular probe for real-time detection of the therapeutic effects of human therapeutic antibodies. It also made it possible for future screening and efficacy evaluation of patients for CLDN18.2-targeted therapies. 89 The references for Example 6 can be found in Table 8.

[0736] The references for Example 6 can be found in Table 8.

[0737] Example 7

[0738] Example 7.1 177 Radioactive Labeling and Quality Control of Lu-18B10

[0739] TZ-DOTA was synthesized with TZ-NHS and TZ-NHS at pH 8.4, and TZ-DOTA reacted with 177 Lu solution at pH 5.5 - 6 at 60 °C for about 10 minutes to obtain TZ- 177 Lu. TCO-NHS was mixed with antibody precursor 18B10 to obtain 18B10-TCO. TZ- 177 Lu and 18B10-TCO were reacted at 37 °C for 5 minutes (300 μg antibody per 1 mCi 177 Lu) and purified by a PD-10 column. The final product 177 Lu-DOTA-18B10 was obtained. Figure 23 A shows 177 the steps and conditions of Lu labeling. 177 The labeling rate, radiochemical purity and yield of Lu-labeled 18B10 were 85.61%, 98.87% and 50% respectively. The product had good stability, and the radiochemical purity was still greater than 95% at 168 h after labeling (Figure 23 B).

[0740] Example 7.2 Small Animal PET Imaging

[0741] Construct BGC823 / AGS CLDN18.2-positive and BGC823 / AGS CLDN18.2-negative nude tumor-bearing mouse models (balb / c nude mice, female, 5 weeks old), and then perform 177 Lu-DOTA-18B10 imaging by microPET (Super Nova PET / CT, China Pingsheng Company). Set up a blocking control group (n = 4) in BGC823 CLDN18.2 mice. The imaging time points are 4 h, 24 h, 48 h, 72 h, and 144 h after injection. Inject 300 μCi 177 Lu-DOTA-18B10 into each mouse. In the positive group, the imaging is good, and the tumor uptake is obvious, reaching the peak at about 72 hours, and can be blocked ( Figure 24A-24B ). There is no significant tumor uptake in the negative group ( Figures 25-26 ). Perform CLDN18.2 immunohistochemical staining on the tumor tissues of the mice dissected at the end of imaging. The CLDN18.2 expression is positive in the positive imaging group and negative in the negative imaging group. The histochemical results are consistent with the imaging results ( Figure 27 ).

[0742] Example 7.3 Cellular Uptake

[0743] Resuspend the digested BGC823 / AGS CLDN18.2 cells and BGC823 / AGS cells with an appropriate concentration of RPMI-1640 medium, and then add them to a 24-well plate, 1 ml per well, and incubate overnight. The next day, add 177 Lu-DOTA-18B10 to each well at 0.037 MBq per well (add 500 μg of unlabeled precursor to the blocking group at the same time, n = 4). Incubate the cells in a 5% CO 2 incubator at 37 °C for 10 minutes, 30 minutes, 60 min, and 120 minutes (60 minutes and 120 minutes for the blocking group), wash and lyse, then collect and test with a gamma counter. The cell uptake results are expressed as the percentage of injected activity (% IA) per 10 5 cells. The results show that BGC823 / AGS CLDN18.2 positive cells have significant uptake of 177 Lu-DOTA-18B10, which is significantly different from the negative group and the blocking group ( Figure 28 ).

[0744] Example 7.4 Saturation Binding Experiment

[0745] The digested and centrifuged AGS CLDN18.2 cells were resuspended in RPMI - 1640 medium to an appropriate concentration and then added to a 24 - well plate, 1 ml per well, and incubated overnight. The next day, 177 Lu - DOTA - 18B10 was added to the wells at different concentration gradients (8 concentrations from 1.25 to 160 nmol / L) (n = 4), and incubated in a 5% CO 2 incubator at 37 °C for 120 minutes. After washing and lysis, the cells were collected and tested with a gamma counter. The values were fitted using GraphPad Prism. The results showed that the Kd constant in the saturation binding test was 12.87, indicating that the probe could specifically target the CLDN18.2 molecule( Figure 29A ).

[0746] Example 7.5 Pharmacokinetics Experiment

[0747] Pharmacokinetic experiments were performed on normal KM mice (female, n = 6) by injecting 0.56 MBq 177 Lu - DOTA - 18B10 into the tail vein. At 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 192 hours after injection, periorbital venous blood was collected through a capillary and radioactivity was detected with a gamma counter. The collected data were analyzed by GraphPad Prism to calculate the pharmacokinetics. The results showed that 177 Lu - DOTA - 18B10 had good pharmacokinetics, with a half - life (slow) of 22.29 h and a half - life (fast) of 0.4512 h( Figure 29B ).

[0748] Example 7.6 Biodistribution

[0749] Lu - DOTA - 18B10 was injected into the mice through the tail vein, and the mice were anesthetized and sacrificed at predetermined time points. The main organs and tissues of the mice, such as blood, stomach, intestine, bone, muscle, heart, lung, spleen, liver, kidney, and tumor, were collected and radioactivity was detected with a gamma counter. The biodistribution results were expressed as a percentage of the injected dose per gram (%ID / g). Each mouse was injected with 0.44 - 0.56 MBq of 177 Lu - DOTA - 18B10, and tissue samples were taken at 8 h, 24 h, 48 h, 72 h, and 144 h after injection (n = 4). In the blocking group, an equal dose of 177 Lu - DOTA - 18B10, and at 177Lu-DOTA-18B10 was injected together with unlabeled 18B10 at 50 μg / g body weight, and samples were taken at the 48 h time point (n = 4). The results showed that the biodistribution was consistent with PET imaging. The tumor distribution was high and could be blocked by the unlabeled precursor ( Figure 30 and 31 ).

