Molecular probes targeting GPC3 and use thereof

Abstract

The present invention relates to the field of molecular imaging, and more particularly, to a molecular probe targeting GPC3. The present invention sets forth a molecular probe targeting GPC3. The molecular probe is a compound having the following sequence, or a pharmaceutically acceptable salt or ester thereof: (GPC3 antibodies, comprising antibody fragments of GPC3 antibodies, or single domain antibodies thereof)m-Rn, wherein R is selected from a group consisting of a linking group, a chelating agent, a radionuclide, another targeting polypeptide or monoclonal antibody, a monoclonal antibody fragment, a single domain antibody, and a small molecule inhibitor, wherein n represents the number of R, n = 0-4, and m is not 0. The probe has good stability in vitro, clear imaging in tumors, and good specificity.

Description

Molecular probes targeting GPC3 and uses thereof Technical Field

[0001] The present invention relates to the field of molecular imaging, and in particular, to a molecular probe targeting GPC3, and more particularly, to a molecular probe comprising a GPC3 antibody or a single-domain antibody thereof. Background Art

[0002] Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is anchored to the cell surface via glycosylphosphatidylinositol (GPI). It is highly expressed in normal embryonic tissues (including the liver and placenta), but is rarely or not expressed in normal adult tissues. GPC3 interacts with heparin-binding proteins, such as extracellular matrix components, cell adhesion molecules, growth factors, enzymes, and enzyme inhibitors, and participates in regulating physiological processes such as cell differentiation, proliferation, adhesion, and migration. Numerous studies have found that GPC3 is specifically highly expressed in liver cancer tissues and is closely associated with liver cancer prognosis. GPC3 plays a crucial role in regulating malignant transformation and promoting liver cancer growth by stimulating the canonical Wnt signaling pathway. Genetic analysis of liver cancer has shown that the majority of cases are associated with upregulation of GPC3. Extensive data indicate that GPC3-positive liver cancer patients have a lower 5-year survival rate than GPC3-negative patients. Therefore, GPC3 is a potential and reliable biomarker for liver cancer and a promising target for precision diagnosis and treatment of liver cancer. Furthermore, more studies have found that GPC3 is associated with various cancers, with expression in non-small cell lung cancer, melanoma, ovarian yolk sac tumor, ovarian clear cell carcinoma, and colon cancer. Studies suggest that GPC3 may participate in multiple signaling pathways and play an important biological role in tumor cell growth.

[0003] Radioimmunoimaging (RII) utilizes radionuclide-labeled monoclonal antibodies, combining the high specificity of antigen-antibody recognition with the high sensitivity of radioactive detection for early tumor diagnosis. RII provides a noninvasive means for studying the distribution (localization), quantity (density), and function (affinity) of antigens in vivo. Compared with full IgG antibodies, antibody Fab fragments have faster blood clearance, better tumor tissue penetration, and significantly reduced nonspecific binding due to interactions with native Fc receptors, thereby improving image quality and enhancing target detection efficiency. Furthermore, radioimmunotherapy (RIT) utilizes monoclonal antibodies or fragments labeled with therapeutic radionuclides that emit α- or β-particles to deliver specific internal irradiation to tumor cells. Compared to the severe toxic side effects of chemotherapy drugs that cause widespread destruction of normal tissue cells and the limitations of radiotherapy, which only irradiates localized areas, RIT combines both systemic and targeted therapeutic approaches and has achieved rapid development in recent years. Currently, most probes targeting RII and RIT are mouse antibodies or humanized human-mouse chimeric antibodies. However, their heterogeneity often causes immune side effects in the body, hindering clinical translation. Producing fully human antibody fragments (Fabs) through antibody library technology can effectively reduce immune side effects, improve safety, and enable further clinical translation.

[0004] Therefore, the development of a molecular probe for non-invasive tracing of GPC3 in the body based on a fully human antibody Fab fragment targeting GPC3 will help in the early real-time dynamic discovery and detection of tissues with high GPC3 expression, such as malignant tumors such as HCC, and has high clinical significance in the diagnosis, staging, and efficacy evaluation of diseases with high GPC3 expression.

[0005] Therefore, there is still a need in the art for a molecular probe targeting GPC3 with high specificity and superior in vivo pharmacokinetic performance, which can be used for the diagnosis and treatment of diseases with high GPC3 expression, particularly solid tumors with high GPC3 expression.

[0006] Summary of the Invention

[0007] In a first aspect, the present invention provides a molecular probe targeting GPC3, wherein the molecular probe is a compound having the following sequence, or a pharmaceutically acceptable salt or ester thereof:

[0008] (GPC3 antibody, including antibody fragment of GPC3 antibody, or single domain antibody thereof) m -R n ,

[0009] wherein R is selected from the group consisting of a linker, a chelator, a radionuclide, another targeting polypeptide or a monoclonal antibody, a monoclonal antibody fragment, a single domain antibody, and a small molecule inhibitor;

[0010] Where n represents the number of R, n = 0-4;

[0011] Here, m is not 0.

[0012] In some embodiments, the molecular probe is a compound having the following general formula (I), or a pharmaceutically acceptable salt or ester thereof: Ab m -(LM z ) n (I)

[0013] wherein Ab is a GPC3 antibody or an antigen-binding fragment thereof; L is a linker; M is a radionuclide; z is 0 or 1; n is an integer of 1-4, preferably 1 or 2; and m is an integer of 1-4, preferably 1 or 2.

[0014] In some embodiments, L is represented by the following general formula (II): Y-Lk-T (II)

[0015] Wherein, Y is a linker, Lk is a coupling group, and T is a chelating moiety;

[0016] Preferably, Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C1-C 20 Alkenylene, optionally substituted C1-C 20 Alkynylidene, optionally substituted C1-C 20 Heteroalkylene, optionally substituted C3-C 20 Cycloalkylene, optionally substituted C3-C 20 Heterocycloalkylene, optionally substituted C5-C 20 Arylene, optionally substituted C5-C 20 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, polypeptides (such as valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), polyglycine (Gly) r , wherein r is an integer from 1 to 10), polyethylene glycol (such as (PEG) r, wherein r is an integer from 1 to 10), polypropylene glycol, polyoxyalkylene, a copolymer of polyethylene glycol and polypropylene glycol, or a combination thereof; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, N-hydroxysuccinimide ester, maleimide, 2,3,5,6-tetrafluorophenol ester, N-succinimidyl-S-acetylthioacetate, amide, or a combination thereof;

[0017] Preferably, Lk is independently selected from a single bond, p-isothiocyanatophenyl, N-hydroxysuccinimide ester, maleimide, 2,3,5,6-tetrafluorophenol ester, N-succinimidyl-S-acetylthioacetate, amide, or a combination thereof;

[0018] Preferably, T is independently selected from a single bond or a ligand derived from any one of the following structures: deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), ethylenediaminetetraacetic acid (EDTA), ), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), triacetylfusine C (TAFC), ferric streptomycin E (FOXE), ferric streptomycin B (FOXB), ferric chromogen A (FCHA), or a combination thereof;

[0019] Optionally, a spacer group is further included between Lk and T, and the spacer group is independently selected from a single bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C1-C 20 Alkenylene, optionally substituted C1-C 20 Alkynylidene, optionally substituted C1-C 20 Heteroalkylene, optionally substituted C3-C 20 Cycloalkylene, optionally substituted C3-C 20 Heterocycloalkylene, optionally substituted C5-C 20 Arylene, optionally substituted C5-C 20Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, polypeptides (such as valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), polyglycine (Gly) r , wherein r is an integer from 1 to 10), polyethylene glycol ((PEG) r , wherein r is an integer from 1 to 10), polypropylene glycol, polyoxyalkylene, a copolymerization group of polyethylene glycol and polypropylene glycol, or a combination thereof.

[0020] In some embodiments, Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, an optionally substituted C1-C 10 Alkylene, optionally substituted C1-C 10 Alkenylene, optionally substituted C1-C 10 Alkynylidene, optionally substituted C1-C 10 Heteroalkylene, optionally substituted C3-C 10 Cycloalkylene, optionally substituted C3-C 10 Heterocycloalkylene, optionally substituted C3-C 10 Arylene, optionally substituted C3-C 10 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, -(PEG) r -, Valine-Citrulline (Val-Cit), Valine-Alanine (Val-Ala), Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), Phenylalanine-Lysine (Phe-Lys), Alanine-Alanine-Asparagine (Ala-Ala-Asn), -(Gly) n - or a combination thereof, wherein n is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof;

[0021] Lk is independently selected from a single bond, p-isothiocyanatophenyl, an amide group or a maleimide group; and

[0022] T is independently selected from a single bond or a ligand derived from any of the following structures: deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), or 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP);

[0023] Optionally, a spacer group is further included between Lk and T, and the spacer group is independently selected from a single bond, an optionally substituted C1-C 10 Alkylene, optionally substituted C1-C 10 Alkenylene, optionally substituted C1-C 10 Alkynylidene, optionally substituted C1-C 10 Heteroalkylene, optionally substituted C3-C 10 Cycloalkylene, optionally substituted C3-C 10 Heterocycloalkylene, optionally substituted C3-C 10 Arylene, optionally substituted C3-C 10 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, -(PEG) r -, Valine-Citrulline (Val-Cit), Valine-Alanine (Val-Ala), Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), Phenylalanine-Lysine (Phe-Lys), Alanine-Alanine-Asparagine (Ala-Ala-Asn), -(Gly) n - or a combination thereof, wherein n is an integer from 1 to 10.

[0024] In some embodiments, Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof;

[0025] Lk is independently selected from a single bond, p-isothiocyanatophenyl or amide; and

[0026] T is independently selected from a single bond or a ligand derived from any of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA);

[0027] Optionally, a spacer group is further included between Lk and T, and the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10.

[0028] In some embodiments, Lk is independently selected from p-isothiocyanatophenyl or amide; Y is a single bond; and T is independently selected from a ligand derived from any one of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

[0029] In some embodiments, Lk is p-isothiocyanatophenyl; Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, a maleimide group, an amide group, or a combination thereof; and T is independently selected from a ligand derived from any one of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA) or diethylenetriaminepentaacetic acid (DTPA).

[0030] In some embodiments, Lk is p-isothiocyanatophenyl; Y is a single bond; and T is independently selected from a ligand derived from any one of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), or diethylenetriaminepentaacetic acid (DTPA).

[0031] In some embodiments, Lk is an amide group; Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is linked to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, a maleimide group, an amide group, or a combination thereof; and T is independently selected from a ligand derived from any one of the following structures: mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

[0032] In some embodiments, Lk is an amide; Y is a single bond; and T is independently selected from a ligand derived from any one of the following structures: mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

[0033] In some embodiments, L is independently selected from a ligand derived from any of the following structures:

[0034] The linker comprises a reactive portion and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanate phenyl, maleimide, amide, or a combination thereof; preferably, the linker is a single bond.

[0035] In some embodiments, M is a beta or alpha particle emitting nuclide, a positron emitting nuclide, a single photon emitting nuclide, or a paramagnetic metal; preferably, M is selected from a diagnostic radionuclide or a therapeutic radionuclide;

[0036] Preferably, the diagnostic radionuclide is selected from 68 Ga, 18 F-Al, 64 Cu, 99m Tc, 89 Zr, 44 Sc, 111 In, 45 Ti, 52 Mn, 59 Fe, 94m Tc,67 Ga, 71 As、 72 As、 82m Rb, 86 Y. 131 I, Gd, Mn or a combination thereof;

[0037] Preferably, the therapeutic radionuclide is selected from 177 Lu, 90 Y. 153 Sm, 67 Cu, 89 Sr. 166 Ho, 177 Yb, 47 Sc, 186 Re、 188 Re、 212 Bi, 213 Bi, 149 Pm, 212 Pb, 211 At 223 Ra, 225 Ac, 227 Th or a combination thereof;

[0038] More preferably, M is selected from 68 Ga, 18 F-Al, 99m Tc, 177 Lu, 131 I, or a combination thereof.

[0039] In some embodiments, LM is selected from the following structures:

[0040] or -Connector- 131 I,

[0041] wherein the linker comprises a reactive portion and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof;

[0042] Preferably, the linker is a single bond.

[0043] In some embodiments, Ab is a GPC3 antibody or an antigen-binding fragment thereof; preferably, Ab is a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; preferably, Ab is selected from a monoclonal antibody (mAb), Fab, Fab', F(ab')2, Fv, scFv, scFv-Fc, or a single-domain antibody.

[0044] In some embodiments, Ab comprises a heavy chain or fragment thereof comprising HCDR1, HCDR2, and HCDR3,

[0045] The HCDR1 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and most preferably an amino acid sequence of SEQ ID NO: 1;

[0046] The HCDR2 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 2, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2, and most preferably an amino acid sequence of SEQ ID NO: 2;

[0047] The HCDR3 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and most preferably an amino acid sequence shown in SEQ ID NO: 3.

[0048] In some embodiments, Ab comprises a heavy chain variable region VH, which comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; preferably comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; more preferably comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; most preferably, the heavy chain variable region VH is the amino acid sequence shown in SEQ ID NO: 7.

[0049] In some embodiments, Ab comprises a heavy chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; preferably comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; more preferably comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; most preferably, the heavy chain is the amino acid sequence shown in SEQ ID NO: 9.

[0050] In some embodiments, Ab comprises a light chain or fragment thereof comprising LCDR1, LCDR2, and LCDR3,

[0051] The LCDR1 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 4, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, and most preferably an amino acid sequence of SEQ ID NO: 4;

[0052] The LCDR2 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 5, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, and most preferably an amino acid sequence of SEQ ID NO: 5;

[0053] The LCDR3 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, and most preferably an amino acid sequence shown in SEQ ID NO: 6.

[0054] In some embodiments, Ab comprises a light chain variable region VL, which comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; preferably comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; more preferably comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; most preferably, the light chain variable region VL is the amino acid sequence shown in SEQ ID NO: 8.

[0055] In some embodiments, Ab comprises a light chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; preferably comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; more preferably comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; most preferably, the light chain is the amino acid sequence shown in SEQ ID NO: 10.

[0056] In some embodiments, Ab is a Fab, Fab' or F(ab')2 fragment, preferably a Fab fragment,

[0057] Preferably, the Ab comprises a heavy chain amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 11, preferably comprises a heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 11, more preferably comprises a heavy chain amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 11, and most preferably comprises a heavy chain amino acid sequence of SEQ ID NO: 11; and

[0058] Preferably, the Ab comprises a light chain amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, preferably comprises a light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, more preferably comprises a light chain amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, and most preferably comprises a light chain amino acid sequence shown in SEQ ID NO: 10.

[0059] In some embodiments, the Ab is a scFv fragment,

[0060] Preferably, the Ab comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; preferably comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; more preferably comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; most preferably, the Ab is the amino acid sequence shown in SEQ ID NO: 12.

[0061] In some embodiments, the molecular probe is selected from the following structures:

[0062] wherein the linker comprises a reactive portion and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanate phenyl, maleimide, amide, or a combination thereof; preferably, the linker is a single bond.

[0063] In another aspect, the present invention provides an antibody conjugate having a structure represented by the following general formula (III): Ab m -L' n (III)

[0064] wherein L' is represented by the following general formula (IV): Y-Lk-T' (IV)

[0065] wherein Lk and T' optionally further comprise a spacer group,

[0066] wherein Ab, Y, Lk, spacer, m and n have the same meanings as above, and

[0067] T' is independently selected from a single bond, deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10- Tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonic acid) (DOTP), 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonic acid) (NOTP), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), triacetylfusine C (TAFC), streptomycin E (FOXE), streptomycin B (FOXB), ferric chromogen A (FCHA), or a combination thereof; preferably selected from Deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene more preferably selected from a single bond, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

[0068] In another aspect, the present invention provides a pharmaceutical composition comprising: the aforementioned molecular probe or the aforementioned antibody conjugate; and a pharmaceutically acceptable carrier.

[0069] In another aspect, the present invention provides a kit comprising: the above-mentioned antibody conjugate; and the above-mentioned radionuclide M.

[0070] In another aspect, the present invention provides a method for preparing the molecular probe targeting GPC3, comprising reacting the antibody conjugate with a radionuclide M.

[0071] In another aspect, the present invention provides use of the molecular probe targeting GPC3, the antibody conjugate, the pharmaceutical composition or the kit in preparing a reagent for detecting GPC3.

[0072] In another aspect, the present invention provides use of the molecular probe targeting GPC3, the antibody conjugate, the pharmaceutical composition or the kit in preparing a kit for molecular imaging.

[0073] In another aspect, the present invention provides a method of imaging, comprising:

[0074] administering the molecular probe targeting GPC3 or the pharmaceutical composition to a subject in need thereof; and

[0075] The presence of the molecular probe targeting GPC3 was detected in vivo by imaging.

[0076] In some embodiments, the presence of the molecular probe targeting GPC3 is detected by single photon emission computed tomography (SPECT), positron emission tomography (PET) imaging, or magnetic resonance imaging (MRI).

[0077] In another aspect, the present invention provides use of the molecular probe targeting GPC3, the antibody conjugate, the pharmaceutical composition or the kit in preparing a kit for cancer diagnosis.

[0078] In another aspect, the present invention provides use of the molecular probe targeting GPC3, the antibody conjugate, the pharmaceutical composition or the kit in preparing an agent for cancer treatment.

[0079] In another aspect, the present invention provides a method for treating cancer, comprising administering the molecular probe targeting GPC3 or the pharmaceutical composition to a subject in need thereof.

[0080] In some embodiments, the subject is GPC3 positive.

[0081] In some embodiments, the cancer is a GPC3-high-expressing cancer, preferably a solid tumor, more preferably selected from liver cancer, non-small cell lung cancer, melanoma, ovarian yolk sac tumor, ovarian clear cell carcinoma and colon cancer, and most preferably liver cancer.

[0082] In some specific embodiments, the small molecule inhibitor can be a small molecule drug.

[0083] In some specific embodiments, the small molecule inhibitor can be a drug for treating cancer.

[0084] In some specific embodiments, the small molecule inhibitor can be doxorubicin, cisplatin, vinblastine, camptothecin, paclitaxel, etc.

[0085] In some specific embodiments, the small molecule inhibitor can be MK-0429, sorafenib, imatinib, gefitinib, etc.