[0750] Example 7.7 Therapeutic Experiment

[0751] Three groups of BGC823 / AGS CLDN18.2-positive tumor-bearing mice (n = 6) were prepared, with the tumor volume being approximately 100 mm 3 , and separately, 300 μCi / 150 μCi 177 Lu-DOTA-18B10 was injected into the tail vein as the treatment group, and an equal volume of PBS was injected as the blank control group. The tumor volume and mouse body weight were measured and recorded every other day until 16 to 17 days after injection. The results showed that for both BGC823 CLDN18.2 or AGS CLDN18.2 model mice, 177 Lu-DOTA-18B10 had a significant therapeutic effect ( Figure 32 ). The efficacy of the 300 μCi group was better than that of the 150 μCi group, and both were better than the blank control group ( Figure 32 ). HE staining of the tumor tissues sampled on the last day also clearly showed obvious tumor tissue damage in the treatment group ( Figure 34 ). In addition, HE staining of all important organs (heart, liver, spleen, lung, kidney, stomach, intestine, and muscle) on the last day and the weight records of all three groups showed that 177 Lu-DOTA-18B10 treatment had no significant effect on the body weight and normal organs of the mice ( Figure 35 ).

[0752] Example 8

[0753] Example 8.1 Abstract

[0754] Claudin 18.2 (CLDN18.2) has made breakthrough progress in clinical research due to its highly selective expression in tumor cells and is expected to be incorporated into routine tumor diagnosis and treatment. Methods: In this study, a molecular probe for PET imaging and treatment was developed by labeling the scFv-Fc antibody specifically targeting CLDN18.2 with radionuclides ( 124 I and 177 Lu), and the diagnostic and therapeutic potential in a tumor-bearing model was evaluated. Results: The molecular probes 124 I-SF106 and 177Lu-DOTA-SF106 has high radiochemical purity (RCP, 95.63 ± 2.90% and 97.05 ± 1.1%), and shows good stability in phosphate-buffered saline and 5% human serum albumin (RCP, 92.44 ± 4.68% and 91.03 ± 2.42% at 120 h). In cells expressing CLDN18.2 124 the uptake of 177Lu-SF106 has good targeting and specificity, and the dissociation constant is 17.74 nM. 124 177Lu-SF106 microPET imaging shows the maximum standardized uptake value (SUVmax), reaching peak uptake in CLDN18.2-positive tumors at 48 h after injection, and significantly higher than that in CLDN18.2-negative tumors (1.83 ± 0.02 vs. 1.23 ± 0.04, p < 0.001). The tumors in CLDN18.2-positive BGC823 CLDN18.2 mouse models have significantly higher SUVmax ratios of tumor to muscle and liver than those in CLDN18.2-negative BGC823 mouse models. 124 The 177Lu-SF106 dosimetry study shows that the effective human dose is 0.0705 mSv / MBq, which is the medical safety standard for further clinical applications. The results of the preliminary treatment experiment show that 3 MBq of 177 177Lu-DOTA-SF106 can significantly inhibit tumor growth in tumor-bearing mice expressing CLDN18. Conclusion: These results indicate that the radionuclide-labeled scFv-Fc molecular probe ( 124 177Lu-SF106 and 177 177Lu-DOTA-SF106) provides new possibilities for the diagnosis and treatment of CLDN18.2-positive cancer patients in clinical practice. Figure 36 The graphical abstract is shown.

[0755] Example 8.2 Introduction

[0756] Gastric cancer (GC) is one of the most common malignancies worldwide. In developing countries, approximately 80% of gastric cancer patients are diagnosed at an advanced stage (1 - 3). For these patients, radiotherapy and chemotherapy are the main treatment methods, aiming to improve symptoms and prolong survival time. However, the prognosis is poor, and the median survival time is only 4 - 13 months (4, 5). Targeted therapy is an emerging treatment method, which is expected to improve patient survival rate and has valuable clinical applications (6).

[0757] CLDN18.2 has been identified as a therapeutic antibody target. In the study by Jin Haek et al., 74.4% of the study patients showed expression of CLDN18.2 (7). CLDN18.2 is a subtype of the claudin protein family and is present in the TJ supramolecular complex of normal gastric mucosal cells (8-10). However, malignant tumor cells lose their polarity, expose the CLDN18.2 epitope and allow antibody binding in vivo (11, 12). Zolbetuximab (IMAB362) is a novel chimeric IgG1 antibody that has shown significant improvement in progression-free survival and overall survival in patients with CLDN18.2-positive gastric cancer or gastroesophageal junction cancer when used in combination with chemotherapy (13, 14).

[0758] CLDN18.2 expression in clinical studies and diagnosis is mainly evaluated by immunohistochemistry and has inherent limitations such as the extent of tissue biopsy, the quality of tumor sections, and tumor heterogeneity, which may not fully reflect the true level of CLDN18.2 expression in patients.