[0086] The molecular probe targeting GPC3 of the present invention has good in vitro stability, clear imaging in tumors and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 is (A) SEC-HPLC profile of antibody aGPC3-Fab; (B) 68 ITLC spectrum of Ga-NOTA-aGPC3-Fab;

[0088] Figure 2 68 ITLC profiles of Ga-NOTA-aGPC3-scFv before and after PD-10 purification;

[0089] Figure 3 68 ITLC profiles of Ga-DOTA-aGPC3-scFv before and after PD-10 purification;

[0090] Figure 4 shows 68 ITLC profiles of Ga-DOTA-aGPC3-Fab before and after PD-10 purification;

[0091] Figure 5 (A) 177 ITLC spectrum of Lu-DOTA-aGPC3-Fab; (B) 177 Radiochemical purity of Lu-DOTA-aGPC3-Fab in PBS over time;

[0092] Figure 6 177 ITLC spectrum of Lu-DOTA-aGPC3-scFv;

[0093] Figure 7 (A) 177 ITLC spectrum of Lu-DOTA-aGPC3-mAb; (B) 177 Radiochemical purity of Lu-DOTA-aGPC3-mAb in PBS over time;

[0094] Figure 8 is a Western blot analysis of GPC3 expression in Huh7 and SK-HEP-1 cells (GAPDH was used as an internal reference);

[0095] Figure 9 (A) 68 PET / CT images of Huh7 tumor-bearing mice at 30 minutes, 1 hour, 2 hours, and 4 hours after Ga-NOTA-aGPC3-Fab injection. (Attenuation correction; fused images, coronal and transverse images; arrows indicate Huh7 subcutaneous tumors); (B) Blood, liver, kidney, muscle, and tumor of Huh7 tumor-bearing mice 68 ROI quantitative analysis of Ga-NOTA-aGPC3-Fab uptake; (C) 68 Tumor / blood, tumor / liver, and tumor / muscle ratios at 30 min, 1 h, 2 h, and 4 h after Ga-NOTA-aGPC3-Fab injection;

[0096] Figure 10 (A) 68 PET / CT images of SK-HEP-1 tumor-bearing mice at 1, 2, and 4 hours after Ga-NOTA-aGPC3-Fab injection. (Attenuation correction; fused image; white dotted circle indicates SK-HEP-1 subcutaneous tumor); (B) Blood, liver, kidney, muscle, and tumor of SK-HEP-1 tumor-bearing mice 68 ROI quantitative analysis of Ga-NOTA-aGPC3-Fab uptake;

[0097] Figure 11 (A) 68 PET / CT images of PDX model mice at 1 h, 2 h, and 4 h after Ga-NOTA-aGPC3-Fab injection; (Attenuation correction; fused image; white dotted circle indicates PDX transplanted tumor); (B) Blood, liver, kidney, muscle, and tumor of PDX model mice 68 ROI quantitative analysis of Ga-NOTA-aGPC3-Fab uptake;

[0098] FIG12 shows H&E staining of Huh7 and PDX tumor tissues and immunohistochemical staining analysis of GPC3;

[0099] Figure 13 shows Hep3B tumor-bearing mice 68 Ga-NOTA-aGPC3-scFv PET / CT imaging; (A) Representative PET / CT images of the Hep3B subcutaneous tumor model at different time points; (B) Semi-quantitative analysis of the mean PET signal intensity of the blood pool, liver, kidney, muscle, and tumor in the Hep3B subcutaneous tumor model PET images (n=3); (C) Analysis of the tumor / blood pool (T / B), tumor / liver (T / L), and tumor / muscle (T / M) ratios in the Hep3B subcutaneous tumor model based on PET images (n=3); white arrows indicate tumors;

[0100] Figure 14 shows Hep3B tumor-bearing mice 68Ga-NOTA-aGPC3-scFv and 68 Comparative analysis of Ga-NOTA-aGPC3-Fab PET / CT imaging. (A) Hep3B subcutaneous tumor model injection 68 Representative PET / CT images of Ga-NOTA-aGPC3-scFv 1h, 2h, and 4h after injection; (B) Hep3B subcutaneous tumor model 68 Representative PET / CT images of Ga-NOTA-aGPC3-Fab 1 hour, 2 hours, and 4 hours after treatment; (C) Analysis of tumor / blood pool ratio in Hep3B subcutaneous tumor model based on PET images (n=3); (D) Analysis of tumor / liver ratio in Hep3B subcutaneous tumor model based on PET images (n=3); (E) Analysis of tumor / muscle ratio in Hep3B subcutaneous tumor model based on PET images (n=3); White arrows indicate tumors. *: P<0.05;

[0101] Figure 15 shows the Hep3B liver tumor in situ model mouse 68 Ga-NOTA-aGPC3-scFv, 68 Ga-NOTA-aGPC3-Fab and 18 F-FDG PET / CT imaging comparison. (A) Hep3B liver tumor model mice injected 68 Ga-NOTA-aGPC3-scFv, 68 Ga-NOTA-aGPC3-Fab and 18 Representative static images of small animal PET images 1 hour after F-FDG administration. (B) Anatomical exploration of a hepatic tumor in situ. Li: liver; St: stomach; Tu: tumor; Sp: spleen; SI: small intestine; LI: large intestine. White arrows (A) and yellow arrows (B) indicate tumor locations. Scale bar = 5 mm.

[0102] Figure 16 (A) 177 SPECT / CT images of Hep3B tumor-bearing mice at 2h, 6h, 12h, 24h and 48h after Lu-DOTA-aGPC3-Fab injection (white dotted circle indicates tumor); (B) Blood, liver, muscle and tumor of Hep3B tumor-bearing mice 177 ROI quantitative analysis of Lu-DOTA-aGPC3-Fab uptake;

[0103] Figure 17 (A) 177 SPECT / CT images of Hep3B tumor-bearing mice at 2h, 6h, 12h, 24h and 48h after Lu-DOTA-aGPC3-scFv injection (white dotted circle indicates tumor); (B) Blood, liver, muscle and tumor of Hep3B tumor-bearing mice 177ROI semiquantitative analysis of Lu-DOTA-aGPC3-scFv uptake;

[0104] Figure 18 (A) 177 SPECT / CT images of Hep3B tumor-bearing mice at 2 h, 6 h, 12 h, 1 day, 3 days, 5 days, and 7 days after Lu-DOTA-aGPC3-mAb injection (white dotted circle indicates tumor); (B) Blood, liver, muscle, and tumor of Hep3B tumor-bearing mice 177 Semi-quantitative analysis of ROIs for Lu-DOTA-aGPC3-mAb uptake; (C) Analysis of tumor / blood pool ratio over time in a Hep3B subcutaneous tumor model based on SPECT images; (D) Analysis of tumor / liver ratio over time in a Hep3B subcutaneous tumor model based on SPECT images; (E) Analysis of tumor / muscle ratio over time in a Hep3B subcutaneous tumor model based on SPECT images;

[0105] Figure 19 shows the results of Hep3B tumor-bearing mice injected with normal saline or 177 (A) Changes of tumor volume over time after Lu-DOTA-aGPC3-Fab; (B) Changes of body weight over time;

[0106] Figure 20 shows the results of Hep3B tumor-bearing mice injected with normal saline or 177 After Lu-DOTA-aGPC3-scFv administration, (A) tumor volume changes over time; (B) body weight changes over time;

[0107] Figure 21 shows the results of Hep3B tumor-bearing mice injected with normal saline, aGPC3-mAb, and 11.1 MBq 177 Lu-DOTA-aGPC3-mAb or 5.55 MBq injected twice 177 After Lu-DOTA-aGPC3-mAb, (A) tumor volume changes over time; (B) body weight changes over time. DETAILED DESCRIPTION

[0108] I. Definition

[0109] Unless otherwise defined, all scientific and technical terms used in the specification, drawings and claims have the conventional meanings commonly understood by those of ordinary skill in the art. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification and the definitions included therein shall prevail. If two or more documents incorporated by reference include disclosures that conflict and / or are inconsistent with each other, the document with the later effective date shall prevail. Materials, methods and examples are exemplary only and are not intended to be limiting. Unless otherwise noted, the following terms used in this document (including the specification and claims) have the definitions given below.

[0110] Singular forms such as "a," "an," or "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a single antibody as well as multiple antibodies, whether the same or different.

[0111] As used herein, the word "about" means that a value is within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation according to the practice in the art. The term "about" is also used to indicate that the amount or value under consideration can be a specified value or some other value that is roughly the same. The phrase is intended to convey that similar values ​​promote equivalent results or effects as described herein. In this context, "about" can refer to a range that is higher and / or lower than up to 20% or 10%. Wherever the term "about" is specified for a certain determination or embodiment, this definition is preferred for the specific context.

[0112] Furthermore, it should be understood that slight variations above and below the stated ranges can be used to achieve substantially the same results as values ​​within the ranges. Furthermore, unless otherwise indicated, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values.

[0113] The terms "comprising", "including", "containing", "having" and the like should be understood broadly or openly rather than restrictively. When used in the specification, the term "comprising" includes "consisting of" and "consisting essentially of".

[0114] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. The terms "at least one" and "at least one of" include, for example, one, two, three, four, or five or more elements.

[0115] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a compound comprising at least two amino acid residues covalently linked by at least one peptide bond. There is no limit to the maximum number of amino acids that can constitute the sequence of a peptide. A "peptide" may include, but is not limited to, modified amino acids, non-naturally occurring amino acids, and / or D amino acids. Unless otherwise indicated, a particular peptide sequence also encompasses variants in which at least one amino acid has been replaced by an amino acid characterized by similar structural properties. A "peptide" may be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof. A "peptide" may be, for example, a biologically active fragment, an oligopeptide, a homodimer, a heterodimer, a peptide variant, a modified peptide, a peptide derivative, a peptide analog, a fusion protein, and the like.

[0116] As used herein, the term "amino acid" or "amino acid residue" ("aa") typically refers to naturally occurring amino acids, but may also refer to non-naturally occurring amino acids. The term typically refers to L-amino acids, but may also encompass D-amino acids. Amino acids may or may not be modified as described elsewhere herein. Single-letter codes are used herein to refer to the corresponding amino acids. As used herein, "charged amino acids" are amino acids that are negatively or positively charged. "Negatively charged amino acids" are aspartic acid (D) and glutamic acid (E). "Positively charged amino acids" are arginine (R), lysine (K), and histidine (H). "Polar amino acids" are all amino acids that form hydrogen bonds as donors or acceptors, including all charged amino acids as well as asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine ​​(C). "Polar uncharged amino acids" are asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine ​​(C). "Amphoteric amino acids" are tryptophan (W), tyrosine (Y), and methionine (M). "Aromatic amino acids" are phenylalanine (F), tyrosine (Y), and tryptophan (W). "Hydrophobic amino acids" are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and cysteine. "Small amino acids" are glycine (G), alanine (A), serine (S), proline (P), threonine (T), aspartic acid (D), and asparagine (N).

[0117] The terms "sequence identity", "percent identity" or "percent (%) sequence identity" are numbers that describe how similar a query sequence is to a target sequence, or more precisely, how many characters in each sequence are identical after alignment. The most popular tool for calculating sequence identity is BLAST (Basic Local Alignment Search Tool, NCBI), which performs a comparison between pairs of sequences, searching for areas of local similarity. Suitable alignment methods are known in the art, such as the Needleman-Wunsch algorithm for global-global alignments, using the BLOSUM62 matrix, with a gap opening penalty of 11 and a gap extension penalty of 1. The pairs of identical residues in the alignment can then be counted and divided by the total length of the alignment (including gaps, internal and external) to obtain a percent identity value.

[0118] When "homologous" is used in reference to a protein or polypeptide, it is meant that the inconsistency in the residue position is usually derived from a conservative amino acid substitution. A "conservative amino acid substitution" refers to an amino acid residue that is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions do not substantially change the functional properties of the protein. Where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage of sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art, see, for example, Pearson, 1994, Methods Mol.Biol.24:307-31 and 25:365-89.

[0119] For example, in some cases, the following six groups each contain amino acids that are conservative substitutions for each other: (1) serine, threonine; (2) aspartic acid, glutamic acid; (3) asparagine, glutamine; (4) arginine, lysine; (5) isoleucine, leucine, methionine, alanine, valine; and (6) phenylalanine, tyrosine, tryptophan.

[0120] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating a nucleic acid molecule to which it is linked. The term also encompasses plasmids (non-viral) and viral vectors. Certain vectors are capable of directing the expression of nucleic acids or polynucleotides to which they are operably linked. Such vectors are referred to herein as "expression vectors." Expression vectors for eukaryotic use can be constructed by inserting a polynucleotide sequence encoding at least one protein of interest (POI) into a suitable vector backbone. The vector backbone may contain the necessary elements to ensure the maintenance of the vector and, as desired, provide amplification within the host. For viral vectors (e.g., lentiviral or retroviral vectors), additional virus-specific elements such as structural elements or other elements may be required and are well known in the art. These elements can, for example, be provided in cis (on the same plasmid) or in trans (on a separate plasmid). Viral vectors may require a helper virus or packaging line for large-scale transfection. The vector may contain additional elements, such as, for example, enhancer elements (e.g., viral, eukaryotic), introns, and plasmid replication origins for viruses that replicate in mammalian cells. According to the present invention, the expression vector typically has a promoter sequence that drives POI expression. Expression of the POI and / or selective marker protein can be constitutive or regulated (e.g., inducible by addition or removal of a small molecule inducer). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level expression of the POI in mammalian cells, such as regulatory elements, promoters, and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter Ad LP), or polyoma virus. For further description of viral regulatory elements and their sequences, see, for example, US Pat. No. 5,168,062 to Stinski, US Pat. No. 4,510,245 to Bell et al., and US Pat. No. 4,968,615 to Schaffner et al.

[0121] The term "conjugate" refers to a molecule comprising at least two moieties. For example, but not limited to, these moieties may be linked via a linking group.

[0122] The term "antibody conjugate" refers to a conjugate comprising at least one portion of an antibody or antigen-binding fragment and one or more additional molecules or moieties. The one or more additional molecules or moieties may be selected from, but are not limited to, drugs, chelating moieties, radioactive elements, cytotoxic agents, additional antibodies or antigen-binding fragments.

[0123] The term "linker" refers to any molecule that can achieve direct topological connection of the different parts of a construct or conjugate. For example, a linker can connect a chelating and targeting moiety. In an alternative form, a linker can connect different parts of a chelating moiety. For bispecific antibodies, a linker can connect different antigen-binding moieties. Examples of linkers that establish covalent attachment between different parts include peptide linkers and non-protein polymers, including but not limited to copolymers of polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or polyethylene glycol, polypropylene glycol.

[0124] In the present invention, the term "GPC3" includes any variant or isoform of GPC3 naturally expressed by cells. GPC3 or any variant or isoform thereof can be isolated from cells or tissues naturally expressing them, or produced by recombinant techniques using techniques commonly used in the art and those described herein.

[0125] The term "targeting moiety" (also "tissue targeting group" or "tissue targeting portion") is any chemical structure that binds to a biological target (e.g., GPC3 or a cell expressing GPC3). The targeting moiety positions itself (e.g., as part of a larger structure) at a specific site to exert a desired effect. Thus, the tissue targeting group or portion can localize the molecule or conjugate to at least one desired site in a subject to a greater extent than an equivalent complex that does not include the targeting moiety. According to the present invention, the targeting moiety can be, for example, but not limited to, selected from nucleotides, DNA and RNA fragments, aptamers, peptides, proteins, antibodies or fragments thereof, nanoparticles, or small molecules, or any combination thereof.

[0126] The term "targeting moiety that binds to GPC3" refers to a chemical structure that binds to GPC3. According to a preferred embodiment of the present invention, the targeting moiety may be an antibody or an antigen-binding fragment thereof as described herein.

[0127] The terms "anti-GPC3 antibody," "GPC3 antibody," or "antibody that binds to GPC3" refer to antibodies that bind to GPC3 or a fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting GPC3. In some embodiments, the extent of binding of an antibody that binds to GPC3 to an unrelated protein other than GPC3 is less than about 10%, less than about 5%, or preferably less than about 2%, and most preferably less than about 1% of the binding of the antibody to GPC3, as measured, for example, by surface plasmon resonance (SPR) or another standard method such as an ELISA assay. In some preferred embodiments, the antibody that binds to GPC3 has a binding affinity of ≤1 μM, ≤500 nM, ≤200 nM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or smaller, such as 10-8 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (K D In certain embodiments, the antibody that binds to GPC3 has a specific activity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or smaller, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M) binding activity (EC 50 ).

[0128] The term "antibody" includes, but is not limited to, immunoglobulin molecules (such as, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgE, IgA1, IgA2, mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, IgD, IgE, or IgM, rat IgG1, IgG2a, IgG2b, IgG2c, IgA, IgD, IgE, or IgM, rabbit IgA1, IgA2, IgA3, IgE, IgG, IgM, goat IgA, IgE, IgG1, IgG2, IgE, IgM, or chicken IgY) that bind to a specific antigen. Depending on the context, the term antibody may also refer to a functional fragment of a full-length antibody as disclosed elsewhere herein. The term also includes any protein binding molecule with immunoglobulin-like functions. According to the present invention, preferably, an antibody or fragment thereof is characterized in that its affinity for its target corresponds to less than 10 -7 M, more preferably less than 10 -8 M, even more preferably at 10 -11 M to 10 -9 K in the M range D .

[0129] Naturally produced complete antibodies are tetramers of approximately 150 kDa, consisting of two identical heavy chain polypeptides and two identical light chain polypeptides, which associate with each other to form a structure commonly referred to as a "Y-shaped" structure. Each heavy chain contains at least four domains: an amino-terminal variable (VH) region (located at the top of the Y-shaped structure), followed by three constant regions: CH1, CH2, and CH3 at the carboxyl terminus (located at the base of the Y-shaped structure). The hinge region is located between CH1 and CH2. Each light chain consists of two domains: an amino-terminal variable (VL) region and a carboxyl-terminal constant (CL) region. The heavy and light chains are linked by a single disulfide bond, and two additional disulfide bonds connect the heavy chain hinge regions to each other, thereby forming a tetramer.

[0130] The variable region at the amino terminus of the antibody polypeptide chain is primarily responsible for antigen recognition. The VH and VL regions can be further subdivided into hypervariable regions (also called complementarity determining regions or CDRs), which are separated by more conserved regions called "framework regions" or "FRs." Each VH and VL is typically composed of three CDRs and up to four FRs from the amino terminus to the carboxyl terminus, for example, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0131] The term "CDR" refers to the complementary determining region of an antibody. CDR is crucial for the diversity of antigen specificity. A group of CDRs constitutes a paratope. Three discontinuously arranged CDRs (CDR1, CDR2, and CDR3) are usually present in the amino acid sequence of the variable domain of an antigen receptor. The CDRs are usually tightly bound together by the FR region and, for example, together with the CDRs from another chain, contribute to the formation of the antigen binding site of the antibody. The CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3. The CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. As is known in the art, the amino acid positions / boundaries that describe the hypervariable regions of an antibody can vary, depending on the context and various definitions known in the art. As used herein, the numbering of antibody amino acid residues is carried out according to the immunoglobulin amino acid residue numbering system of IMGT.