[0759] Current clinical studies use immunohistochemistry (IHC) methods to evaluate CLDN18.2 expression and are thus inherently limited in several aspects, including the quality and quantity of the required tumor slides, tumor heterogeneity, and the challenge of performing a therapeutic biopsy. However, it is inherently limited in several aspects, including the size and location of the tissue biopsy sample, the quality and quantity of the required tumor slides, and tumor heterogeneity. Therefore, a real-time controllable detection technique for accurately detecting the expression and distribution of CLDN18.2 throughout the body at baseline and during treatment would be valuable. Positron emission tomography (PET) is a widely used clinical diagnostic method that detects the distribution of target molecules in patients using a radionuclide probe. Previous reports on the use of 124 I-trastuzumab PET radioligand to detect HER2-positive lesions in gastric cancer patients have confirmed its feasibility (15).

[0760] Previously, a molecular probe based on the CLDN18.2-specific antibody 5C9 was reported, using 124I. Modification of Cy5.5 and FD1080. This probe can detect CLDN18.2 expression in solid tumors and guide surgical treatment of CLDN18.2 overexpressing tumors (16). Compared with full-length antibodies, the single-chain variable fragment-crystallizable protein (scFv-Fc) structure provides greater editability, enhances tissue permeability, accelerates blood clearance, and reduces background noise in tumor imaging (17-19). Fusion of the intact Fc region endows scFv-Fc antibodies with pharmacokinetics comparable to those of full antibodies (20). In this study, a novel single-chain fragment variable fragment crystallin antibody (Hu18B10HaLa scFv-Fc, also referred to as SF106 in this study) was successfully developed and radiolabeled with the radionuclide iodine-124 to explore its potential in PET imaging. Additionally, a therapeutic molecular probe 177 Lu-DOTA-SF106 was obtained by labeling with the radionuclide lutetium-177, and its inhibitory effect on tumor growth was preliminarily tested in a CLDN18.2-expressing mouse tumor model.

[0761] Example 8.3 Materials and Methods

[0762] 1) Antibody, cell culture, and tumor-bearing model construction

[0763] The scFv-Fc antibody targeting CLDN18.2 was generated using hybridoma technology. The gastric cancer cell lines BGC823 and AGS, as well as BBGC823 with high-level expression of CLDN18.2 CLDN18.2 and AGS CLDN18.2 cells were mainly used in this study. In vitro cell uptake experiments were performed using unlabeled SF106 antibody to block the uptake of radiolabeled SF106 antibody. Additional information is provided in the supplementary materials.

[0764] 2) Radionuclide labeling and its in vitro studies

[0765] In this study, 124 I-SF106 and 177 Lu-DOTA-SF106 probes were constructed by radiolabeling the SF106 antibody with the radionuclides 124 I and 177 Lu, respectively. The radiochemical purity and in vitro stability of the labeled products were studied. Detailed procedures are provided in the supplementary materials.

[0766] 3) Radioimmunological imaging and radionuclide therapy studies

[0767] All animal experiments were conducted in accordance with the guidelines of the Peking University Institutional Animal Care and Use Committee (EAEC 202201). GBC823 and BGC823 CLDN18.2 model was injected 124 with I-SF106, and PET static imaging was performed at different times after injection. The AGS CLDN18.2 model was randomly divided into an experimental group and a control group. The experimental group was injected 177 with Lu-DOTA-SF106, and the control group was injected with the same volume of PBS for tumor inhibition experiments.

[0768] 4) Statistical analysis

[0769] All data are expressed as mean ± standard deviation (SD). The Mann-Whitney t test was performed for in vitro studies. Two-way ANOVA multiple comparison analysis was used to analyze the data of different groups, and a P value less than 0.05 was considered statistically significant. According to the following values, significance is indicated by asterisks (*): P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

[0770] Example 8.4 Results

[0771] 1) Construction and molecular detection of the SF106 antibody

[0772] The scFv-Fc (SF106) antibody (SEQ ID NO: 49) was constructed by fusing scFv with the human Fc of IgG1. The scFv was designed according to the VH-linker-VL orientation. The VH and VL sequences were from Hu18B10HaLa, and the linker was (GGGGS)4. ( Figure 37A ). The molecular weight of the SF106 antibody was further verified by MALDI-TOF-MS to be 108,303 Da ( Figure 37B ). SDS-page testing showed that SF106 had only one band at 108 kDa. At the same time, a molecular fragment close to 55 kDa was detected after denaturation at 90 °C for 10 minutes ( Figure 42B ). FACS analysis of the binding of SF106 to HEK293-human CLDN18.2-hi cells showed a half-maximal effective concentration (EC50) value of 11.31 nM ( Figure 37C ).