[0132] As used herein, the term "crystallizable fragment region", also known as "Fc domain", "Fc region" or "Fc portion" refers to the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The Fc region can interact with Fc receptors and some proteins of the complement system (e.g., on immune effector cells). The Fc region defines the class of antibody or isotype. For IgG antibodies, the Fc region consists of the CH2 and CH3 domains. The term includes native sequence Fc regions and variant Fc regions.

[0133] In some embodiments, the antibodies are polyclonal; in some embodiments, the antibodies are monoclonal.

[0134] The term "polyclonal antibody" refers to a heterogeneous population of antibodies derived, for example, from the serum of an animal immunized with an antigen or a functional derivative thereof. To produce polyclonal immunoglobulins, one or more different host animals can be immunized by injection with the antigen.

[0135] The term "monoclonal antibody" is a substantially homogeneous population of antibodies that bind to a specific antigen. Monoclonal antibodies can be obtained by methods well known to those skilled in the art (see, e.g., et al., Nature (1975) 256, 495-497, and U.S. Patent No. 4,376,110). Antibodies or fragments having specific binding affinity can be isolated, enriched, or purified from prokaryotic or eukaryotic organisms.

[0136] In some embodiments, the antibody with constant region sequence is an antibody with mouse, rabbit, primate or human antibody characteristics. In some embodiments, the antibody sequence elements are humanized, primatized or chimeric. In some embodiments, the antibody comprises a complete antibody or an immunoglobulin or an antibody fragment.

[0137] "Fragments," "functional fragments," or "antigen-binding fragments" of antibodies are those that retain the ability of an antibody to bind to a specific antigen. Thus, an antibody fragment typically comprises a functional portion of a full-length antibody, generally its antigen-binding or variable region. Preferably, as used herein, an antibody fragment substantially retains the affinity of the full-length antibody. Thus, a suitable fragment of an anti-GPC3 antibody will retain the ability to bind to a target protein (e.g., GPC3). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and scFv fragments, single-chain antibody molecules, diabodies, and single-domain antibodies.

[0138] Fab fragments are monovalent fragments composed of the VL, VH, CL, and CH1 domains. F(ab')2 fragments are bivalent fragments consisting of two Fab fragments linked by a disulfide bond in the hinge region. Fab' fragments are produced by cleaving the disulfide bond at cysteines in the hinge region of F(ab')2 and differ from Fab fragments by the addition of several residues to the carboxyl terminus of the heavy chain CH1 domain. Fab and F(ab')2 fragments lack the Fc region of intact antibodies, allowing for more rapid clearance from animal circulation and potentially less nonspecific tissue binding than intact antibodies.

[0139] The Fv fragment is the smallest fragment of an antibody containing a complete target recognition and binding site. This region is composed of a dimer of a heavy chain and a light chain variable domain in close non-covalent association (VH-VL dimer). In this configuration, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Typically, six CDRs confer antibody antigen binding specificity. However, in some cases, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) may have the ability to recognize and bind antigens, although the affinity is lower than that of the entire binding site.

[0140] Single-chain Fv (scFv) antibody fragments contain the VH and VL domains of an antibody within a single polypeptide chain. Typically, scFvs also include a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. scFvs retain the antigen specificity of the original immunoglobulin, while their smaller molecular size offers advantages such as enhanced tumor penetration, rapid degradation in the blood, and minimal negative feedback loops in the human body.

[0141] Single-domain antibodies or nanobodies are composed of a single VH domain that can specifically bind to an antigen.

[0142] Antibodies or antibody fragments can be produced synthetically or recombinantly. A variety of techniques can be used to produce antibodies. For example, phage antibodies can be used to generate antibodies (Knappik et al., J. Mol. Biol. 296: 57-86, 2000). Another method for obtaining antibodies is to screen DNA libraries from B cells, as described in WO 91 / 17271 and WO 92 / 01047. In these methods, phage libraries are produced, each member of which displays a different antibody on its outer surface. Antibodies are typically displayed as Fv or Fab fragments. Phage-displayed antibodies are selected by affinity enrichment for binding to a selected protein. Antibodies can also be produced using the trioma method (e.g., Oestberg et al., Hybridoma 2: 361-367, 1983; U.S. Patent No. 4,634,664; U.S. Patent No. 4,634,666).

[0143] Antibodies can also be purified from any cell expressing the antibody (including host cells transfected with expression constructs encoding the antibody). Host cells can be cultured under conditions of expressing the antibody. Using methods well known in the art, the purified antibody can be separated from other cellular components such as certain proteins, carbohydrates, or lipids that can associate with the antibody in the cell. Such methods include but are not limited to size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. The purity of the preparation can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. The preparation of the purified antibody can contain more than one type of antibody.

[0144] Alternatively, antibodies according to the present invention can be produced by chemically synthesizing their amino acid sequences, such as by direct peptide synthesis using solid phase techniques (e.g., Merrifield, J. Am. Chem. Soc. 85: 2149-2154, 1963; Roberge et al., Science 269: 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Optionally, fragments of the antibody can be synthesized separately and combined using chemical methods to produce full-length molecules.

[0145] The term "affinity" or "binding affinity" is a term of art used to describe the strength of the non-covalent bond between a single binding site of a molecule and its binding partner. The affinity of an antibody or fragment thereof for a target can be determined using techniques well known in the art, such as ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), flow cytometry, or fluorescence polarization assays. Preferably, affinity is expressed as the dissociation constant K D Provided, in an alternative format, the affinity is expressed as EC 50 Value provided.

[0146] The dissociation constant (K D ) has molar units (M) and corresponds to the concentration of the binding agent (e.g., antibody or fragment) when half of the target protein or bound ligand is occupied at equilibrium. The smaller the dissociation constant, the higher the affinity between the binding agent and its target. D The affinity value can be preferably measured by using a surface plasmon resonance assay using a suitable device, including but not limited to a Biacore instrument such as Biacore T100, Biacore T200, Biacore 2000, Biacore 4000, Biacore 3000 (GE Healthcare Biacore, Inc.), see, for example, Sjolander & Urbaniczky; Anal. Chem. 63: 2338-2345, 1991; Szabo et al., Curr. Opin. Struct. Biol. 5: 699-705, 1995), or a ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.). In an alternative form, affinity can be determined using any method known in the art, including, for example, immunoassays such as enzyme-linked immunosorbent assays (ELISA) and fluorescence-activated cell sorting (FACS), for quantifying the binding of antibodies to cells expressing the antigen. If it is found that the assay conditions affect the determined K, the affinity can be determined using any method known in the art, including, for example, immunoassays such as enzyme-linked immunosorbent assays (ELISA) and fluorescence-activated cell sorting (FACS). D , the assay settings that minimize the standard deviation should be used.

[0147] The half-maximal effective concentration (EC50 ) refers to the concentration of drug, modulator, antibody, fragment, conjugate, or molecule that induces a response intermediate between baseline and maximum after a specified incubation time. Thus, when referring to affinity, EC 50 Reflects the concentration of binding agent (e.g., antibody) required for half-maximal binding. If the inflection point can be determined by mathematical modeling (e.g., nonlinear regression) of the dose-response curve describing the relationship between the concentration of the applied drug, antibody, fragment, conjugate, or molecule and the signal, the EC can be determined. 50 For example, if the dose-response curve follows a sigmoidal shape, the EC 50 In cases where the relevant response is inhibition, EC 50 The half-maximal inhibitory concentration (IC 50 ).

[0148] As used herein, a binding agent or antibody that "specifically binds..." is "specific for / directed against" or "specifically recognizes" an antigen of interest (e.g., GPC3), binds the antigen with sufficient affinity to allow the binding agent or antibody to be used as a therapeutic agent targeting cells or tissues expressing the antigen, and does not significantly cross-react with proteins other than orthologs and variants (e.g., mutant forms, splice variants, or proteolytic truncations) of the aforementioned antigenic target. As used herein, the term "specifically recognizes" can be, for example, achieved by a binding agent, antibody, or antigen-binding fragment thereof exhibiting less than about 10 -4 M, or less than about 10 -5 M, or less than about 10 -6 M, or less than about 10 -7 M, or less than about 10 -8 M, or less than about 10 -9 M, or less than about 10 -10 M, or less than about 10 -11 M, or less than about 10 -12 M, or smaller unit price K D .

[0149] In its most general form, "specific binding" refers to the ability of a binding agent or antibody to distinguish an antigen of interest from an unrelated antigen, as measured, for example, by surface plasmon resonance (SPR), Western blot, ELISA-, RIA-, ECL-, IRMA-tests, or peptide scanning. For example, standard ELISA assays can be performed. Scoring can be performed by standard color development (e.g., secondary antibodies with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide). The reaction in a specific well is scored by, for example, the optical density at 450 nm. Typical background (=negative reaction) can be 0.1 OD; a typical positive reaction can be 1 OD. This means that the positive / negative difference is greater than 5 times, 10 times, 50 times, and preferably greater than 100 times. Typically, the determination of binding specificity is not performed using a single reference antigen, but using a group of about three to five unrelated antigens (e.g., milk powder, BSA, transferrin, etc.).

[0150] A "radionuclide" (also: "radioactive nuclide", "radioisotope" or "radioisotope") is an atom with an unstable nucleus that is characterized by excess energy, which can be transferred to newly created radiating particles or atomic electrons within the nucleus. During this process, the radionuclide undergoes radioactive decay. Radionuclides can occur naturally or be artificially manufactured. Radionuclides vary in their properties (including half-life, energy emission characteristics and decay type), so radionuclides can be selected with a desired mix of properties suitable for diagnostic and / or therapeutic use, for example, gamma emitters are generally used for diagnosis, while beta emitters are generally used for treatment. However, some radionuclides are both gamma and beta emitters, so by varying the radioactivity used, the radionuclide may be suitable for both diagnostic and therapeutic uses. Radionuclides used in this article also include paramagnetic metals used for MRI imaging.

[0151] The term "diagnostic radionuclide" refers to a radionuclide that can be attached to an antibody and that provides a detectable signal. Diagnostic radionuclides include radionuclides that can be detected by radioactive decay or other imaging techniques. Diagnostic radionuclides can be detected by various imaging techniques, such as PET, SPECT, or MRI imaging. Exemplary diagnostic radionuclides include, but are not limited to: 68 Ga, 18 F-Al, 64 Cu, 99m Tc, 89 Zr, 44 Sc, 111 In, 45 Ti, 52 Mn, 59 Fe, 94m Tc,131 I. 67 Ga, 71 As、 72 As、 82m Rb, 86 Y, Gd, Mn or any combination thereof.

[0152] The term "therapeutic radionuclide" refers to a radionuclide that can be attached to an antibody and whose function is to deliver a cytotoxic dose of radiation to a target site (e.g., a tumor). Suitable therapeutic radionuclides include those that emit beta particles, alpha particles, or Auger electrons. Exemplary therapeutic radionuclides include, but are not limited to: 177 Lu, 90 Y. 153 Sm, 67 Cu, 89 Sr. 131 I. 166 Ho, 177 Yb, 47 Sc, 186 Re、 188 Re、 212 Bi, 213 Bi, 149 Pm, 212 Pb, 211 At 223 Ra, 225 Ac, 227 Th or any combination thereof.

[0153] "Treating" a disease in a subject or "treating" a subject having a disease means subjecting the subject to drug therapy, e.g., administration of a drug, such that at least one symptom of the disease is alleviated or prevented from worsening. When referring to a specific disease, treating means: (1) ameliorating the disease or one or more biological manifestations of the disease; (2) interfering with one or more points in the biological cascade that causes or contributes to the disease; (3) alleviating one or more symptoms, effects, or side effects associated with the disease; or (4) slowing the progression of the disease.

[0154] The term "prevent" or "preventing" refers to reducing the likelihood of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition but is at risk of developing the disease, disorder, or condition or is susceptible to developing the disease, disorder, or condition.

[0155] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount sufficient to achieve a particular biological result, or to modulate or improve symptoms, or the duration of symptom onset, in a subject, typically by at least about 10%; usually by at least about 20%, preferably by at least about 30%, or more preferably by at least about 50%.

[0156] The term "pharmaceutically acceptable" refers to a substance that is not physiologically or otherwise undesirable. For example, the substance can be added to a pharmaceutical composition and administered to a patient without producing any significant undesirable physiological effects, nor does the substance interact in a deleterious manner with any other ingredients contained in the composition.

[0157] "Pharmaceutically acceptable salts" refers to inorganic or organic acid addition salts of the compounds disclosed herein. Suitable pharmaceutically acceptable salts of the compounds disclosed herein can be, for example, but not limited to, acid addition salts of the compounds disclosed herein that are sufficiently basic with nitrogen atoms in the chain or ring, such as acid addition salts with inorganic acids or "mineral acids" such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, bisulfuric acid, phosphoric acid, or nitric acid, or with organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, Acid addition salts of cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, or thiocyanic acid. In addition, another suitable pharmaceutically acceptable salt of the compounds disclosed herein that is sufficiently acidic is an alkali metal salt (e.g., sodium or potassium salt), an alkaline earth metal salt (e.g., calcium, magnesium or strontium salt), or an aluminum or zinc salt, or an ammonium salt derived from ammonia or an organic primary, secondary or tertiary amine having 1 to 20 carbon atoms, such as ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, diethylaminoethanol, tris(hydroxymethyl)aminomethane, procaine, dibenzylamine, N-methylmorpholine, arginine , lysine, 1,2-ethylenediamine, N-methylpiperidine, N-methyl-glucamine, N,N-dimethyl-glucamine, N-ethyl-glucamine, 1,6-hexanediamine, glucosamine, sarcosine, serinol, 2-amino-1,3-propanediol, 3-amino-1,2-propanediol, 4-amino-1,2,3-butanetriol, or a salt with a quaternary ammonium ion having 1 to 20 carbon atoms, such as tetramethylammonium, tetraethylammonium, tetra(n-propyl)ammonium, tetra(n-butyl)ammonium, N-benzyl-N,N,N-trimethylammonium, choline or benzalkonium.

[0158] II. Radiolabeled GPC3 Antibody Conjugate

[0159] The present invention provides a radiolabeled molecular probe that binds to Glypican-3 (GPC3), comprising a targeting moiety that binds to GPC3, a linker group, and a radionuclide.

[0160] 2.1. Targeting moieties that bind to GPC3

[0161] The targeting moiety that binds to GPC3 according to the present invention can be selected from, but not limited to, peptides, proteins, antibodies or antigen-binding fragments, nanoparticles, polynucleotides, DNA or RNA fragments, aptamers, or small molecules that specifically bind to GPC3.

[0162] In some embodiments, the targeting moieties provided herein bind to GPC3 with a K of about 1.0 pM to 200 nM. D Binding to GPC3. In some embodiments, the targeting moieties provided herein that bind to GPC3 have an IC of about 0.01-250 nM. 50 Inhibit cell proliferation.

[0163] In some embodiments, the targeting moiety that binds to GPC3 provided herein is a GPC3 antibody or an antigen-binding fragment thereof. In some embodiments, the GPC3 antibody or antigen-binding fragment thereof is a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0164] In some embodiments, the GPC3 antibody or antigen-binding fragment thereof is selected from a monoclonal antibody (mAb), Fab, Fab', F(ab')2, Fv, scFv, scFv-Fc, or a single domain antibody.

[0165] In some embodiments, the GPC3 antibody or antigen-binding fragment thereof is of IgG, IgM, IgA, IgE, IgD, or IgY type.

[0166] In some preferred embodiments, the GPC3 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, five, or six CDR sequences that have at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0167] In a preferred embodiment, the GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain or fragment thereof, wherein the heavy chain or fragment thereof comprises a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR1 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, preferably the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, preferably the amino acid sequence of SEQ ID NO: 2; and the HCDR3 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, preferably the amino acid sequence of SEQ ID NO: 3.

[0168] In a preferred embodiment, the heavy chain or fragment thereof comprises a heavy chain variable region VH, which comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:7; preferably, the heavy chain variable region VH is the amino acid sequence shown in SEQ ID NO:7.

[0169] In a more preferred embodiment, the heavy chain comprises an amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:9; preferably, the heavy chain is the amino acid sequence shown in SEQ ID NO:9.

[0170] In a preferred embodiment, the GPC3 antibody or antigen-binding fragment thereof comprises a light chain or fragment thereof, wherein the light chain or fragment thereof comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, preferably the amino acid sequence of SEQ ID NO:4; the LCDR2 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:5, preferably the amino acid sequence of SEQ ID NO:5; and the LCDR3 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:6, preferably the amino acid sequence of SEQ ID NO:6.

[0171] In a preferred embodiment, the light chain or fragment thereof comprises a light chain variable region VL, which comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:8; preferably, the light chain variable region VL is the amino acid sequence shown in SEQ ID NO:8.

[0172] In a more preferred embodiment, the light chain comprises an amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; preferably, the light chain is the amino acid sequence shown in SEQ ID NO: 10.

[0173] In some embodiments, the GPC3 antibody or antigen-binding fragment thereof is a Fab, F(ab') or F(ab')2 fragment, preferably a Fab fragment.

[0174] In some preferred embodiments, the Fab, F(ab') or F(ab')2 fragment comprises at least one, two, three, four, five, or six CDR sequences, and the CDR sequences have at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with at least one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.

[0175] In a preferred embodiment, the Fab, F(ab') or F(ab')2 fragment comprises a heavy chain or fragment thereof, wherein the heavy chain or fragment thereof comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, preferably the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, preferably the amino acid sequence of SEQ ID NO: 2; and the HCDR3 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, preferably the amino acid sequence of SEQ ID NO: 3.

[0176] In a preferred embodiment, the heavy chain or fragment thereof comprises a heavy chain variable region VH, which comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:7; preferably, the heavy chain variable region VH is the amino acid sequence shown in SEQ ID NO:7.

[0177] In a more preferred embodiment, the heavy chain comprises an amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; preferably, the heavy chain is the amino acid sequence shown in SEQ ID NO: 11.

[0178] In a preferred embodiment, the Fab, F(ab') or F(ab')2 fragment comprises a light chain or fragment thereof, wherein the light chain or fragment thereof comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, preferably the amino acid sequence of SEQ ID NO:4; the LCDR2 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:5, preferably the amino acid sequence of SEQ ID NO:5; the LCDR3 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:6, preferably the amino acid sequence of SEQ ID NO:6.