[0773] 2) 124 Radioactive labeling and in vitro stability of I-SF106

[0774] Figure 38 Small figure A shows the 124 I-labeling process of the SF106 antibody. The test was performed by non-radioactive natural iodine ( nat I) labeling, and the product was tested by MALDI-TOF, with an average molecular weight of 108963 Da, an increase of approximately 360 Da relative to the SF106 antibody. At the same time, the labeled antibody molecules were verified by SDS-PAGE ( Figure 42A and 42B ). 124 The labeling rate of 124 I-SF106 was 95.63 ± 2.90% (n = 3), and after purification, the radiochemical purity (RCP) of Figure 42C and 42D I-SF106 was 98.18 ± 0.93% (n = 3) ( 124 The stability experiment showed that the radiochemical purities of Figure 37B I-SF106 incubated in 0.01 M PBS and 5% HSA solution for 120 h were 92.44 ± 4.68% (n = 3) and 91.03 ± 2.42% (n = 3), respectively (

[0775] 3) 124 In vitro cellular uptake and biological evaluation of

[0776] Flow cytometry results showed that 71.0% of BGC823 CLDN18.2 cells had a positive signal for the CLDN18.2 antibody (1D5) ( Figure 37C ). At all selected time points in this experiment, the uptake of CLDN18.2 I-SF106 in BGC823 124 cells was significantly higher than the uptake of 124 I-SF106 in BGC823 cells (0.47 ± 0.02% vs. 0.2 ± 0.01% at 10 minutes, 0.67 ± 0.03% vs. 0.20 ± 0.05% at 30 minutes, 0.93 ± 0.02% vs. 0.17 ± 0.02% at 60 minutes, 0.96 ± 0.04% vs. 0.22 ± 0.09% at 120 minutes, P < 0.001).

[0777] The uptake of CLDN18.2 I-SF106 in BGC823 124 cells increased with incubation time, and there was no significant change in BGC823 cells. After pre-incubation for 120 minutes, the uptake of CLDN18.2 I-SF106 in BGC823 124The uptake of I-SF106 was significantly reduced (untreated group vs. blocked group: 0.96 ± 0.04% vs. 0.21 ± 0.02%, P < 0.001) (Figure 37D).

[0778] Using BGC823 CLDN18.2 Cells were determined by cell saturation binding assay 124 The binding constant (K d of I-SF106 to the CLDN18.2 receptor was 17.74 nmol / L ( Figure 42E ).

[0779] 4) 124 Dosimetry estimation of I-SF106

[0780] The study showed that in vivo 124 the half-life of I-SF106 in the elimination phase was 27.68 h, and the half-life in the distribution phase was 0.6354 h ( Figure 42F ). The organ radiation dosimetry in humans was estimated using the OLINDA / EXM 2.0 software package based on 124 the biodistribution data of I-SF106. According to Table 6, osteoblasts received the highest dose (0.513 mSv / MBq), followed by the lungs (0.234 mSv / MBq). The radiation doses to the kidneys and liver were 0.137 mSv / MBq and 0.187 mSv / MBq, respectively, and the effective dose was 0.0873 mSv / MBq, which is acceptable in nuclear medicine research.

[0781] 5) In tumor-bearing mice 124 MicroPET / CT imaging of I-SF106

[0782] The distribution and metabolic characteristics of I-SF106 were evaluated by microPET / CT imaging at different time points (4, 24, 48, 72, 96, and 120 h) after injection in a tumor-bearing mouse model 124 of I-SF106. Figure 39 The maximum intensity projection (MIP) images of microPET / CT are shown. Significant accumulation of I-SF106 was observed in CLDN18.2 the BGC823 tumors expressing CLDN18.2 124 showing good imaging results from 24 to 120 h after injection. In the BGC823 CLDN18.2 tumor-bearing mouse model 124 the signal of I-SF106 had peak uptake at 48 h, and the highest SUV maximum was 1.83 ± 0.02. In contrast, BGC823 tumor-bearing mice without CLDN18.2 expression showed lower 124The concentration of I-SF106, where the maximum SUV value at 48 h was 1.23 ± 0.04. In addition, the blocking group was injected with unlabeled SF106 antibody, which effectively reduced BGC823 expressing CLDN18.2 CLDN18.2 In the tumors of tumor-bearing mice 124 The concentration of I-SF106( Figure 40 Panel A; Figure 43 Panel A, Panel B and Panel C). In normal tissues, 124 I-SF106 was mainly detected in the liver and blood. At the same time, the 124 I-SF106 and BGC823 in KM mice CLDN18.2 in the tumor-bearing mouse model 125 The biodistribution of I-SF106. The results showed that the molecular probe was mainly distributed in the blood and gradually decreased over time, and 125 the metabolic trend of I-SF106 in tumors was consistent with the imaging results( Figure 43 Panel D and Panel E).

[0783] For imaging, the anti-CLDN18.2 monoclonal antibody Hu18B10HaLa specifically targeting CLDN18.2 was co-injected for target blocking, while 124 I-IgG was used as a control group( Figure 44 Panel A). Co-injection of Hu18B10HaLa significantly reduced the tumor uptake of 124 I-SF106. The maximum SUV value in tumors at each time point was significantly lower than 124 the maximum SUV value in the I-SF106 group, while in 124 the I-IgG group, the maximum SUV value in tumors gradually decreased with the imaging time( Figure 44 Panel B).

[0784] In addition, at each imaging time, the tumor-to-muscle SUV maximum ratio of LDN18.2 I-SF...

Claims

1. A method for detecting or visualizing CLDN18.2 protein at a site of interest in an object, the method comprising: a) administering to the object a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate; and b) performing radionuclide imaging on the object at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain an image; and c) determining or visualizing the presence of the CLDN18.2 protein in the site of interest of the object based on the image, wherein the presence and / or location of radionuclide uptake above background indicates the presence and / or location of the CLDN18.2 protein, and wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

2. The method according to claim 1, wherein the object has not been treated with a CLDN18.2-targeted therapy or has not received the CLDN18.2-targeted therapy.