[0179] In a preferred embodiment, the light chain or fragment thereof comprises a light chain variable region VL, which comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:8; preferably, the light chain variable region VL is the amino acid sequence shown in SEQ ID NO:8.

[0180] In a more preferred embodiment, the light chain comprises an amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; preferably, the light chain is the amino acid sequence shown in SEQ ID NO: 10.

[0181] In some embodiments, the GPC3 antibody or antigen-binding fragment thereof is a scFv.

[0182] In some preferred embodiments, the scFv comprises at least one, two, three, four, five, or six CDR sequences that have at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6.

[0183] In a preferred embodiment, the scFv comprises a heavy chain variable region VH, wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, preferably the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, preferably the amino acid sequence of SEQ ID NO: 2; and the HCDR3 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, preferably the amino acid sequence of SEQ ID NO: 3.

[0184] In a preferred embodiment, the heavy chain variable region VH comprises an amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:7; preferably, the heavy chain variable region VH is the amino acid sequence shown in SEQ ID NO:7.

[0185] In a preferred embodiment, the scFv comprises a light chain variable region, VL, comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4, preferably the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5, preferably the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, preferably the amino acid sequence of SEQ ID NO: 6.

[0186] In a preferred embodiment, the light chain variable region VL comprises an amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:8; preferably, the light chain variable region VL is the amino acid sequence shown in SEQ ID NO:8.

[0187] In a more preferred embodiment, the scFv comprises an amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; preferably, the scFv is the amino acid sequence shown in SEQ ID NO: 12.

[0188] The proteins, peptides and / or amino acid sequences involved in the present invention should also be understood to include variants or homologs that have the same or similar functions as the proteins, peptides and / or amino acid sequences.

[0189] In the present invention, the variant can be a protein or peptide obtained by substituting, deleting, or inserting one or more amino acids relative to the amino acid sequence of the protein and / or peptide (e.g., an antibody or fragment thereof that specifically binds to GPC3). For example, a functional variant can include a protein or peptide having a modification (e.g., substitution, deletion, and / or insertion) of at least one amino acid (e.g., 1-30, 1-20, 1-10, or, for example, 1, 2, 3, 4, or 5 amino acids). A functional variant can substantially retain the biological activity of the original protein or peptide that has not been modified (e.g., subjected to amino acid substitution, deletion, and / or insertion). For example, a functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the original protein or peptide. For example, the substitution can be a conservative amino acid substitution.

[0190] 2.2. Radionuclides and linking groups

[0191] In the radiolabeled antibody conjugate according to the present invention, a radionuclide is conjugated directly or indirectly to a GPC3 targeting moiety (eg, a GPC3 antibody or an antigen-binding fragment thereof) to form a radiolabeled antibody conjugate.

[0192] Suitable radionuclides include, but are not limited to, those that form stable complexes with the chelating moiety and have physical half-lives suitable for imaging and / or therapeutic purposes. Suitable radionuclides also include those that are directly bonded to the GPC3 targeting moiety (e.g., 76 Br and 131 I) and those introduced by prosthetic groups (e.g. 18 F).

[0193] Examples of chelating moieties include, but are not limited to, structures that form stable complexes with radionuclides, such as deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), triacetylfusine C (TAFC), ferric streptomycin E (FOXE), ferric streptomycin B (FOXB), ferric pigment A (FCHA), etc.

[0194] In some embodiments, the chelating moiety is covalently bonded to the GPC3 targeting moiety (e.g., a GPC3 antibody or antigen-binding fragment thereof) via a linker that covalently links the chelating moiety to the GPC3 targeting moiety. In some embodiments, these linkers are formed by the reaction between a reactive moiety of the GPC3 targeting moiety (e.g., a cysteine ​​or lysine of the antibody) and a reactive moiety linked to the chelating moiety (e.g., a p-isothiocyanatophenyl or amide group) and a reactive moiety provided in the conjugation method described below. In addition, the linker optionally may also contain a chemical group ("spacer") for adjusting polarity, solubility, steric interaction, rigidity, and / or length between the chelating moiety and the GPC3 targeting moiety, such as a single bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C1-C 20 Alkenylene, optionally substituted C1-C 20 Alkynylidene, optionally substituted C1-C 20 Heteroalkylene, optionally substituted C3-C 20 Cycloalkylene, optionally substituted C3-C 20 Heterocycloalkylene, optionally substituted C5-C 20 Arylene, optionally substituted C5-C 20 Heteroarylene group, keto group, -S-, -(C=S)-, -O-, -NH-, -NHCO-, polypeptide (such as valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), polyglycine, etc.), polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol.

[0195] The radionuclides described herein include diagnostic radionuclides and therapeutic radionuclides. Exemplary diagnostic radionuclides include, but are not limited to: 68 Ga, 18 F-Al, 64 Cu, 99m Tc, 89 Zr, 44 Sc, 111 In, 45 Ti, 52 Mn, 59 Fe, 94m Tc, 67 Ga, 71 As、 72 As、 82m Rb, 86 Y. 131I, Gd, Mn or any combination thereof. Exemplary therapeutic radionuclides include, but are not limited to: 177 Lu, 90 Y. 153 Sm, 67 Cu, 89 Sr. 166 Ho, 177 Yb, 47 Sc, 131 I. 186 Re、 188 Re、 212 Bi, 213 Bi, 149 Pm, 212 Pb, 211 At 223 Ra, 225 Ac, 227 Th or any combination thereof.

[0196] 2.3. Preparation of radiolabeled conjugates

[0197] Radiolabeled GPC3 conjugates can be prepared by: (1) reacting a GPC3 targeting moiety (e.g., an antibody or antigen-binding fragment thereof) with a molecule containing a chelating moiety and a moiety capable of reacting with the desired conjugation site of the GPC3 targeting moiety; and (2) loading with the desired radionuclide.

[0198] Suitable conjugation sites include, but are not limited to, lysine and cysteine, which can be, for example, natural or synthetic, and can, for example, be present in the heavy or light chain of an antibody. Cysteine ​​conjugation sites include, but are not limited to, structures obtained by mutation, insertion, or reduction of antibody disulfide bonds. Methods for preparing cysteine ​​engineered antibodies include, but are not limited to, those disclosed in WO2011 / 056983. Site-specific conjugation methods can also be used to guide the conjugation reaction to the specific site of the antibody, achieve the desired stoichiometry, and / or achieve the desired drug to antibody (DAR) ratio. Such conjugation methods are well known to those of ordinary skill in the art, including, but not limited to, cysteine ​​engineering and enzymatic and chemo-enzymatic methods, including, but not limited to, glutamine conjugation, Q295 conjugation, and transglutaminase-mediated conjugation, as well as those described in J.Clin.Immunol., 36:100 (2016), the entire contents of which are incorporated herein by reference. Suitable moieties that react with the desired conjugation site are generally capable of efficiently and easily coupling the GPC3 targeting moiety (e.g., an antibody or antigen-binding fragment thereof) to the chelating moiety of the radionuclide. Moieties that react with lysine and cysteine ​​sites include electrophilic groups, which are well known to those of ordinary skill in the art. In certain aspects, when the desired conjugation site is lysine, the reactive moiety is an isothiocyanate (e.g., p-isothiocyanatophenyl or a reactive ester). In certain aspects, when the desired conjugation site is cysteine, the reactive moiety is a maleimide.

[0199] In some embodiments, suitable reactive moieties include, but are not limited to, p-isothiocyanatophenyl, N-hydroxysuccinimide esters, maleimido, 2,3,5,6-tetrafluorophenol esters, N-succinimidyl-S-acetylthioacetate, etc. In some embodiments, molecules comprising a chelating moiety and a moiety reactive with a conjugation site include, but are not limited to, p-isothiocyanatophenyl-NOTA (p-SCN-Bn-NOTA), p-isothiocyanatophenyl-DOTA (p-SCN-Bn-DOTA), p-isothiocyanatophenyl-deferoxamine (p-SCN-Bn-DFO), or a tetrafluorophenyl ester derivative of RESCA (H3RESCA-TFP).

[0200] Loading of the radionuclide is achieved by incubating the GPC3 targeting moiety and chelating moiety conjugate with the radionuclide for a period of time sufficient to allow the radionuclide to be coordinated to the chelating moiety, for example, by performing the methods described in the Examples provided herein, or substantially similar methods.

[0201] 2.4. Exemplary Embodiments of Conjugates

[0202] In some embodiments, the radiolabeled molecular probe provided by the present invention is a conjugate having the following structure, or a pharmaceutically acceptable salt or ester thereof: Ab m -R n (I)

[0203] Wherein, Ab is a targeting moiety that binds to GPC3, such as a GPC3 antibody, including an antigen-binding fragment, scFv, or single-domain antibody thereof; R is selected from the group consisting of a linker, a chelating moiety, a radionuclide, other targeting polypeptides or antibodies, antibody fragments, single-domain antibodies, and small molecule inhibitors; n is an integer from 0 to 4; m is not 0, but is preferably an integer from 1 to 4, and more preferably 1 or 2.

[0204] In some embodiments, the molecular probe provided by the present invention is a compound having the following structure, or a pharmaceutically acceptable salt or ester thereof: Ab m -(LM z ) n (II)

[0205] wherein Ab is a targeting moiety that binds to GPC3; L is a linking group; M is a radionuclide, z is 0 or 1; n is an integer of 1-4, preferably 1 or 2; and m is an integer of 1-4, preferably 1 or 2.

[0206] In some embodiments, L is represented by the formula: Y-Lk-T (III)

[0207] Wherein, Y is a linker, Lk is a coupling group, and T is a chelating moiety.

[0208] In some embodiments, Lk is independently selected from a single bond, p-isothiocyanatophenyl, N-hydroxysuccinimide ester, maleimide, 2,3,5,6-tetrafluorophenol ester, N-succinimidyl-S-acetylthioacetate, amide, or a combination thereof. In some embodiments, Lk is independently selected from a single bond, p-isothiocyanatophenyl, amide, or maleimide. In some embodiments, Lk is p-isothiocyanatophenyl or amide. In some embodiments, Lk is p-isothiocyanatophenyl.

[0209] In some embodiments, Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C1-C 20 Alkenylene, optionally substituted C1-C 20 Alkynylidene, optionally substituted C1-C 20 Heteroalkylene, optionally substituted C3-C 20 Cycloalkylene, optionally substituted C3-C 20Heterocycloalkylene, optionally substituted C5-C 20 Arylene, optionally substituted C5-C 20 heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, polypeptides (such as valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), polyglycine), polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, copolymers of polyethylene glycol and polypropylene glycol, or combinations thereof; the reactive portion is linked to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, N-hydroxysuccinimide ester, maleimide, 2,3,5,6-tetrafluorophenol ester, N-succinimidyl-S-acetylthioacetate, amide, or combinations thereof.

[0210] In some embodiments, Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, an optionally substituted C1-C 10 Alkylene, optionally substituted C1-C 10 Alkenylene, optionally substituted C1-C 10 Alkynylidene, optionally substituted C1-C 10 Heteroalkylene, optionally substituted C3-C 10 Cycloalkylene, optionally substituted C3-C 10 Heterocycloalkylene, optionally substituted C5-C 10 Arylene, optionally substituted C5-C 10 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, -(CH2CH2O) r -(also known as -(PEG) r -)、-(CH2CH2CH2O) r -, Valine-Citrulline (Val-Cit), Valine-Alanine (Val-Ala), Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), Phenylalanine-Lysine (Phe-Lys), Alanine-Alanine-Asparagine (Ala-Ala-Asn), -(Gly) r -, or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, N-hydroxysuccinimide ester, maleimide, 2,3,5,6-tetrafluorophenol ester, N-succinimidyl-S-acetylthioacetate, amide, or a combination thereof.

[0211] In some embodiments, Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof.

[0212] In some embodiments, T is independently selected from a single bond or a ligand derived from any of the following structures: deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), ethylenediaminetetraacetic acid (EDTA), DTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), triacetylfusine C (TAFC), ferric streptomycin E (FOXE), ferric streptomycin B (FOXB), ferric chromogen A (FCHA), or a combination thereof.

[0213] In some embodiments, T is independently selected from a single bond or a ligand derived from any of the following structures: deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP), or a combination thereof.

[0214] In some embodiments, T is independently selected from a single bond or a ligand derived from any of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), or a combination thereof.

[0215] In some embodiments, T is independently selected from a ligand derived from any one of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), or a combination thereof.

[0216] In some embodiments, Lk and T may further comprise a spacer group, wherein the spacer group is independently selected from a single bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C1-C 20 Alkenylene, optionally substituted C1-C 20 Alkynylidene, optionally substituted C1-C 20 Heteroalkylene, optionally substituted C3-C 20 Cycloalkylene, optionally substituted C3-C 20 Heterocycloalkylene, optionally substituted C5-C 20 Arylene, optionally substituted C5-C 20 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, polypeptides (such as valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), polyglycine (Gly) r , wherein r is an integer from 1 to 10), polyethylene glycol ((PEG) r , wherein r is an integer of 1-10), polypropylene glycol, polyoxyalkylene, a copolymer of polyethylene glycol and polypropylene glycol, or a combination thereof; preferably selected from single bonds, optionally substituted C1-C 10 Alkylene, optionally substituted C1-C 10 Alkenylene, optionally substituted C1-C 10 Alkynylidene, optionally substituted C1-C10 Heteroalkylene, optionally substituted C3-C 10 Cycloalkylene, optionally substituted C3-C 10 Heterocycloalkylene, optionally substituted C5-C 10 Arylene, optionally substituted C5-C 10 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, -(CH2CH2O) r -(also known as -(PEG) r -)、-(CH2CH2CH2O) r -, Valine-Citrulline (Val-Cit), Valine-Alanine (Val-Ala), Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), Phenylalanine-Lysine (Phe-Lys), Alanine-Alanine-Asparagine (Ala-Ala-Asn), -(Gly) r -, or a combination thereof, wherein r is an integer from 1 to 10; more preferably selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10.

[0217] In some embodiments, Lk is independently selected from a single bond, p-isothiocyanatophenyl, an amide group, or a maleimide group; Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, an optionally substituted C1-C 10 Alkylene, optionally substituted C1-C 10 Alkenylene, optionally substituted C1-C 10 Alkynylidene, optionally substituted C1-C 10 Heteroalkylene, optionally substituted C3-C 10 Cycloalkylene, optionally substituted C3-C 10 Heterocycloalkylene, optionally substituted C5-C 10 Arylene, optionally substituted C5-C 10 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, -(PEG) r -, Valine-Citrulline (Val-Cit), Valine-Alanine (Val-Ala), Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), Phenylalanine-Lysine (Phe-Lys), Alanine-Alanine-Asparagine (Ala-Ala-Asn), -(Gly) r- or a combination thereof, wherein r is an integer from 1 to 10, the reactive portion is linked to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof; and T is independently selected from a single bond or a ligand derived from any one of the following structures: deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA ), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), or 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP).

[0218] In some embodiments, Lk is independently selected from a single bond, p-isothiocyanatophenyl, or an amide group; Y comprises a reactive portion and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10, the reactive portion is linked to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, a maleimide group, an amide group, or a combination thereof; and T is independently selected from a single bond or a ligand derived from any one of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

[0219] In some embodiments, Lk is independently selected from p-isothiocyanatophenyl or amide; Y is a single bond; and T is independently selected from a ligand derived from any one of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

[0220] In some embodiments, Lk is p-isothiocyanatophenyl; Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10, the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, a maleimide group, an amide group, or a combination thereof; and T is independently selected from or derived from a ligand of any one of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA) or diethylenetriaminepentaacetic acid (DTPA).

[0221] In some embodiments, Lk is p-isothiocyanatophenyl; Y is a single bond; and T is independently selected from or derived from a ligand of any of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), or diethylenetriaminepentaacetic acid (DTPA).

[0222] In some embodiments, Lk is an amide group; Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10, the reactive portion is linked to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, a maleimide group, an amide group, or a combination thereof; and T is independently selected from or derived from a ligand having any one of the following structures: mercaptoacetyl triglycine (MAG3) or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

[0223] In some embodiments, Lk is an amide group; Y is a single bond; and T is independently selected from a ligand derived from any one of the following structures: mercaptoacetyl triglycine (MAG3) or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

[0224] In some embodiments, L is independently selected from a ligand derived from any of the following structures:

[0225] The linker comprises a reactive portion and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly)r - or a combination thereof, wherein r is an integer from 1 to 10, the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanate phenyl, maleimide, amide, or a combination thereof, preferably, the linker is a single bond.

[0226] In some embodiments, M is selected from a diagnostic radionuclide or a therapeutic radionuclide. In some embodiments, the diagnostic radionuclide is selected from 68 Ga, 18 F-Al, 64 Cu, 99m Tc, 89 Zr, 44 Sc, 111 In, 45 Ti, 52 Mn, 59 Fe, 94m Tc, 67 Ga, 71 As、 72 As、 82m Rb, 86 Y. 131 I, Gd, Mn or a combination thereof. In some embodiments, the therapeutic radionuclide is selected from 177 Lu, 90 Y. 153 Sm, 67 Cu, 89 Sr. 131 I. 166 Ho, 177 Yb, 47 Sc, 186 Re、 188 Re、 212 Bi, 213 Bi, 149 Pm, 212 Pb, 211 At 223 Ra, 225 Ac, 227 Th or a combination thereof.

[0227] In some embodiments, M is selected from 68 Ga, 18 F-Al, 99m Tc, 177 Lu, 131 I, or a combination thereof.

[0228] In some embodiments, LM is selected from the following structures: or -Connector- 131 I,

[0229] wherein the linker comprises a reactive portion and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10, the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanate phenyl, maleimide, amide, or a combination thereof, preferably, the linker is a single bond.

[0230] In some embodiments, the Ab is a GPC3 antibody or an antigen-binding fragment thereof. In some embodiments, the GPC3 antibody or antigen-binding fragment thereof is a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the GPC3 antibody or antigen-binding fragment thereof is selected from a monoclonal antibody (mAb), Fab, Fab', F(ab')2, Fv, scFv, scFv-Fc, or a single-domain antibody.

[0231] In some embodiments, Ab is a monoclonal antibody, a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, or a scFv fragment.

[0232] In some embodiments, the Ab comprises at least one, two, three, four, five, or six CDR sequences that have at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6.