3. A method for diagnosing an object with a CLDN18.2-related disease, the method comprising: a) administering to the object a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate; and b) performing radionuclide imaging on the object at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain an image; c) diagnosing the object's CLDN18.2-related disease based on the presence and / or location of the CLDN18.2 protein in the site of interest of the object as determined from the image, wherein the presence and / or location of radionuclide uptake above background indicates the presence and / or location of the CLDN18.2 protein, and wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

4. The method according to claim 3, wherein the object has not been treated with a CLDN18.2-targeted therapy or has not received the CLDN18.2-targeted therapy.

5. A method for identifying an object in need who may respond to a CLDN18.2-targeted therapy, the method comprising: a) administering to the object a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate; and b) performing radionuclide imaging on the object at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain an image; c) identifying that the object may respond to the CLDN18.2-targeted therapy based on the presence and / or location of the CLDN18.2 protein in the site of interest of the object as determined from the image, wherein the presence and / or location of radionuclide uptake above background indicates the presence and / or location of the CLDN18.2 protein, and wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

6. The method according to claim 5, wherein the subject has not been treated with a CLDN18.2 - targeted therapy or has not received the CLDN18.2 - targeted therapy.

7. The method according to any one of the preceding claims, wherein the diagnostic radionuclide is selected from the group consisting of: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154 ", 1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac.

8. The method according to any one of the preceding claims, wherein the method further comprises: d) administering a therapeutically effective amount of a CLDN18.2 - targeted therapy to the subject identified as having the CLDN18.2 protein at the site of interest, diagnosed with a CLDN18.2 - related disease, and / or identified as likely to respond to a CLDN18.2 - targeted therapy.

9. The method according to claim 8, wherein the CLDN18.2 - targeted therapy comprises a therapy selected from the group consisting of anti - CLDN18.2 antibody therapy, CLDN18.2 - targeted compounds, CLDN18.2 - targeted nucleic acid therapy, CLDN18.2 - targeted peptides, CLDN18.2 - targeted cell therapy, and CLDN18.2 - targeted gene therapy.

10. The method according to claim 9, wherein the anti-CLDN18.2 antibody therapy comprises an anti-CLDN18.2 antibody-therapeutic radionuclide conjugate, and the therapeutic radionuclide is selected from the group consisting of: 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 124 I, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb.

11. The method according to claim 10, wherein the therapeutic radionuclide is 177 Lu or 124 I.

12. The method according to claim 10 or 11, wherein the therapeutic radionuclide is the same as the diagnostic radionuclide.

13. The method according to claim 12, wherein both the therapeutic radionuclide and the diagnostic radionuclide are 177 Lu or 124 I.

14. The method according to claim 10, wherein the anti - CLDN18.2 antibody - diagnostic radionuclide conjugate is the same as the anti - CLDN18.2 antibody - therapeutic radionuclide.

15. The method according to any one of claims 8 to 14, wherein the therapeutically effective amount is at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% of the detectable effective amount.

16. A method of monitoring the progression of a CLDN18.2 - related disease in a subject during a monitoring period, the method comprises: d) administering a detectable effective amount of an anti - CLDN18.2 antibody - radionuclide conjugate to the subject after the monitoring period; e) performing radionuclide imaging on the subject at a subsequent time point after administering the anti - CLDN18.2 antibody - radionuclide conjugate to obtain a post - monitoring image; and f) comparing the post - monitoring image with a pre - monitoring image to determine a change in the level of the CLDN18.2 protein during the monitoring period based on the images, wherein the level of the radionuclide conjugate above background indicates the level of the CLDN18.2 protein, wherein the change indicates the presence or absence of disease progression, wherein the anti - CLDN18.2 antibody - radionuclide conjugate comprises an anti - CLDN18.2 antibody - diagnostic radionuclide conjugate.

17. The method according to claim 16, wherein the pre - monitoring image is obtained from the subject before the monitoring period by: administering an effective amount of the anti - CLDN18.2 antibody - radionuclide conjugate, and then performing radionuclide imaging on the subject at a subsequent time point after administering the anti - CLDN18.2 antibody - radionuclide conjugate to obtain the pre - monitoring image.

18. The method according to claim 16, wherein an increase in the CLDN18.2 level during the monitoring time period indicates disease progression, and the absence of such an increase in the CLDN18.2 level during the monitoring time period indicates the absence of disease progression.

19. The method according to claim 16, wherein the subject has not been treated with or has not received a CLDN18.2-targeted therapy.

20. The method according to claim 3 or 16, wherein the disease is a tumor.

21. The method according to claim 16, wherein the progression is metastasis of the tumor.

22. The method according to claim 16, wherein the level of the CLDN18.2 protein comprises the amount, distribution, and / or location of the CLDN18.2 protein.