[0233] In some embodiments, Ab comprises a heavy chain or a fragment thereof, wherein the heavy chain or fragment thereof comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, preferably the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, preferably the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, preferably the amino acid sequence of SEQ ID NO: 3.

[0234] In some embodiments, Ab comprises a heavy chain variable region VH, which comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:7; preferably, the heavy chain variable region VH is the amino acid sequence shown in SEQ ID NO:7.

[0235] In some embodiments, Ab comprises a heavy chain amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:9; preferably, Ab comprises the heavy chain amino acid sequence shown in SEQ ID NO:9.

[0236] In some embodiments, Ab comprises a light chain or a fragment thereof, wherein the light chain or fragment thereof comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, preferably the amino acid sequence of SEQ ID NO:4; the LCDR2 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:5, preferably the amino acid sequence of SEQ ID NO:5; the LCDR3 comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:6, preferably the amino acid sequence of SEQ ID NO:6.

[0237] In some embodiments, Ab comprises a light chain variable region VL, which comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; preferably, the light chain variable region VL is the amino acid sequence shown in SEQ ID NO: 8.

[0238] In some embodiments, Ab comprises a light chain amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 10; preferably, Ab comprises the light chain amino acid sequence shown in SEQ ID NO: 10.

[0239] In some embodiments, Ab is a Fab fragment, which comprises a heavy chain amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 11, preferably comprises the heavy chain amino acid sequence shown in SEQ ID NO: 11; and the Fab fragment comprises a light chain amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, preferably comprises the light chain amino acid sequence shown in SEQ ID NO: 10.

[0240] In some embodiments, Ab is a scFv and comprises an amino acid sequence that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 12; preferably, Ab is the amino acid sequence shown in SEQ ID NO: 12.

[0241] In some embodiments, the radiolabeled molecular probe provided by the present invention is selected from the following structures:

[0242] wherein the linker comprises a reactive portion and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10, the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanate phenyl, maleimide, amide, or a combination thereof, preferably, the linker is a single bond.

[0243] III. Methods Using Radiolabeled GPC3 Molecular Probes

[0244] In some aspects, the present invention provides diagnostic and therapeutic methods using the radiolabeled molecular probes of the invention.

[0245] According to one aspect, the present invention provides a method for detecting GPC3 in a tissue, the method comprising administering to the tissue a radiolabeled molecular probe provided herein; and visualizing GPC3 expression by single photon emission computed tomography (SPECT), positron emission tomography (PET), or magnetic resonance imaging (MRI). In some embodiments, the tissue comprises a cell or cell line. In some embodiments, the tissue is present in a subject, wherein the subject is a mammal. In some embodiments, the subject is a human subject. In some embodiments, the subject suffers from a disease or condition selected from the group consisting of cancer, an infectious disease, and an inflammatory disease. In one embodiment, the subject suffers from cancer.

[0246] According to one aspect, the present invention provides a method for measuring a response to a treatment, wherein the response to the treatment is measured by measuring inflammation. According to this aspect, the method comprises administering a radiolabeled molecular probe provided herein to a subject in need thereof, and visualizing GPC3 expression by SPECT, PET, or MRI. In some embodiments, inflammation is present in a tumor in the subject. In some embodiments, increased GPC3 expression correlates with increased inflammation in the tumor.

[0247] According to one aspect, the present invention provides a method for determining whether a patient is suitable for anti-tumor therapy comprising a GPC3 inhibitor, the method comprising selecting a patient having a solid tumor, administering a radiolabeled molecular probe of the present invention, and localizing the administered radiolabeled molecular probe in the tumor by SPECT, PET or MRI, wherein the presence of the radiolabeled molecular probe in the tumor indicates that the patient is suitable for anti-tumor therapy comprising a GPC3 inhibitor.

[0248] According to one aspect, the present invention provides a method for detecting a GPC3-positive tumor in a subject. According to this aspect, the method comprises selecting a subject having a solid tumor, administering to the subject a radiolabeled molecular probe of the present invention, and determining the localization of the radiolabeled molecular probe by SPECT, PET, or MRI, wherein the presence of the radiolabeled molecular probe in the tumor indicates that the tumor is GPC3-positive.

[0249] According to one aspect, the present invention provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a radiolabeled molecular probe of the present invention. The molecular probe can be administered by any suitable route known to those skilled in the art, including intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, or intratumoral injection. In some embodiments, the method of treatment of the present invention comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises the radiolabeled molecular probe of the present invention and a pharmaceutically acceptable carrier.

[0250] As used in this application, the expression "object in need thereof" refers to a human or non-human mammal that exhibits one or more symptoms or indications of cancer, and / or has been diagnosed with cancer (including solid tumors) and needs to be treated. In many embodiments, the term "object" can be used interchangeably with the term "patient". For example, a human subject can be diagnosed with a primary or metastatic tumor and / or have one or more symptoms or indications, including but not limited to unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, abdominal distension, chest pain / pressure, splenomegaly, and elevated levels of cancer-related biomarkers. This statement includes objects with primary or established tumors. In specific embodiments, this statement includes human subjects suffering from and / or needing to be treated for solid tumors (e.g., colon cancer, breast cancer, lung cancer, prostate cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, cervical cancer, pancreatic cancer, head and neck cancer, and brain cancer). This term includes objects suffering from primary or metastatic tumors (advanced malignancies). In some embodiments, the phrase "a subject in need thereof" includes patients with solid tumors that are resistant to, difficult to treat, or inadequately controlled by previous therapies (e.g., treatment with anticancer agents). For example, the phrase includes subjects who have been treated with one or more previous therapies, such as chemotherapy (e.g., carboplatin or docetaxel). In some embodiments, the phrase "a subject in need thereof" includes patients with solid tumors that have been treated with one or more previous therapies but have subsequently relapsed or metastasized.

[0251] In some embodiments, the method of the present invention is used for the object suffering from solid tumor.Term " tumor ", " cancer " and " malignant tumor " are used interchangeably in this application.As used in this application, term " solid tumor " refers to the abnormal tissue mass that does not usually contain cyst or liquid area.Solid tumor can be benign (not cancer) or malignant (cancer).For purposes of the present invention, term " solid tumor " refers to malignant solid tumor.This term includes different types of solid tumors named after the cell type that forms it, i.e. sarcoma, carcinoma and lymphoma.In some embodiments, term " solid tumor " includes cancer, and it includes but is not limited to colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.

[0252] IV. Examples

[0253] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0254] Example 1. Preparation of mAbs, Fabs, and scFvs targeting GPC3

[0255] The variable region VH and VL sequences of mAb, Fab, and scFv antibodies targeting GPC3 are derived from the variable region VH and VL sequences of P17-8E12 in patent application PCT / US2022 / 077807, the entire contents of which are incorporated herein by reference.

[0256] The variable regions (VH and VL) of P17-8E12 were cloned into a mammalian IgG1 expression vector that provides the constant regions of human IgG1, including CL, CH1-CH2-CH3. The final sequence of the light chain of the GPC3-targeting mAb is shown in SEQ ID NO: 10, and the final sequence of the heavy chain is shown in SEQ ID NO: 9. The mAb was expressed in Expi293F cells using a standard transient transfection protocol and purified from the cell culture supernatant using MabSelect Prism A to obtain a GPC3-targeting mAb (aGPC3-mAb).

[0257] The complete sequence of the GPC3-targeting scFv (SEQ ID NO: 12) was obtained by linking the variable region sequences VH and VL of P17-8E12 with a linker (G4S)3 and introducing six histidine residues at the C-terminus. This sequence was cloned into a mammalian expression vector. The scFv was expressed in Expi293F cells using standard transient transfection protocols and purified from the cell culture supernatant using Ni Sepharose High Performance to obtain the GPC3-targeting scFv antibody (aGPC3-scFv).

[0258] The P17-8E12 variable region sequence was cloned into a mammalian Fab expression vector that provides the human Fab constant regions, including CL and CH1. The final sequence of the light chain of the GPC3-targeting Fab is shown in SEQ ID NO: 10, and the final sequence of the heavy chain is shown in SEQ ID NO: 11. The GPC3-targeting Fab was expressed in ExpiCHO-S cells using a standard transient transfection protocol and purified from the cell culture supernatant using MabSelect Prism A to obtain a GPC3-targeting Fab antibody (aGPC3-Fab).

[0259] Example 2. 68 Preparation of Ga-NOTA-aGPC3-Fab

[0260] 2.1 Preparation of NOTA-aGPC3-Fab

[0261] The antibody aGPC3-Fab was purified using a PD-10 desalting column. The purity of the antibody aGPC3-Fab was confirmed by size exclusion chromatography (SEC-HPLC). 20 nmol of the purified antibody aGPC3-Fab was adjusted to a pH of 8.5-9.0 with Na2CO3 (0.1 M, pH 9.0). A 20-30-fold molar ratio of p-SCN-Bn-NOTA (dissolved in a small amount of DMSO) was added and the reaction was allowed to proceed overnight at 4°C in the dark to synthesize NOTA-aGPC3-Fab. Excess unreacted NOTA was removed by purification using a PD-10 desalting column. The purified antibody was concentrated using an ultrafiltration centrifuge tube (Millipore 0.5 mL / 3Kd) at 4°C, 13,000 × g, for 30 minutes and then stored in a refrigerator at 4°C. The concentrated antibody solution is stable at 4°C for at least 3 months.

[0262] 2.2 68 Radiolabeling with Ga

[0263] Elute with 4 mL of 0.05 M HCl 68 Ge / 68 Ga generator, using sodium acetate (0.25 M, pH 6.8) 68 The pH of GaCl3 was adjusted to 4.0-4.5. The purified NOTA-aGPC3-Fab (3 nmol) was mixed with 68 GaCl3 (111 MBq, 3 mCi) was thoroughly mixed and reacted at 37°C for 20 min. The labeled mixture was purified using a PD-10 desalting column. The radiochemical purity of the product was determined by radioactive instant thin-layer chromatography (ITLC) using citric acid-sodium citrate buffer (pH 4.0) as the developing solvent.

[0264] 2.3 68 Radiochemical characterization of Ga-NOTA-aGPC3-Fab

[0265] Take 3.70MBq 68 Ga-NOTA-aGPC3-Fab was added to 100 μL PBS and incubated at room temperature for 1 hour. A PBS sample was directly taken and subjected to SEC-HPLC to determine radiochemical purity. The elution conditions were the same as those in Example 2.1.

[0266] Prepared 68 Ga-NOTA-aGPC3-Fab was identified and characterized by analytical SEC-HPLC. SEC-HPLC results showed that the purity of antibody aGPC3-Fab was greater than 99% (Figure 1A). 68The radiochemical purity of Ga-NOTA-aGPC3-Fab was greater than 99% ( FIG. 1B ).

[0267] Example 3. 68 Preparation of Ga-NOTA-aGPC3-scFv

[0268] 3.1 Preparation of NOTA-aGPC3-scFv

[0269] The antibody aGPC3-scFv was purified using a PD-10 desalting column. The purity of the antibody aGPC3-scFv was confirmed by size exclusion chromatography (SEC-HPLC). 20 nmol of the purified antibody aGPC3-scFv was adjusted to a pH of 8.5-9.0 with Na2CO3 (0.1 M, pH 9.0). A 15-fold molar ratio of p-SCN-Bn-NOTA (dissolved in a small amount of DMSO) was added and the reaction was allowed to proceed overnight at 4°C in the dark to synthesize NOTA-aGPC3-scFv. Excess unreacted NOTA was removed by purification using a PD-10 desalting column. The purified antibody was concentrated using an ultrafiltration centrifuge tube (Millipore 0.5 mL / 3 kd) at 4°C, 13,000 × g, for 30 min, and then stored in a refrigerator at 4°C.

[0270] 3.2 68 Radiolabeling with Ga

[0271] By using 4 mL of 0.05 M HCl solution from 68 Ge / 68 The Ga radionuclide was eluted from a Ga radionuclide generator (ITM Medical Isotopes GmbH, Germany). 68 For radiolabeling, 740 MBq of GaCl3 was added to the solution with 0.25 M sodium acetate. 68 GaCl3 was adjusted to pH 4.0, and then 5 nmol of the anti-GPC3 antibody NOTA-aGPC3-scfv was added. The reaction mixture was stirred at 35°C for 30 minutes and then purified using a PD-10 desalting column (GE, USA). Radioactive transient thin-layer chromatography (ITLC) using 0.5 M EDTA as the mobile phase was used to assess the labeling efficiency of PD-10 before purification and the radiochemical purity after purification.

[0272] 3.3 68 Radiochemical characterization of Ga-NOTA-aGPC3-scFv

[0273] Take 3.70MBq 68Ga-NOTA-aGPC3-scFv was added to 100 μL FBS and incubated at room temperature for 2 h. An FBS sample was directly taken for radiochemical purity by SEC-HPLC, using the same elution conditions as in Example 2.

[0274] Prepared 68 The labeling efficiency of Ga-NOTA-aGPC3-scfv before purification and the radiochemical purity after purification were evaluated by radioactive transient thin layer chromatography (ITLC) using 0.5 M EDTA as the mobile phase. The ITLC results (Figure 2) indicate that the PET probe was successfully prepared. 68 The labeling efficiency of Ga-NOTA-aGPC3-scfv was approximately 67.2%. SEC-HPLC results showed that the radiochemical purity of the labeled mixture after purification on a PD-10 desalting column was greater than 98%.

[0275] Example 4. 68 Preparation of Ga-DOTA-aGPC3-scFv

[0276] 4.1 Preparation of DOTA-aGPC3-scFv

[0277] To synthesize DOTA-aGPC3-scfv, 50 nmol of aGPC3-scfv antibody was adjusted to pH 8.5-9.0 with 0.1 M Na2CO3. A 20-fold molar ratio of p-SCN-Bn-DOTA (dissolved in a small amount of DMSO) was added and allowed to react at 25°C in the dark for 4 h. Excess unreacted DOTA was removed using a PD-10 desalting column. The purified antibody was concentrated using an ultrafiltration centrifuge (Millipore 0.5 mL / 3Kd) at 4°C, 13,000 × g, for 30 min and then stored in a refrigerator at 4°C. The concentrated antibody solution is stable at 4°C for at least 3 months.

[0278] 4.2 68 Radiolabeling with Ga

[0279] By using 4 mL of 0.05 M HCl solution from 68 Ge / 68 The Ga radionuclide was eluted from a Ga radionuclide generator (ITM Medical Isotopes GmbH, Germany). 68 For radiolabeling, 740 MBq of GaCl3 was added to the solution with 0.25 M sodium acetate. 68The pH was adjusted to 4.0 with GaCl₃, followed by the addition of 30 nmol of the anti-GPC3 antibody DOTA-aGPC3-scfv. The mixture was shaken at 35°C for 30 minutes and then purified using a PD-10 desalting column (GE, USA). Radioactive transient thin-layer chromatography (ITLC) using 0.5 M EDTA as the mobile phase was used to assess the PD-10 labeling efficiency before purification and the radiochemical purity after purification.

[0280] 4.3 68 Radiochemical characterization of Ga-DOTA-aGPC3-scFv

[0281] Prepared 68 The labeling efficiency of Ga-DOTA-aGPC3-scfv before PD-10 purification and the radiochemical purity after purification were assessed by radioactive transient thin-layer chromatography (ITLC) using 0.5 M EDTA as the mobile phase.

[0282] Prepared 68 Ga-NOTA-aGPC3-Fab was identified and characterized by analytical SEC-HPLC. The results showed that the PET probe was successfully prepared. 68 Ga-DOTA-aGPC3-scfv, ITLC (Figure 3) showed a labeling efficiency of approximately 28.78%. SEC-HPLC showed that after purification of the labeling mixture using a PD-10 desalting column, the radiochemical purity was greater than 90%.

[0283] Example 5. 68 Preparation of Ga-DOTA-aGPC3-Fab

[0284] 5.1 Preparation of DOTA-aGPC3-Fab

[0285] To synthesize NOTA-aGPC3-Fab, 50 nmol of aGPC3-Fab antibody was adjusted to pH 8.5-9.0 with 0.1 M Na2CO3. A 20-fold molar ratio of p-SCN-Bn-DOTA (dissolved in a small amount of DMSO) was added and allowed to react at 25°C in the dark for 4 h. Excess unreacted NOTA was removed using a PD-10 desalting column. The purified antibody was concentrated using an ultrafiltration centrifuge (Millipore 0.5 mL / 3Kd) at 4°C, 13,000 × g, for 30 min, and then stored in a refrigerator at 4°C. The concentrated antibody solution is stable at 4°C for at least 3 months.

[0286] 5.2 68 Radiolabeling with Ga

[0287] By using 4 mL of 0.05 M HCl solution from 68Ge / 68 The Ga radionuclide was eluted from a Ga radionuclide generator (ITM Medical Isotopes GmbH, Germany). 68 For radiolabeling, 740 MBq of GaCl3 was added to the solution with 0.25 M sodium acetate. 68 The pH was adjusted to 4.0 with GaCl3, and then 30 nmol of the anti-GPC3 antibody DOTA-aGPC3-Fab was added. The reaction mixture was shaken at 35°C for 30 minutes and then purified using a PD-10 desalting column (GE, USA). Radioactive transient thin-layer chromatography (ITLC) using 0.5 M EDTA as the mobile phase was used to assess the PD-10 labeling efficiency before purification and the radiochemical purity after purification.

[0288] 5.3 68 Radiochemical characterization of Ga-DOTA-aGPC3-Fab

[0289] Prepared 68 The labeling efficiency of Ga-DOTA-aGPC3-Fab before PD-10 purification and the radiochemical purity after purification were evaluated by thin layer chromatography (TLC) using 0.5 M EDTA as the mobile phase.

[0290] The results showed that the PET probe was successfully prepared 68 Ga-DOTA-aGPC3-Fab, ITLC showed that the labeling rate was about 67.41% (Figure 4), and SEC-HPLC results showed that the radiochemical purity was greater than 99%.

[0291] Example 6. 18 Preparation of FAl-H3RESCA-aGPC3-scFv

[0292] 6.1 Preparation of H3RESCA-aGPC3-scFv

[0293] The antibody aGPC3-scFv was purified using a PD-10 desalting column. The purity of the antibody aGPC3-scFv was confirmed by size exclusion chromatography (SEC-HPLC). To 20 nmol of the purified antibody aGPC3-scFv, a 12-fold molar ratio of (±)-H3RESCA-TFP (dissolved in a small amount of DMSO) was added. The pH of the reaction solution was adjusted to 8.5-9.0 with a small amount of Na2CO3 solution (0.1 M). The reaction was shaken at room temperature in the dark for 2 h to synthesize H3RESCA-aGPC3-scFv. Excess unreacted (±)-H3RESCA-TFP was removed using a PD-10 desalting column. The equilibration and eluent were both 0.1 M ammonium acetate buffer (pH = 4.4-4.6). The purified antibody was concentrated using an ultrafiltration centrifuge tube (Millipore 0.5 mL / 3KD) at 4°C, 13,000 × g, for 30 min, and stored in a refrigerator at 4°C.

[0294] 6.2 18 Radiolabeling of F

[0295] A batch of high-activity fluoride water (>37MBq) was treated with an activated QMA column and eluted with metal-free saline. 18 F ions, add an appropriate amount of 2mM AlCl3 solution (per 1.1-2.2GBq 18 F- Add 20 nmol AlCl3) and incubate at room temperature for 5 min. Then add 20 nmol H3RESCA-aGPC3-scFv and shake at room temperature for 15 min. Purify the product using PD-10 desalting column, and use physiological saline as the equilibration and elution buffer to obtain 18 The labeling efficiency and radiochemical purity of FAl-H3RESCA-aGPC3-scFv were calculated using radioactive transient thin-layer chromatography (ITLC) (with normal saline as the mobile phase).

[0296] 6.3 18 Radiochemical characterization of FAl-H3RESCA-aGPC3-scFv

[0297] Prepared 18 FAl-H3RESCA-aGPC3-scFv was identified and characterized by analytical SEC-HPLC. SEC-HPLC results showed that the purity of antibody aGPC3-scFv was greater than 99%. 18 The radiochemical purity of FAl-H3RESCA-aGPC3-scFv was greater than 99%. After purification of the labeling mixture using a PD-10 desalting column, the radiochemical purity was greater than 95%.

[0298] Example 7. 177Preparation of Lu-DOTA-aGPC3-Fab

[0299] 7.1 Preparation of DOTA-aGPC3-Fab

[0300] The antibody aGPC3-Fab was purified using a PD-10 desalting column. The purity of the antibody aGPC3-Fab was confirmed by size exclusion chromatography (SEC-HPLC). 20 nmol of the purified antibody aGPC3-Fab was adjusted to pH 8.5-9.0 with 0.1 M Na2CO3-NaHCO3 (pH 9.0). A 20-30-fold molar ratio of p-SCN-Bn-DOTA (dissolved in a small amount of DMSO) was added and the reaction was allowed to proceed overnight at 4°C in the dark to synthesize DOTA-aGPC3-Fab. Excess unreacted DOTA was removed by purification using a PD-10 desalting column. The purified antibody was concentrated using an ultrafiltration centrifuge tube (Millipore 0.5 mL / 30 kd) at 4°C, 8000 × g, for 10 min. For short-term storage, the antibody was stored in a 4°C freezer. For long-term storage, the antibody was stored in aliquots at -80°C.

[0301] 7.2 177 Radiolabeling of Lu

[0302] Pick 177 LuCl3 solution (185 MBq) was adjusted to pH 5.5 by adding 500 μL of ammonium acetate (0.1 M, pH 5.5). Approximately 200 μg of DOTA-aGPC3-Fab was then added, mixed thoroughly, and reacted at 37°C for 1 hour. The reaction product was purified using a PD-10 desalting column, and its radiochemical purity was determined by radioactive instant thin-layer chromatography (ITLC) using citric acid-sodium citrate buffer (pH 4.0) as the developing solvent.

[0303] 7.3 177 Radiochemical characterization of Lu-DOTA-aGPC3-Fab

[0304] Prepared 177 Lu-DOTA-aGPC3-Fab was identified and characterized by analytical Radio-ITLC. A new immuno-SPECT probe was successfully prepared. 177 Lu-DOTA-aGPC3-Fab, radioactive ITLC results showed that the labeling rate was about 82.5% (Figure 5A), and SEC-HPLC results showed 177 The radiochemical purity of Lu-DOTA-aGPC3-Fab was greater than 97%, and the stability in PBS for 24 h was greater than 85% ( FIG5B ).

[0305] Example 8. 177 Preparation of Lu-DOTA-aGPC3-scFv

[0306] 8.1 Preparation of DOTA-aGPC3-scFv

[0307] The antibody aGPC3-scFv was purified using a PD-10 desalting column. The purity of the antibody aGPC3-scFv was confirmed by size exclusion chromatography (SEC-HPLC). 20 nmol of the purified antibody aGPC3-scFv was adjusted to pH 8.5-9.0 with 0.1 M Na2CO3-NaHCO3 (pH 9.0). A 20-fold molar ratio of p-SCN-Bn-DOTA (dissolved in a small amount of DMSO) was added and the reaction was allowed to proceed overnight at 4°C in the dark to synthesize DOTA-aGPC3-scFv. Excess unreacted DOTA was removed using a PD-10 desalting column. The purified antibody was concentrated using an ultrafiltration centrifuge tube (Millipore 0.5 mL / 10Kd) at 4°C, 10,000 × g, for 15 minutes. For short-term storage, the antibody was stored in a 4°C freezer. For long-term storage, the antibody was stored in aliquots at -80°C.

[0308] 8.2 177 Radiolabeling of Lu

[0309] Pick 177 LuCl3 solution (185 MBq) was adjusted to pH 5.5 by adding 500 μL of ammonium acetate (0.1 M, pH 5.5). Approximately 400 μg of DOTA-aGPC3-scFv was then added, mixed thoroughly, and reacted at 37°C for 1 hour. The reaction product was purified using a PD-10 desalting column, and its radiochemical purity was determined by radioactive instant thin-layer chromatography (ITLC) using citric acid-sodium citrate buffer (pH 4.0) as the developing solvent.

[0310] 8.3 177 Radiochemical characterization of Lu-DOTA-aGPC3-scFv

[0311] Prepared 177 Lu-DOTA-aGPC3-scFv was identified and characterized by analytical radio-ITLC. A new immuno-SPECT probe was successfully prepared. 177 Lu-DOTA-aGPC3-scFv, radioactive iTLC results showed that the labeling rate was about 73.1% (Figure 6), and SEC-HPLC results showed 177 The radiochemical purity of Lu-DOTA-aGPC3-scFv was greater than 95%.

[0312] Example 9. 177 Preparation of Lu-DOTA-aGPC3-mAb

[0313] 9.1 Preparation of DOTA-aGPC3-mAb

[0314] The antibody aGPC3-mAb was purified using a PD-10 desalting column. The purity of the antibody aGPC3-mAb was confirmed by size exclusion chromatography (SEC-HPLC). 20 nmol of the purified antibody aGPC3-mAb was adjusted to a pH of 8.5-9.0 using 0.1 M Na2CO3-NaHCO3 (pH 9.0). A 60-fold molar ratio of p-SCN-Bn-DOTA (dissolved in a small amount of DMSO) was added and the reaction was allowed to proceed overnight at 4°C in the dark to synthesize DOTA-aGPC3-mAb. Excess unreacted DOTA was removed using a PD-10 desalting column. The purified antibody was concentrated using an ultrafiltration centrifuge tube (Millipore 0.5 mL / 30 Kd) at 4°C, 10,000 × g, for 10 min. For short-term storage, the antibody was stored in a 4°C refrigerator. For long-term storage, the antibody was stored in aliquots at -80°C.

[0315] 9.2 177 Radiolabeling of Lu

[0316] Pick 177 LuCl3 solution (185 MBq) was adjusted to pH 5.5 by adding 500 μL of ammonium acetate (0.1 M, pH 5.5). Approximately 1 mg of DOTA-aGPC3-mAb was then added, mixed thoroughly, and reacted at 37°C for 1 hour. The reaction product was purified using a PD-10 desalting column, and its radiochemical purity was determined by radioactive instant thin-layer chromatography (ITLC) using citric acid-sodium citrate buffer (pH 4.0) as the developing solvent.

[0317] 9.3 177 Radiochemical characterization of Lu-DOTA-aGPC3-mAb

[0318] Prepared 177 Lu-DOTA-aGPC3-mAb was identified and characterized by analytical radio-ITLC. A new immuno-SPECT probe was successfully prepared. 177 Lu-DOTA-aGPC3-mAb, radioactive ITLC results showed that the labeling rate was about 80.4% (Figure 7A), and SEC-HPLC results showed 177 The radiochemical purity of Lu-DOTA-aGPC3-mAb was greater than 97%, and the stability in PBS for 72 h was greater than 80% ( FIG. 7B ).

[0319] Example 10. Analysis of Cellular GPC3 Expression

[0320] Human hepatoma cell lines Huh7 (GPC3 positive) and SK-HEP-1 (GPC3 negative) were cultured in complete MEM or DMEM medium (containing 10% FBS and 1% penicillin-streptomycin) in a 5% CO2, 37°C cell culture incubator.

[0321] Huh7 and SK-HEP-1 cells were cultured in culture dishes. When the cells reached 75% to 80% confluence, total protein was extracted. Protein concentration was determined using a BCA assay kit. SDS-PAGE stacking and separating gels were then prepared according to standard ratios. The prepared protein samples and markers were added to the sample wells, with 40 μg of total protein per sample loaded. SDS-PAGE electrophoresis was performed. After electrophoresis, the membrane was transferred and blocked with a PVDF membrane. The primary rabbit anti-Glypican 3 monoclonal antibody was diluted 1:1000 and incubated with the PVDF membrane overnight at 4°C. The membrane was then incubated with a secondary antibody on a shaker at room temperature for 1 hour before development. The grayscale values ​​of the bands were determined using Image J software. GPC3 expression in human hepatocellular carcinoma cell lines was assessed by Western blot. As shown in Figure 8, the Huh7 cell line was positive for GPC3 expression, while the SK-HEP-1 cell line was negative.

[0322] Example 11. 68 PET / CT imaging of subcutaneous tumor model using Ga-NOTA-aGPC3-Fab probe

[0323] All animal studies were conducted in accordance with the guidelines of the Laboratory Animal Use and Care Committee of Tongji Medical College, Huazhong University of Science and Technology. Female Balb / c nude mice (4–5 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. and maintained in an SPF-grade environment in the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology.

[0324] Huh7 and SK-HEP-1 cells were collected in the logarithmic phase of growth and digested with conventional trypsin. The cell pellets were collected and counted. Subsequently, they were washed twice with pre-chilled PBS and the cell pellets were collected by centrifugation. The cells were resuspended in a 1:1 volume ratio of PBS to Matrigel. The cells were thoroughly pipetted and then inoculated subcutaneously in the right upper limb of mice near the axilla. Each mouse was inoculated with 1×10 7 cells (volume 125 μL / mouse). When the tumor grows to 8-10 mm in diameter, it is used for subsequent experiments.

[0325] Huh7 tumor-bearing nude mice in the experimental group, SK-HEP-1 tumor-bearing nude mice in the control group, and liver cancer PDX models were injected into the tail vein 68PET / CT scans were performed at 0.5, 1, 2, and 4 hours after the administration of Ga-NOTA-aGPC3-Fab (3.7-5.55 MBq, 100-150 μCi). Each scan took approximately 15 minutes. Following completion of the scan, PET and CT images were reconstructed after attenuation correction, and regions of interest (ROIs) were delineated using Amide software to calculate uptake values ​​(%ID / g).

[0326] For evaluation 68 In vivo targeting specificity of Ga-NOTA-aGPC3-Fab was observed in Huh7 tumor-bearing mice injected via the tail vein 68 PET / CT static scans were performed at 30 min, 1 h, 2 h and 4 h after the injection of Ga-NOTA-aGPC3-Fab. 68 Tumors became visible after 30 minutes of Ga-NOTA-aGPC3-Fab application, with tumor uptake reaching 1.89% ID / g. Thereafter, tumor uptake remained stable, reaching 1.87% ID / g and 2.04% ID / g at 1 and 2 hours, respectively. At 4 hours, the tumor outline remained clear, with a tumor uptake value of 1.97% ID / g, indicating high affinity for the probe and good tumor retention. The probe was rapidly cleared from the blood circulation and liver, with images displaying a high tumor-to-background ratio at 2 hours. ROI quantitative analysis revealed that cardiac uptake decreased from 3.06% ID / g at 30 minutes to 0.96% ID / g, and liver uptake decreased from 2.76% ID / g to 1.35% ID / g. At 4 hours, the tumor-to-heart ratio was 3.06, the tumor-to-liver ratio was 1.42, and the tumor-to-muscle ratio was 6.60. In contrast, tumors in the SK-HEP-1 tumor-bearing mice in the control group showed no tumor imaging at any time point (Figure 10). Tumor uptake values ​​at 1, 2, and 4 hours were 0.29% ID / g, 0.37% ID / g, and 0.42% ID / g, respectively, significantly lower than those in the Huh7 tumor-bearing mice at each time point. Significant radioactivity accumulation was observed in the non-target organs, the kidneys and bladder, in both groups of tumor-bearing mice, indicating that the probe is primarily metabolized by the kidneys.

[0327] Example 12 PET / CT imaging of PDX model

[0328] Collection of patient basic information: All patient information and tumor sample collection were approved by the Clinical Ethics Committee of Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology. The sampling inclusion criteria were patients with suspected liver cancer before surgery and who were assessed to have a 200mm 3Patients with tumor tissues above 300 μg / cm2 were selected and informed consent was obtained from the patients before surgery. The basic clinical information of the patients was collected, including general information, clinical data, tumor markers, biochemical indicators, imaging examinations, and other test and examination data, as well as postoperative pathological data.

[0329] Acquisition and transportation of fresh tumor tissue: Liver tumor surgical specimens were immediately placed in a sterile tray after excision and cut into 200-500mm pieces using a sterile scalpel. 3 Place fresh tumor tissue in a 15mL sample tube filled with sterile PBS. Place the tumor sample in a 4°C refrigerator and prepare an ice box for sampling. To preserve tissue viability, the sample should be kept in an ice-water mixture or surrounded by ice packs throughout transportation. The time from sampling to transport to the laboratory for PDX model generation should not exceed 2 hours.

[0330] Tumor transplantation: The entire tumor transplantation process is performed in a sterile operating table. First, the tumor tissue is poured into a cell culture dish and transferred to PBS containing a penicillin-streptomycin mixture using sterile ophthalmic forceps for washing. Then, necrotic tumor tissue is removed using sterile ophthalmic scissors, and active tumor tissue pieces are retained as much as possible. The washed tumor tissue is transferred to a cell culture dish containing sterile DMEM medium and the tissue is trimmed into approximately 2 mm pieces with tissue scissors. 3 The size of the tumor is reserved. And take 50-100mm 3 Tumor tissue is used for fixation and embedding in paraffin blocks, frozen storage and biopsy. Healthy NSG mice were fasted for 10 hours and deprived of water for 4 hours before surgery. Mice underwent surgery under isoflurane inhalation anesthesia. The surgical area was depilated and the skin was routinely disinfected with povidone-iodine solution before surgery. An incision of about 2-3 mm was made in the middle at the level of the shoulder blade. The tumor tissue was gently pushed into the bilateral armpits of the mouse with a trocar, and then the skin was sutured with absorbable sutures. The health status of the mice was monitored for 5 consecutive days after surgery, and attention was paid to observe whether there was any sign of wound dehiscence or infection. When the tumor volume reached 1000-1500mm 3 When used for imaging studies.

[0331] Liver tumor tissue samples were collected from patients undergoing surgical resection to establish PDX models. The patient's clinical data and tumor histological characteristics are listed in Table 1. A bilateral PDX model was successfully established. PDX model mice were injected with 68 After the addition of Ga-NOTA-aGPC3-Fab, small animal PET / CT static imaging was performed at 1, 2, and 4 hours, respectively ( Figure 11 ). 68Following injection of Ga-NOTA-aGPC3-Fab, the tumors were clearly visualized, with average bilateral tumor uptake of 0.98% ID / g, 0.93% ID / g, and 0.99% ID / g at 1, 2, and 4 hours, respectively, indicating stable tumor retention of the probe. Similar to the subcutaneous tumor model, probe uptake in the heart and liver decreased over time, while radioactivity accumulated in the kidneys and bladder, suggesting that the probe is primarily metabolized by the kidneys.

[0332] Table 1 Clinical information of patients and tumor histological characteristics

[0333] Example 13 Histological Identification

[0334] Tumor tissues were fixed with 4% paraformaldehyde, dehydrated with graded ethanol, transparentized with xylene, infiltrated with paraffin, and embedded in paraffin to prepare paraffin tissue blocks, which were then sliced ​​(5 μm) using a tissue slicer.

[0335] H&E staining: Use Harris hematoxylin staining solution for 4 minutes, rinse with tap water for 2 minutes until no excess stain is removed from the slice. Differentiate with 0.8% hydrochloric acid alcohol for 2 seconds, rinse with tap water, or use lithium carbonate aqueous solution to return to blue, then rinse with water for 2 minutes. Stain with eosin staining solution (alcohol-soluble) for 20 seconds, without washing with water, directly color in 95% ethanol for 5 seconds, dehydrate in anhydrous ethanol (1) and anhydrous ethanol (2) for 2 minutes, then transparentize with environmentally friendly transparent agent, seal, and examine under a microscope.

[0336] GPC3 immunohistochemical staining: After antigen retrieval, block endogenous peroxidase activity and incubate sections with 10% serum from the same source as the secondary antibody at 37°C for 30 minutes to reduce nonspecific staining. Discard the serum and dilute the primary antigen solution with 10% serum to prepare the primary antibody working solution. Then, add the diluted primary antibody working solution (anti-Glypican 3 monoclonal antibody, 1:500 dilution) and incubate overnight at 4°C. The next day, let the sections rest at room temperature for 15 minutes (rewarming). Rinse three times with TBST and then dip three times for 3 minutes each. Dilute the secondary antigen solution with TBST to prepare the secondary antibody working solution. Add the secondary antibody working solution to each section and incubate at 37°C for 45 minutes. Rinse three times with TBST and then dip three times for 3 minutes each. Discard the TBST and add the tyramine salt working solution to each section. Incubate at room temperature for 10 minutes in the dark. Rinse three times with TBST and then dip three times for 3 minutes each. Freshly prepared tyramine working solution was then added to each section for color development. After counterstaining with hematoxylin, the sections were then blued. The sections were mounted and examined under a microscope.

[0337] Immunohistochemical analysis of tumor tissue was used to determine GPC3 expression in Huh7 subcutaneous tumor and PDX models. As shown in Figure 12, Huh7 tumor tissue showed extensive and significant GPC3 expression, while PDX tumor tissue (cholangiocarcinoma) showed lower GPC3 expression, which corresponded to the imaging results, indicating that the probe can effectively image tumors with different GPC3 expression levels.