23. The method according to claim 16, wherein the subject is at risk of metastasis.

24. A method of monitoring the therapeutic efficacy of a subject having a CLDN18.2-related disease and who has been treated with a therapy for a certain treatment time period, the method comprising: d) administering to the subject a detectable effective amount of an anti-CLDN18.2 antibody-radionuclide conjugate after the treatment time period; e) performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain a post-treatment image; and f) comparing the post-treatment image with a pre-treatment image to determine a change in the level of the CLDN18.2 protein during the treatment time period based on the images, wherein the level of the radionuclide conjugate above background indicates the level of the CLDN18.2 protein, wherein the change indicates the presence or absence of the therapeutic efficacy, wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

25. The method according to claim 24, wherein the pre-treatment image is obtained from the subject prior to the treatment time period by: administering an effective amount of the anti-CLDN18.2 antibody-radionuclide conjugate, and subsequently performing radionuclide imaging on the subject at a subsequent time point after administering the anti-CLDN18.2 antibody-radionuclide conjugate to obtain the pre-treatment image.

26. The method according to claim 24 or 25, wherein an increase in the CLDN18.2 level during the treatment time period indicates the absence of therapeutic efficacy or poor therapeutic efficacy, and / or wherein the absence of such an increase in the CLDN18.2 level during the treatment time period indicates the presence of therapeutic efficacy or positive therapeutic efficacy.

27. The method according to claim 26, wherein the method further comprises: a) when determining poor therapeutic efficacy, increasing the dose of the therapy or stopping the therapy; or b) when determining positive therapeutic efficacy, advising the subject to continue the therapy.

28. The method according to claim 24, wherein the therapy comprises a CLDN18.2-targeted therapy.

29. The method according to claim 28, wherein the CLDN18.2-targeted therapy comprises a therapy selected from the group consisting of: anti-CLDN18.2 antibody therapy, CLDN18.2-targeted compounds, CLDN18.2-targeted nucleic acid therapy, CLDN18.2-targeted peptides, CLDN18.2-targeted cell therapy, and CLDN18.2-targeted gene therapy.

30. The method according to claim 28, wherein the method further comprises recommending that the subject switch to a therapy other than the CLDN18.2-targeted therapy when the CLDN18.2 level after treatment is lower than the corresponding reference level or is reduced by at least 40% (or at least 50%, 60%, 70%, 80%, 90%, or 95%) relative to the CLDN18.2 level before treatment.

31. The method according to claim 24, wherein the therapy is not a CLDN18.2-targeted therapy.

32. The method according to any one of claims 1 to 31, wherein the diagnostic radionuclide is selected from the group consisting of: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154 " 1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac.

33. The method according to claim 32, wherein the diagnostic radionuclide is 124 I, 123 I, 131 I, 89 Zr or 177 Lu.

34. The method according to claim 33, wherein the anti-CLDN18.2 antibody-radionuclide conjugate is administered at a dose in the range of 0.5 mCi to 10 mCi (18.5 MBq to 370 MBq).

35. The method according to claim 33 or 34, wherein the radionuclide imaging is performed 2 hours after administration of the anti-CLDN18.2 antibody-radionuclide conjugate to the subject, or at a time point between 2 hours and 144 hours after administration of the anti-CLDN18.2 antibody-radionuclide conjugate to the subject.

36. The method according to any one of the preceding claims, wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises heavy chain HCDR1, HCDR2, and HCDR3 sequences and / or light chain LCDR1, LCDR2, and LCDR3 sequences, wherein: the HCDR1 sequence comprises GYNMN (SEQ ID NO:1) or a homologous sequence having at least 80% sequence identity therewith; The HCDR2 sequence includes X 1 IDPYYX 2 X 3 TX 4 YNQKFX 5 G (SEQ ID NO:32) or a homologous sequence having at least 80% sequence identity thereto; The HCDR3 sequence includes X 6 X 7 X 8 GNAFDY (SEQ ID NO:33) or a homologous sequence having at least 80% sequence identity thereto; The LCDR1 sequence includes KSSQX 9 LX 10 NX 11 GNX 12 KNYLT (SEQ ID NO:34) or a homologous sequence having at least 80% sequence identity thereto; The LCDR2 sequence includes WASTRX 13 S (SEQ ID NO: 35) or a homologous sequence having at least 80% sequence identity thereto; The LCDR3 sequence includes QNDYSX 15 PX 16 T (SEQ ID NO:36) or a homologous sequence having at least 80% sequence identity therewith; where X 1 is N or Y or H, X 2 is G or V, X 3 is A or G or T, X 4 is R or T or S, X 5 is K or R, X 6 is S or M, X 7 is Y or F, X 8 is Y or H, X 9 is S or N, X 10 is L or F, X 11 is S or N, X 12 is Q or L, X 13 is E or K, X 15 is F or Y and X 16 is F or L.

37. The method according to any one of the preceding claims, wherein the CLDN18.2-related disease is a CLDN18.2-positive tumor or a CLDN18.2-positive non-cancerous lesion (e.g., a gastric lesion).

38. The method according to claim 20, wherein the tumor is gastric cancer, ovarian cancer, pancreatic cancer, cholangiocarcinoma, colorectal cancer, lung cancer, or esophageal adenocarcinoma.

39. The method according to claim 37, wherein the gastric lesion is a gastric ulcer.

40. The method according to any one of claims 1 to 6 and 19, wherein the CLDN18.2-targeted therapy comprises a therapy that specifically targets CLDN18.2 or cells expressing CLDN18.

2.