[0338] Example 14 68 PET / CT imaging of subcutaneous tumor model using Ga-NOTA-aGPC3-scFv probe

[0339] The human hepatoma cell line Hep3B (GPC3 positive) was cultured in complete MEM medium (containing 10% FBS and 1% penicillin-streptomycin) and the cells were cultured in a cell culture incubator at 5% CO 2 and 37° C.

[0340] Hep3B cells were collected in the logarithmic phase of growth and digested with trypsin. The cell pellet was collected and counted. Subsequently, the cells were washed twice with pre-chilled PBS and centrifuged to collect the cell pellet. The cells were resuspended in a 1:1 volume ratio of PBS:Matrigel. The cells were thoroughly pipetted and then inoculated subcutaneously in the right upper limb of mice near the axilla. Each mouse was inoculated with 5×10 6 cells (volume 100 μL / mouse). When the tumor grows to 8-10 mm in diameter, it is used for subsequent experiments.

[0341] Hep3B cells growing in the logarithmic phase were collected, digested with routine trypsin, and the cell pellets were collected and counted. Subsequently, the cells were washed twice with pre-cooled PBS and the cell pellets were collected by centrifugation. The cell resuspension was prepared according to the volume ratio of PBS: Matrigel = 1:1 for use. Healthy BALB / c-nu mice were fasted for 10 hours and deprived of water for 4 hours before surgery. In the clean bench, the mice were continuously anesthetized using a small animal gas anesthesia system (2% isoflurane). The mice were fixed in a supine position, and the skin was routinely disinfected with povidone iodine solution. An incision of about 1 cm was made below the xiphoid process in the middle of the upper abdomen. The skin and peritoneum were cut layer by layer, and a gauze soaked in saline was placed under the incision. The sides of the costal arch were gently squeezed to expose the left lobe of the mouse liver to the wet gauze. The injection needle was advanced obliquely, with the needle tip at 20° to the horizontal plane, and penetrated the liver about 1 cm. The syringe needle core was gently pushed, and 0.05 mL of cell suspension was slowly injected, about 5×10 5 Hep3B cells. Slowly withdraw the needle and apply gentle pressure with a sterile cotton swab for a moment before gently returning the liver to the abdominal cavity. Close the abdomen layer by layer. Disinfect the skin again and close the surgery. Do not eat or drink for 4 hours after surgery.

[0342] Tail vein injection of Hep3B tumor-bearing nude mice and Hep3B liver cancer orthotopic tumor models 68PET / CT scans were performed 1, 2, and 4 hours after the administration of Ga-NOTA-aGPC3-scFv (3.7-5.55 MBq, 100-150 μCi) under isoflurane inhalation anesthesia. Each scan lasted approximately 15 minutes. After completion of the scan, PET and CT images were reconstructed after attenuation correction, and regions of interest (ROIs) were delineated using Amide software to calculate uptake values ​​(%ID / g).

[0343] Hep3B tumor-bearing mice were injected subcutaneously with 68 PET / CT static scans were performed at 1 hour, 2 hours and 4 hours after Ga-NOTA-aGPC3-scFv injection. 68 Tumors were clearly visualized after 1 hour of Ga-NOTA-aGPC3-scFv administration, and the images exhibited a high tumor-to-background ratio. ROI delineation and semiquantitative analysis of PET images (Figures 13B and C) revealed that tumor uptake continued to increase over time, reaching 3.00±1.37% ID / g at 1, 2, and 4 hours, respectively. The probe was rapidly cleared from the bloodstream. At 1 hour, the tumor-to-blood pool ratio was 4.91±1.84, the tumor-to-liver ratio was 2.25±0.78, and the tumor-to-muscle ratio was 8.92±5.72. These ratios continued to increase over time. Significant radioactivity accumulation was observed in the kidneys and bladder, suggesting that the probe is primarily metabolized by the kidneys. The long retention of the probe at the tumor site and its rapid clearance from the background demonstrate its high affinity to the target and good potential for early imaging after injection.

[0344] exist 68 Ga-NOTA-aGPC3-scFv vs. 68 In the comparative analysis of PET imaging of Hep3B subcutaneous tumor model with Ga-NOTA-aGPC3-Fab (Figure 14A-B), both probes showed high uptake in the tumor site 1 hour after injection, and the tumor uptake gradually increased with time. 68 Ga-NOTA-aGPC3-scFv has a shorter blood half-life and can be cleared faster from the blood and non-target organs such as the liver, thus showing better tumor / background contrast at various time points. Semi-quantitative analysis of PET images showed (Figure 14C-E) that at various time points 68 The tumor / blood pool ratio and tumor / liver ratio of Ga-NOTA-aGPC3-scFv were significantly higher than those of 68Ga-NOTA-aGPC3-Fab, tumor / muscle ratio is slightly higher than 68 Ga-NOTA-aGPC3-Fab.

[0345] Example 15 Hep3B liver tumor in situ model mice 68 Ga-NOTA-aGPC3-scFv, 68 Ga-NOTA-aGPC3-Fab and 18 F-FDG PET / CT imaging comparison

[0346] Use 68 Ga-NOTA-aGPC3-scFv, 68 Ga-NOTA-aGPC3-Fab and 18 F-FDG was used to image the Hep3B orthotopic tumor model in the same group to compare the detection capabilities of the three methods for liver orthotopic tumors. As shown in Figure 15A, 68 Ga-NOTA-aGPC3-scFv and 68 Ga-NOTA-aGPC3-Fab can clearly show the location of liver in situ tumor 1 hour after injection. 18 F-FDG is highly absorbed by the abdominal intestine, making it difficult to distinguish tumors from normal organs. The anatomical exploration of the liver in situ tumor showed (Figure 15B) that the tumor grew in the left lobe of the mouse liver, with a diameter of about 5 mm. The tumor site was 68 Ga-NOTA-aGPC3-scFv and 68 Ga-NOTA-aGPC3-Fab PET imaging showed the same 68 Ga-NOTA-aGPC3-scFv and 68 Ga-NOTA-aGPC3-Fab can clearly detect lesions of 5 mm.

[0347] Example 16 177 SPECT / CT imaging of subcutaneous tumor model using Lu-DOTA-aGPC3-Fab probe

[0348] The human hepatocellular carcinoma cell line Hep3B (GPC3 positive) was cultured in complete MEM medium (containing 10% FBS and 1% penicillin-streptomycin double antibody) and the cells were cultured in a cell culture incubator at 5% CO2 and 37°C. Hep3B cells growing in the logarithmic phase were collected, routinely trypsinized, and the cell pellets were collected and counted. Subsequently, they were washed twice with pre-cooled PBS and the cell pellets were collected by centrifugation. The cell resuspension was prepared according to the volume ratio of PBS: Matrigel = 1:1, the cells were thoroughly blown, and then inoculated subcutaneously in the right upper limb of the mouse near the armpit, with 5×10 cells per mouse inoculated. 6The tumors were grown to about 8-10 mm in diameter and used for subsequent experiments.

[0349] Injection of Hep3B subcutaneous tumor into nude mice 177 SPECT / CT scans were performed 2, 6, 12, 24, and 48 hours after administration of Lu-DOTA-aGPC3-Fab (18.5 MBq, 500 μCi), with each scan taking approximately 20–30 minutes. Anesthesia was maintained with 1.0–1.5% isoflurane throughout the scan. SPECT data were corrected for attenuation and scatter, and images were reconstructed using the three-dimensional ordered subsets expectation maximization (OSEM) algorithm. Regions of interest (ROIs) were delineated and used for quantitative analysis of SPECT images. Tissue radioactivity uptake was expressed as the percentage of injected dose per gram of tissue (%ID / g).

[0350] [Corrected 17.10.2024 according to Rule 91] Hep3B subcutaneous tumor-bearing mice were injected via tail vein 177 SPECT / CT static scans were performed at 2, 6, 12, 24, and 48 hours after Lu-DOTA-aGPC3-Fab injection. 177 Lu-DOTA-aGPC3-Fab clearly visualized the tumor 2 hours after administration, and the images showed a high tumor-to-background ratio. ROI delineation and semiquantitative analysis of SPECT images (Figure 16B) revealed that tumor uptake remained stable from 2 to 6 hours (2 hours: 3.55±0.76% ID / g, 6 hours: 3.46±0.37% ID / g). After 6 hours, tumor uptake gradually decreased, remaining at 1.50±0.67% ID / g at 48 hours. The probe was rapidly cleared from the bloodstream, with a tumor-to-blood pool ratio of 3.34±1.66 at 2 hours and gradually increasing thereafter, reaching a peak of 6.08±2.44 at 24 hours. The tumor-to-liver and tumor-to-muscle ratios peaked at 6 hours, reaching 1.95±0.55 and 5.37±1.17, respectively. Significant radioactivity accumulation was observed in the kidneys and bladder among non-target organs, suggesting that the kidneys are the primary organ of probe metabolism. ROI delineation and semiquantitative analysis of the kidneys revealed a rapid decrease in renal radioactivity within 48 hours. The rapid uptake and long-lasting retention of the probe in tumors, along with its rapid clearance from non-target organs, suggest its potential for further application in radioimmunotherapy.

[0351] Example 17. 177 SPECT / CT imaging of subcutaneous tumor model using Lu-DOTA-aGPC3-scFv probe

[0352] The human hepatocellular carcinoma cell line Hep3B (GPC3 positive) was cultured in complete MEM medium (containing 10% FBS and 1% penicillin-streptomycin double antibody) and the cells were cultured in a cell culture incubator at 5% CO2 and 37°C. Hep3B cells growing in the logarithmic phase were collected, routinely trypsinized, and the cell pellets were collected and counted. Subsequently, they were washed twice with pre-cooled PBS and the cell pellets were collected by centrifugation. The cell resuspension was prepared according to the volume ratio of PBS: Matrigel = 1:1, the cells were thoroughly blown, and then inoculated subcutaneously in the right upper limb of the mouse near the armpit, with each mouse inoculated with 7×10 6 The tumors were grown to about 8-10 mm in diameter and used for subsequent experiments.

[0353] Injection of Hep3B subcutaneous tumor into nude mice 177 SPECT / CT scans were performed 2, 6, 12, 24, and 48 hours after administration of Lu-DOTA-aGPC3-scFv (18.5 MBq, 500 μCi), with each scan taking approximately 20–30 minutes. Anesthesia was maintained with 1.0–1.5% isoflurane throughout the scan. SPECT data were corrected for attenuation and scatter, and images were reconstructed using the three-dimensional ordered subsets expectation maximization (OSEM) algorithm. Regions of interest (ROIs) were delineated and used for quantitative analysis of SPECT images. Tissue radioactivity uptake was expressed as the percentage of injected dose per gram of tissue (%ID / g).

[0354] [Corrected 17.10.2024 according to Rule 91] Hep3B subcutaneous tumor-bearing mice were injected via tail vein 177 SPECT / CT static scans were performed at 2, 6, 12, 24, and 48 hours after Lu-DOTA-aGPC3-scFv administration. 177Lu-DOTA-aGPC3-scFv clearly visualized tumors 2 hours after administration, and images demonstrated high tumor-to-background ratios. ROI delineation and semiquantitative analysis of SPECT images (Figure 17B) revealed that tumor uptake remained stable from 2 to 6 hours (2 hours: 3.99±0.15% ID / g, 6 hours: 3.77±0.32% ID / g). After 6 hours, tumor uptake gradually decreased, reaching 0.94±0.61% ID / g at 48 hours. The probe was rapidly cleared from the mid-circulation, with high tumor-to-background ratios from 2 to 24 hours. The tumor-to-blood pool, tumor-to-liver, and tumor-to-muscle ratios reached their highest values ​​at 6 hours, reaching 10.72±3.93, 2.89±0.80, and 7.36±2.08, respectively. Significant radioactivity accumulation was observed in the kidneys and bladder among non-target organs, suggesting that the kidneys are the primary organ of probe metabolism. ROI delineation and semiquantitative analysis of the kidneys revealed a rapid decrease in renal radioactivity uptake within 24 hours. The rapid uptake and long-lasting retention of the probe in tumors, along with its rapid clearance from non-target organs, suggest its potential for further application in radioimmunotherapy.

[0355] Example 18. 177 SPECT / CT imaging of subcutaneous tumor model using Lu-DOTA-aGPC3-mAb probe

[0356] The human hepatocellular carcinoma cell line Hep3B (GPC3 positive) was cultured in complete MEM medium (containing 10% FBS and 1% penicillin-streptomycin double antibody) and the cells were cultured in a cell culture incubator at 5% CO2 and 37°C. Hep3B cells growing in the logarithmic phase were collected, routinely trypsinized, and the cell pellets were collected and counted. Subsequently, they were washed twice with pre-cooled PBS and the cell pellets were collected by centrifugation. The cell resuspension was prepared according to the volume ratio of PBS: Matrigel = 1:1, the cells were thoroughly blown, and then inoculated subcutaneously in the right upper limb of the mouse near the armpit, with 5×10 cells per mouse inoculated. 6 cells (volume 100 μL / mouse). When the tumor grows to about 8-10 mm in diameter, it is used for subsequent experiments.

[0357] Injection of Hep3B subcutaneous tumor into nude mice 177SPECT / CT scans were performed 2, 6, and 12 hours, and 3, 5, and 7 days after administration of Lu-DOTA-aGPC3-mAb (18.5 MBq, 500 μCi). Each scan took approximately 20–30 minutes. Anesthesia was maintained with 1.0–1.5% isoflurane throughout the scan. SPECT data were corrected for attenuation and scatter, and images were reconstructed using the three-dimensional ordered subsets expectation maximization (OSEM) algorithm. Regions of interest (ROIs) were delineated and analyzed quantitatively for SPECT images. Tissue radioactivity uptake was expressed as the percentage of injected dose per gram of tissue (%ID / g).

[0358] Hep3B tumor-bearing mice were injected via tail vein 177 SPECT / CT static scans were performed at 2, 6, and 12 hours and at 1, 3, 5, and 7 days after Lu-DOTA-aGPC3-mAb administration. 177 After Lu-DOTA-aGPC3-mAb administration, the probe was primarily in the blood circulation for 2-6 hours. Tumor uptake began to appear at 6 hours, followed by a gradual increase in tumor uptake. The probe was gradually cleared from the circulation, and the images exhibited good tumor / background contrast at 24 hours. ROI delineation and semi-quantitative analysis of SPECT images (Figure 18B) showed that the probe maintained sustained and stable retention within the tumor for 24-72 hours (24 hours: 11.34±0.98% ID / g, 72 hours: 10.40±1.13% ID / g). Uptake then slowly decreased, remaining at 4.55±0.64% ID / g on day 7. The probe was gradually cleared from the mid-circulation, and the tumor / blood pool and tumor / liver ratios continued to increase (Figures 18C-D), reaching their highest values ​​on day 7, at 10.08±2.42 and 4.11±1.37, respectively. The tumor / muscle ratio peaked at 9.13±1.53 on day 5 (Figure 18E). The long-term retention of the probe in the tumor suggests its potential for further application in radioimmunotherapy.

[0359] Example 19. 177 Radioimmunotherapy of subcutaneous tumor models using Lu-DOTA-aGPC3-Fab probe

[0360] The human hepatocellular carcinoma cell line Hep3B (GPC3 positive) was cultured in complete MEM medium (containing 10% FBS and 1% penicillin-streptomycin double antibody) and the cells were cultured in a cell culture incubator at 5% CO2 and 37°C. Hep3B cells growing in the logarithmic phase were collected, routinely trypsinized, and the cell pellets were collected and counted. Subsequently, they were washed twice with pre-cooled PBS and the cell pellets were collected by centrifugation. The cell resuspension was prepared according to the volume ratio of PBS: Matrigel = 1:1, the cells were thoroughly blown, and then inoculated subcutaneously in the right upper limb of the mouse near the armpit, with each mouse inoculated with 7×10 6 cells (volume 100 μL / cell).

[0361] When the tumor volume reaches approximately 150–200 mm 3 The tumor-bearing mice were randomly divided into two groups: normal saline group and 177 Lu-DOTA-aGPC3-Fab group (n=6). The living conditions of mice were observed every 3 days, and their body weight was measured (normalized body weight: body weight on day n / body weight on day 0 × 100%). The length and width of the tumor were measured with a vernier caliper, and the tumor volume was calculated (tumor volume = length × width × width / 2).

[0362] On day 0, the two groups of mice were injected into the tail vein with 150 μL of normal saline and an equal volume of 11.1 MBq 177 Lu-DOTA-aGPC3-Fab. Figure 19 shows the changes in tumor volume in each group of mice after 12 days of treatment. 177 The tumor volume of the Lu-DOTA-aGPC3-Fab treatment group was significantly smaller than that of the saline group (P<0.05). Both groups of mice showed a certain amount of weight loss after treatment, with no statistical difference. The above results show that the injection of 11.1MBq 177 Radioimmunotherapy with Lu-DOTA-aGPC3-Fab can significantly slow down the tumor growth rate in tumor-bearing mice.

[0363] Example 20. 177 Radioimmunotherapy of subcutaneous tumor models using Lu-DOTA-aGPC3-scFv probe

[0364] The human hepatocellular carcinoma cell line Hep3B (GPC3 positive) was cultured in complete MEM medium (containing 10% FBS and 1% penicillin-streptomycin double antibody) and the cells were cultured in a cell culture incubator at 5% CO2 and 37°C. Hep3B cells growing in the logarithmic phase were collected, routinely trypsinized, and the cell pellets were collected and counted. Subsequently, they were washed twice with pre-cooled PBS and the cell pellets were collected by centrifugation. The cell resuspension was prepared according to the volume ratio of PBS: Matrigel = 1:1, the cells were thoroughly blown, and then inoculated subcutaneously in the right upper limb of the mouse near the armpit, with each mouse inoculated with 7×10 6cells (volume 100 μL / cell).

[0365] When the tumor volume reaches approximately 150–200 mm 3 The tumor-bearing mice were randomly divided into two groups: normal saline group and 177 Lu-DOTA-aGPC3-scFv group (n=6). The living conditions of mice were observed every 3 days, and their body weight was measured (normalized body weight: body weight on day n / body weight on day 0 × 100%). The length and width of the tumor were measured with a vernier caliper, and the tumor volume was calculated (tumor volume = length × width × width / 2).

[0366] On day 0, the two groups of mice were injected into the tail vein with 150 μL of normal saline and an equal volume of 11.1 MBq 177 Lu-DOTA-aGPC3-scFv. Figure 20 shows the changes in tumor volume in each group of mice after 12 days of treatment. On the 12th day, 177 The tumor volume of the Lu-DOTA-aGPC3-scFv treatment group was significantly smaller than that of the saline group (P<0.05). Both groups of mice showed a certain amount of weight loss after treatment, with no statistical difference. The above results show that the injection of 11.1MBq 177 Radioimmunotherapy with Lu-DOTA-aGPC3-scFv can significantly slow down the tumor growth rate in tumor-bearing mice.

[0367] Example 21. 177 Radioimmunotherapy of subcutaneous tumor model using Lu-aGPC3-mAb probe

[0368] The human hepatocellular carcinoma cell line Hep3B (GPC3 positive) was cultured in complete MEM medium (containing 10% FBS and 1% penicillin-streptomycin double antibody) and the cells were cultured in a cell culture incubator at 5% CO2 and 37°C. Hep3B cells growing in the logarithmic phase were collected, routinely trypsinized, and the cell pellets were collected and counted. Subsequently, they were washed twice with pre-cooled PBS and the cell pellets were collected by centrifugation. The cell resuspension was prepared according to the volume ratio of PBS: Matrigel = 1:1, the cells were thoroughly blown, and then inoculated subcutaneously in the right upper limb of the mouse near the armpit, with each mouse inoculated with 7×10 6 cells (volume 100 μL / cell).

[0369] When the tumor volume reaches approximately 150–200 mm 3 The tumor-bearing mice were randomly divided into 4 groups: normal saline group, aGPC3-mAb group, 11.1MBq 177 Lu-DOTA-aGPC3-mAb group and 5.55MBq×2 177Lu-DOTA-aGPC3-mAb group (n=6). The living conditions of mice were observed every 3 days, and their body weight was measured (normalized body weight: body weight on day n / body weight on day 0 × 100%). The length and width of the tumor were measured with a vernier caliper, and the tumor volume was calculated (tumor volume = length × width × width / 2).

[0370] On day 0, the four groups of mice were injected into the tail vein with 150 μL of normal saline, an equal volume of aGPC3-mAb (100 μg), and 11.1 MBq 177 Lu-DOTA-aGPC3-mAb, for 5.55 MBq × 2 177 Lu-DOTA-aGPC3-mAb group was given an equal volume of 5.55 MBq on day 0 and day 7, respectively. 177 Lu-DOTA-aGPC3-mAb. Figure 21 shows the changes in tumor volume in each group of mice after 21 days of treatment. On the 21st day, 11.1MBq 177 Lu-DOTA-aGPC3-mAb group and 5.55MBq×2 177 The tumor volume of the Lu-DOTA-aGPC3-mAb group was significantly reduced compared with the saline group and the aGPC3-mAb group (P < 0.01). All four groups of mice showed a certain amount of weight loss after treatment, and there was no statistical difference between each group and the saline group. 177 Lu-DOTA-aGPC3-mAb was administered once with 11.1 MBq 177 Lu-DOTA-aGPC3-mAb treatment had similar effects, but 11.1 MBq was given once 177 The weight loss in the Lu-DOTA-aGPC3-mAb group was more significant than that in the split-dose group, especially on the 6th and 11th days, but there was no statistical significance on the 21st day. 177 Radioimmunotherapy with Lu-DOTA-aGPC3-mAb can significantly slow down the tumor growth rate in tumor-bearing mice.

[0371] The information of the antibodies used in the present invention is shown in Table 2 below.

[0372] Table 2

Claims

1. A molecular probe targeting GPC3, wherein the molecular probe is a compound having the following sequence, or a pharmaceutically acceptable salt or ester thereof: (GPC3 antibody, including antibody fragment of GPC3 antibody, or single domain antibody thereof) m -R n , in, R is selected from the group consisting of a linker, a chelator, a radionuclide, another targeting peptide or a monoclonal antibody, a monoclonal antibody fragment, a single domain antibody, and a small molecule inhibitor; Where n represents the number of R, n = 0-4; Wherein, m is not 0.

2. The molecular probe targeting GPC3 according to claim 1, wherein the molecular probe is a compound having the following general formula (I), or a pharmaceutically acceptable salt or ester thereof: Ab m -(LM z ) n (I) in, Ab is a GPC3 antibody or an antigen-binding fragment thereof; L is a linking group; M is a radionuclide; z is 0 or 1; n is an integer of 1-4, preferably 1 or 2; and m is an integer of 1-4, preferably 1 or 2.

3. The molecular probe targeting GPC3 according to claim 2, wherein L is represented by the following general formula (II): Y-Lk-T (II) in, Y is a linker, Lk is a coupling group, and T is a chelating moiety; Preferably, Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C1-C 20 Alkenylene, optionally substituted C1-C 20 Alkyne, optionally substituted C1-C 20 Heteroalkylene, optionally substituted C3-C 20 Cycloalkylene, optionally substituted C3-C 20 Heterocycloalkylene, optionally substituted C5-C 20 Arylene, optionally substituted C5-C 20 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, polypeptides (such as valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), polyglycine (Gly) r , wherein r is an integer from 1 to 10), polyethylene glycol (such as (PEG) r , wherein r is an integer of 1-10), polypropylene glycol, Polyoxyalkylene, copolymer groups of polyethylene glycol and polypropylene glycol, or combinations thereof; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, N-hydroxysuccinimide ester, maleimide, 2,3,5,6-tetrafluorophenol ester, N-succinimidyl-S-acetylthioacetate, amide, or combinations thereof; Preferably, Lk is independently selected from a single bond, p-isothiocyanatophenyl, N-hydroxysuccinimide ester, maleimide, 2,3,5,6-tetrafluorophenol ester, N-succinimidyl-S-acetylthioacetate, amide, or a combination thereof; Preferably, T is independently selected from a single bond or a ligand derived from any of the following structures: deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyltriglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), ethylenediaminetetraacetic acid (EDTA), ), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), triacetylfusine C (TAFC), ferric streptomycin E (FOXE), ferric streptomycin B (FOXB), ferric chromogen A (FCHA), or a combination thereof; Optionally, a spacer group is further included between Lk and T, and the spacer group is independently selected from a single bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C1-C 20 Alkenylene, optionally substituted C1-C 20 Alkyne, optionally substituted C1-C 20 Heteroalkylene, optionally substituted C3-C 20 Cycloalkylene, optionally substituted C3-C 20 Heterocycloalkylene, optionally substituted C5-C 20 Arylene, optionally substituted C5-C 20 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, polypeptides (such as valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), polyglycine (Gly) r , wherein r is an integer from 1 to 10), polyethylene glycol ((PEG) r , wherein r is an integer of 1-10), polypropylene glycol, polyoxyalkylene, a copolymer group of polyethylene glycol and polypropylene glycol, or a combination thereof.

4. The molecular probe targeting GPC3 according to claim 3, wherein: Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, an optionally substituted C1-C 10 Alkylene, optionally substituted C1-C 10 Alkenylene, optionally substituted C1-C 10 Alkyne, optionally substituted C1-C 10 Heteroalkylene, optionally substituted C3-C 10 Cycloalkylene, optionally substituted C3-C 10 Heterocycloalkylene, optionally substituted C3-C 10 Arylene, optionally substituted C3-C 10 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, -(PEG) r -, valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), -(Gly) n - or a combination thereof, wherein n is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof; Lk is independently selected from a single bond, p-isothiocyanatophenyl, an amide group or a maleimide group; and T is independently selected from a single bond or a ligand derived from any of the following structures: deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) (DOTP), or 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP); Optionally, a spacer group is further included between Lk and T, and the spacer group is independently selected from a single bond, an optionally substituted C1-C 10 Alkylene, optionally substituted C1-C 10 Alkenylene, optionally substituted C1-C 10 Alkyne, optionally substituted C1-C 10 Heteroalkylene, optionally substituted C3-C 10 Cycloalkylene, optionally substituted C3-C 10 Heterocycloalkylene, optionally substituted C3-C 10 Arylene, optionally substituted C3-C 10 Heteroarylene, keto, -S-, -(C=S)-, -O-, -NH-, -NHCO-, -(PEG) r -, valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), phenylalanine-lysine (Phe-Lys), alanine-alanine-asparagine (Ala-Ala-Asn), -(Gly) n - or a combination thereof, wherein n is an integer from 1 to 10.

5. The molecular probe targeting GPC3 according to claim 3 or 4, wherein: Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof; Lk is independently selected from a single bond, p-isothiocyanatophenyl or an amide group; and T is independently selected from a single bond or a ligand derived from any of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyltriglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA); Optionally, a spacer group is further included between Lk and T, and the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10.

6. The molecular probe targeting GPC3 according to any one of claims 3 to 5, wherein: Lk is independently selected from p-isothiocyanatophenyl or amide; Y is a single bond; and T is independently selected from a ligand derived from any one of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyltriglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

7. The molecular probe targeting GPC3 according to any one of claims 3 to 6, wherein: Lk is p-isothiocyanatophenyl; Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof; and T is independently selected from a ligand derived from any of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), or diethylenetriaminepentaacetic acid (DTPA).

8. The molecular probe targeting GPC3 according to any one of claims 3 to 6, wherein: Lk is p-isothiocyanatophenyl; Y is a single bond; and T is independently selected from a ligand derived from any of the following structures: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), or diethylenetriaminepentaacetic acid (DTPA).

9. The molecular probe targeting GPC3 according to any one of claims 3 to 6, wherein: Lk is an amide group; Y comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof; and T is independently selected from a ligand derived from any of the following structures: mercaptoacetyl triglycine (MAG3) or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

10. The molecular probe targeting GPC3 according to any one of claims 3 to 6, wherein: Lk is an amide group; Y is a single bond; and T is independently selected from a ligand derived from any of the following structures: mercaptoacetyl triglycine (MAG3) or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA).

11. The molecular probe targeting GPC3 according to any one of claims 2 to 10, wherein L is independently selected from a ligand derived from any one of the following structures: The linker comprises a reactive portion and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof; preferably, the linker is a single bond.

12. The molecular probe targeting GPC3 according to any one of the preceding claims, wherein M is a nuclide that releases beta or alpha particles, a positron-emitting nuclide, a single-photon nuclide, or a paramagnetic metal; preferably, M is selected from a diagnostic radionuclide or a therapeutic radionuclide; Preferably, the diagnostic radionuclide is selected from 68 Ga, 18 F-Al, 64 Cu, 99m Tc, 89 Zr, 44 Sc, 111 In, 45 Ti, 52 Mn, 59 Fe, 94m Tc, 67 Ga, 71 As, 72 As, 82m Rb, 86 Y. 131 I, Gd, Mn or a combination thereof; Preferably, the therapeutic radionuclide is selected from 177 Lu, 90 Y. 131 I. 153 Sm, 67 Cu, 89 Sr. 166 Ho, 177 Yb, 47 Sc, 186 Re, 188 Re, 212 Bi, 213 Bi, 149 Pm, 212 Pb, 211 At 223 Ra, 225 Ac, 227 Th or a combination thereof; More preferably, M is selected from 68 Ga, 18 F-Al, 99m Tc, 177 Lu, 131 I, or a combination thereof.

13. The molecular probe targeting GPC3 according to any one of the preceding claims, wherein LM is selected from the following structures: or -Connector- 131 I, in, The linker comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is linked to Ab, and are independently selected from a single bond, p-isothiocyanatophenyl, a maleimide group, an amide group, or a combination thereof; Preferably, the linker is a single bond.

14. The molecular probe targeting GPC3 according to any one of the preceding claims, wherein Ab is a GPC3 antibody or an antigen-binding fragment thereof; preferably, Ab is a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody or a fully human antibody; preferably, Ab is selected from a monoclonal antibody (mAb), Fab, Fab', F(ab')2, Fv, scFv, scFv-Fc or a single domain antibody.

15. The molecular probe targeting GPC3 according to any one of the preceding claims, wherein Ab comprises a heavy chain or a fragment thereof, wherein the heavy chain or the fragment thereof comprises HCDR1, HCDR2 and HCDR3, The HCDR1 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and most preferably an amino acid sequence of SEQ ID NO: 1; The HCDR2 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 2, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2, and most preferably an amino acid sequence of SEQ ID NO: 2; The HCDR3 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and most preferably an amino acid sequence shown in SEQ ID NO:

3.

16. The molecular probe targeting GPC3 according to claim 15, wherein Ab comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; preferably comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; more preferably comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; most preferably, the heavy chain variable region VH is the amino acid sequence shown in SEQ ID NO:

7.

17. The molecular probe targeting GPC3 according to claim 16, wherein Ab comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; preferably comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; more preferably comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; most preferably, the heavy chain is the amino acid sequence shown in SEQ ID NO:

9.

18. The molecular probe targeting GPC3 according to any one of the preceding claims, wherein Ab comprises a light chain or a fragment thereof, wherein the light chain or the fragment thereof comprises LCDR1, LCDR2 and LCDR3, The LCDR1 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:4, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:4, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:4, and most preferably an amino acid sequence of SEQ ID NO:4; The LCDR2 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:5, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:5, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:5, and most preferably an amino acid sequence of SEQ ID NO:5; The LCDR3 comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:6, preferably an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:6, more preferably an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:6, and most preferably an amino acid sequence shown in SEQ ID NO:

6.

19. The molecular probe targeting GPC3 according to claim 18, wherein Ab comprises a light chain variable region VL, and the light chain variable region VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; preferably comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; more preferably comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; most preferably, the light chain variable region VL is the amino acid sequence shown in SEQ ID NO:

8.

20. The molecular probe targeting GPC3 according to claim 19, wherein Ab comprises a light chain, wherein the light chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; preferably comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; more preferably comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; most preferably, the light chain is the amino acid sequence shown in SEQ ID NO:

10.

21. The molecular probe targeting GPC3 according to any one of the preceding claims, wherein Ab is a Fab, Fab' or F(ab')2 fragment, preferably a Fab fragment, Preferably, the Ab comprises a heavy chain amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 11, preferably comprises a heavy chain amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 11, more preferably comprises a heavy chain amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 11, and most preferably comprises a heavy chain amino acid sequence shown in SEQ ID NO: 11; and Preferably, the Ab comprises a light chain amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, preferably comprises a light chain amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, more preferably comprises a light chain amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, and most preferably comprises a light chain amino acid sequence shown in SEQ ID NO:

10.

22. The molecular probe targeting GPC3 according to any one of claims 1 to 20, wherein Ab is a scFv fragment, Preferably, the Ab comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; preferably, it comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; more preferably, it comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; most preferably, the Ab comprises the amino acid sequence shown in SEQ ID NO:

12.

23. The molecular probe targeting GPC3 according to any one of the preceding claims, which is selected from the following structures: in, The linker comprises a reactive moiety and a spacer group, wherein the spacer group is independently selected from a single bond, -(PEG) r -、-(Gly) r - or a combination thereof, wherein r is an integer from 1 to 10; the reactive portion is connected to Ab and is independently selected from a single bond, p-isothiocyanatophenyl, maleimide, amide, or a combination thereof; preferably, the linker is a single bond.

24. An antibody conjugate having a structure represented by the following general formula (III): Ab m -L' n (III) Wherein L' is represented by the following general formula (IV): Y-Lk-T' (IV) Wherein Lk and T' optionally further comprise a spacer group, wherein Ab, Y, Lk, the spacer, m and n have the same meanings as defined in any one of the preceding claims 1 to 21, and T' is independently selected from a single bond, deferoxamine (DFO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyltriglycine (MAG3), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), ethylenediaminetetraacetic acid (EDTA ... 4,7,10-tetrakis(methylenephosphonic acid) (DOTP), 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonic acid) (NOTP), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), triacetylfusine C (TAFC), streptomycin E (FOXE), streptomycin B (FOXB), ferric pigment A (FCHA), or a combination thereof; preferably selected from single bond deferoxamine (D FO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyl triglycine (MAG3), 6-hydrazinonicotinic acid (HYNIC), 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid) )(DOTP), or 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP); more preferably selected from a single bond, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tricarboxylic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetyltriglycine (MAG3), or 4-[[[(1R,2R)-2-[bis(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino]methyl]-phenylacetic acid (H3RESCA). 25 . A pharmaceutical composition comprising: the molecular probe targeting GPC3 according to any one of claims 1 to 23 or the antibody conjugate according to claim 24 ; and a pharmaceutically acceptable carrier.

26. A kit comprising: the antibody conjugate of claim 24; and a radionuclide M, wherein M has the same meaning as defined in any one of the preceding claims 1-23. 27 . A method for preparing a molecular probe targeting GPC3 according to any one of claims 1 to 23 , comprising reacting the antibody conjugate according to claim 24 with a radionuclide M.

28. Use of the molecular probe targeting GPC3 according to any one of claims 1 to 23, the antibody conjugate according to claim 24, the pharmaceutical composition according to claim 25, or the kit according to claim 26 in the preparation of a reagent for detecting GPC3.

29. Use of the molecular probe targeting GPC3 according to any one of claims 1 to 23, the antibody conjugate according to claim 24, the pharmaceutical composition according to claim 25, or the kit according to claim 26 in the preparation of a kit for molecular imaging.

30. An imaging method comprising: Administering the molecular probe targeting GPC3 according to any one of claims 1 to 23 or the pharmaceutical composition according to claim 24 to a subject in need thereof; and The presence of the molecular probe targeting GPC3 is detected in vivo by imaging.

31. The imaging method of claim 30, wherein the presence of the molecular probe targeting GPC3 is detected by single photon emission computed tomography (SPECT), positron emission tomography (PET) imaging, or magnetic resonance imaging (MRI).

32. Use of the molecular probe targeting GPC3 according to any one of claims 1 to 23, the antibody conjugate according to claim 24, the pharmaceutical composition according to claim 25 or the kit according to claim 26 in the preparation of a kit for cancer diagnosis.

33. Use of the molecular probe targeting GPC3 according to any one of claims 1 to 23, the antibody conjugate according to claim 24, the pharmaceutical composition according to claim 25 or the kit according to claim 26 in the preparation of an agent for cancer treatment. 34 . A method for treating cancer, comprising administering the molecular probe targeting GPC3 according to any one of claims 1 to 23 or the pharmaceutical composition according to claim 25 to a subject in need thereof.

35. The method according to claim 34, wherein the subject is GPC3 positive.

36. The method according to any one of claims 32 to 34, wherein the cancer is a GPC3-high expressing cancer, preferably a solid tumor, more preferably selected from liver cancer, non-small cell lung cancer, melanoma, ovarian yolk sac tumor, ovarian clear cell carcinoma and colon cancer, most preferably liver cancer.

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