41. The method according to any one of claims 1 to 6 and 19, wherein the CLDN18.2-targeted therapy comprises a therapy selected from the group consisting of: anti-CLDN18.2 antibody therapy, CLDN18.2-targeted compound, CLDN18.2-targeted nucleic acid therapy, CLDN18.2-targeted peptide, CLDN18.2-targeted cell therapy, and CLDN18.2-targeted gene therapy.

42. The method according to claim 41, wherein the anti-CLDN18.2 antibody therapy comprises an anti-CLDN18.2 antibody-therapeutic radionuclide conjugate, and the therapeutic radionuclide is selected from the group consisting of: 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 124 I, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb.

43. The method according to claim 42, wherein the therapeutic radionuclide is 177 Lu or 124 I.

44. The method according to any one of the preceding claims, wherein the CLDN18.2-targeted cell therapy comprises CAR T cells, TCR T cells, or CAR NK cells that target CLDN18.

2.

45. The method according to any one of the preceding claims, wherein the site of interest has or is suspected of having a tumor or gastric lesion.

46. The method according to any one of the preceding claims, wherein the site of interest is the whole body.

47. The method according to any one of the preceding claims, wherein the radionuclide imaging comprises positron emission tomography (PET) or SPECT.

48. The method according to any one of the preceding claims, wherein the radionuclide imaging is combined with CT, MR, or ultrasound.

49. A kit for the method according to any one of the preceding claims, the kit comprising an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

50. A kit for: 1) diagnosing a subject with a CLDN18.2-related disease; 2) identifying a subject in need who may respond to a CLDN18.2-targeted therapy; 3) monitoring the progression of a CLDN18.2-related disease in a subject during a monitoring period; and / or 4) monitoring the therapeutic efficacy of a subject with a CLDN18.2-related disease who has been treated with a therapy for a certain treatment period, the kit comprising an anti-CLDN18.2 antibody-diagnostic radionuclide conjugate.

51. The kit according to claim 49 or 50, further comprising a therapeutically effective amount of an anti-CLDN18.2 antibody-therapeutic radionuclide conjugate.

52. The kit according to claim 51, wherein the therapeutic radionuclide is 177 Lu or 124 I.

53. The kit according to claim 51 or 52, wherein the therapeutic radionuclide is the same as the diagnostic radionuclide.

54. The kit according to claim 53, wherein both the therapeutic radionuclide and the diagnostic radionuclide are 177 Lu or 124 I.

55. The kit according to any one of claims 49 to 54, wherein the kit further comprises instructions for providing a detectable effective amount of the anti-CLDN18.2 antibody-diagnostic radionuclide conjugate and a therapeutically effective amount of the anti-CLDN18.2 antibody-therapeutic radionuclide conjugate.

56. The kit according to claim 55, wherein the therapeutically effective amount is at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% of the detectable effective amount.

57. The method according to any one of claims 1 to 48 or the kit according to any one of claims 49 to 56, wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises: a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:3, and an HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:7, and an HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:2, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:8; a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:9, and an HCDR3 comprising the sequence of SEQ ID NO:11; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:10, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:6; a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1 an HCDR2 comprising the sequence of SEQ ID NO:19, and an HCDR3 comprising the sequence of SEQ ID NO:21; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:16, and an LCDR3 comprising the sequence of SEQ ID NO:18; or a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO:1 an HCDR2 comprising the sequence of SEQ ID NO:22, and an HCDR3 comprising the sequence of SEQ ID NO:5; the light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO:20, an LCDR2 comprising the sequence of SEQ ID NO:4, and an LCDR3 comprising the sequence of SEQ ID NO:

6.

58. The method according to any one of claims 1 to 48 or the kit according to any one of claims 49 to 56, wherein the heavy chain variable region further comprises one or more of heavy chain HFR1, HFR2, HFR3, and HFR4, and / or the light chain variable region further comprises one or more of light chain LFR1, LFR2, LFR3, and LFR4, wherein: The HFR1 includes QVQLVQSGAEVKKPGASVKVSCKASGYX 17 FT (SEQ ID NO:54) or a homologous sequence having at least 80% sequence identity thereto; The HFR2 includes WVX 18 QAPGQGLEWX 19 G (SEQ ID NO:55) or a homologous sequence having at least 80% sequence identity thereto; The HFR3 sequence includes RVTX 20 TIDKSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 56) or a homologous sequence having at least 80% sequence identity thereto; the HFR4 comprises WGQGTTVTVSS (SEQ ID NO:57) or a homologous sequence having at least 80% sequence identity therewith; The LFR1 includes DIVMTQSPDSLAVSLGERATX 21 NC (SEQ ID NO:58) or a homologous sequence having at least 80% sequence identity thereto; the LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO:59) or a homologous sequence having at least 80% sequence identity therewith; The LFR3 includes GVPDRFX 22 GSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:60) or a homologous sequence having at least 80% sequence identity therewith; and the LFR4 comprises FGGGTKVEIK (SEQ ID NO:61) or a homologous sequence having at least 80% sequence identity therewith; where X 17 is T or S, X 18 is R or K, X 19 is M or I, X 20 is M or L, X 21 is I or M, and X 22 is S or T.

59. The method according to claim 58, wherein: the HFR1 comprises a sequence selected from the group consisting of SEQ ID NO:62 and 63, the HFR2 comprises a sequence selected from the group consisting of SEQ ID NO:64 and 65, the HFR3 comprises a sequence selected from the group consisting of SEQ ID NO:66 and 67, the HFR4 comprises the sequence of SEQ ID NO:57, the LFR1 comprises a sequence from the group consisting of SEQ ID NO:68 and 69, the LFR2 comprises the sequence of SEQ ID NO:59, the LFR3 comprises a sequence selected from the group consisting of SEQ ID NO:70 and 71, and the LFR4 comprises the sequence of SEQ ID NO:

61.

60. The method according to claim 58 or 59, wherein the heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:45, and SEQ ID NO:47, and homologous sequences having at least 80% sequence identity therewith but retaining the specific binding affinity for CLDN18.

2.

61. The method according to any one of claims 58 to 60, wherein the light chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, and homologous sequences having at least 80% sequence identity therewith but retaining the specific binding affinity for CLDN18.

2.

62. The method according to any one of claims 58 to 61, wherein: The heavy chain variable region comprises the sequence of SEQ ID NO:23, and the light chain variable region comprises the sequence of SEQ ID NO:24; The heavy chain variable region comprises the sequence of SEQ ID NO:25, and the light chain variable region comprises the sequence of SEQ ID NO:26; The heavy chain variable region comprises the sequence of SEQ ID NO:27, and the light chain variable region comprises the sequence of SEQ ID NO:28; The heavy chain variable region comprises the sequence of SEQ ID NO:29, and the light chain variable region comprises the sequence of SEQ ID NO:26 or 28; The heavy chain variable region comprises the sequence of SEQ ID NO:37, and the light chain variable region comprises the sequence of SEQ ID NO:38; The heavy chain variable region comprises the sequence of SEQ ID NO:39, and the light chain variable region comprises the sequence of SEQ ID NO:40; The heavy chain variable region comprises the sequence of SEQ ID NO:41, and the light chain variable region comprises the sequence of SEQ ID NO:42; The heavy chain variable region comprises the sequence of SEQ ID NO:45, and the light chain variable region comprises the sequence of SEQ ID NO:46; or The heavy chain variable region comprises the sequence of SEQ ID NO:47, and the light chain variable region comprises the sequence of SEQ ID NO:

48.

63. The method according to any one of claims 58 to 62, wherein the anti-CLDN18.2 antibody-radionuclide conjugate further comprises an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG.

64. The method according to any one of claims 58 to 63, wherein the constant region comprises a constant region of human IgG1, IgG2, IgG3 or IgG4.

65. The method according to any one of claims 58 to 64, wherein the constant region of human IgG1 comprises SEQ ID NO:49 or a homologous sequence having at least 80% sequence identity therewith.

66. The method according to any one of claims 58 to 65, wherein the anti-CLDN18.2 antibody-radionuclide conjugate is humanized.

67. The method according to any one of claims 58 to 66, wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises a bifunctional antibody, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibody (ds bifunctional antibody), single-chain antibody molecule (scFv), scFv-Fc antibody, scFv dimer (bivalent bifunctional antibody), multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, and bivalent domain antibody.

68. A composition comprising the anti-CLDN18.2 antibody-radionuclide conjugate as defined in any one of the preceding claims and one or more pharmaceutically acceptable carriers, wherein the anti-CLDN18.2 antibody-radionuclide conjugate has at least one of the following properties: a) having a radiochemical purity of at least 90% (such as 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%); b) having a radiolabeling rate of at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%); c) capable of specifically binding to CLDN18.2 with a Kd value of no more than 15 nM (such as no more than 14 nM, no more than 13 nM, no more than 12 nM, no more than 10 nM, no more than 8 nM, no more than 6 nM, no more than 4 nM or no more than 2 nM); and d) capable of specifically binding to CLDN18.2 with an EC50 value of no more than 1.0 nM (such as no more than 0.8 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM or no more than 0.3 nM), as measured by ELISA.

69. The composition according to claim 68, wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises a therapeutic radionuclide selected from the group consisting of: 111 In, 111m In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 124 I, 125 I, 131 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 199 Au and 211 Pb, or a diagnostic radionuclide selected from the group consisting of: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 ln, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154 " 1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac.

70. The composition according to claim 69, wherein the therapeutic radionuclide is 177 Lu or 124 I.

71. The composition according to claim 69, wherein the diagnostic radionuclide is 124 I, 89 Zr or 177 Lu.

72. The composition according to any one of claims 69 to 71, wherein the anti-CLDN18.2 antibody-radionuclide conjugate further comprises a chelating agent.

73. The composition according to claim 72, wherein the chelating agent is DFO or DOTA.

74. The composition according to claim 73, wherein the chelating agent is DFO and the radionuclide is 89 Zr.

75. The composition according to claim 73, wherein the chelating agent is DOTA and the radionuclide is 177 Lu.

76. The composition according to claim 74, wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises a compound of formula (I): Among them A is the anti-CLDN18.2 antibody or an antigen-binding fragment thereof as defined in any one of the foregoing claims, and k is 1 - 40 (such as 3 - 5, 5 - 35, 10 - 30, 15 - 25 or 20).

77. The composition according to claim 75, wherein the anti-CLDN18.2 antibody-radionuclide conjugate comprises a moiety of a compound of formula (II):

Images