Anti-GPC3 antibodies and their radioconjugates

Abstract

This invention discloses novel antibodies or antigen-binding fragments thereof that bind to GPC3. These anti-GPC3 antibodies or fragments thereof preferably do not contain lysine residues in their CDR, thus making them particularly suitable for conjugation. This disclosure also relates to conjugates targeting radionuclides, such as actinium-225-targeting conjugates (TACs). Preferably, the TAC comprises an anti-GPC3 antibody or fragment thereof of formula (I) of this disclosure. The antibody or antigen-binding fragment thereof, and / or the antibody-conjugate, and / or the TAC can be used for diagnosis and / or treatment, preferably for treatment, more preferably for the treatment of cancer, and most preferably for the treatment of hepatocellular carcinoma (HCC). Pharmaceutical compositions comprising these antibodies, functional fragments, and conjugates and kits with instructions are provided. This disclosure also provides methods and tools for preparing these antibodies, functional fragments, and conjugates.

Description

[0001] This application is a divisional application of Chinese patent application filed on October 11, 2024, with application number 202480066910.1 and invention title "Anti-GPC3 Antibody and its Radioconjugate". Technical Field

[0002] This invention relates to novel monoclonal antibodies or antigen-binding fragments thereof capable of binding to phosphatidylinositol proteoglycan-3 (GPC3), and conjugates comprising these anti-GPC3 antibodies or fragments thereof. The invention also relates to conjugates targeting radionuclides, such as actinium-225 (Ac-225) conjugates (TACs). Preferably, the actinium-targeting conjugate of formula (I) comprises the anti-GPC3 antibody or fragment thereof of the invention. The antibody or antigen-binding fragment thereof, and / or the antibody conjugate, and / or the actinium-targeting conjugate can be used for diagnosis and / or treatment, preferably for treatment, more preferably for treatment of hyperproliferative diseases (e.g., cancer), and most preferably for treatment of hepatocellular carcinoma (HCC).

[0003] Pharmaceutical compositions comprising the antibodies, functional fragments, and conjugates of the present invention, as well as kits with instructions for use, are provided.

[0004] The present invention also provides methods and tools for preparing the antibodies, functional fragments, and conjugates of the present invention. For example, polynucleotides encoding the above-described antibodies or fragments, vectors containing them, and cells for production are provided. Background Technology

[0005] Phosphatidylinositol proteoglycan-3 (GPC3) is an oncoemulsification protein expressed in a variety of tumors, such as hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), gastric cancer, ovarian cancer, melanoma, and embryonal tumors in children. GPC3 expression is particularly high in HCC, limited in adults, and absent in viral hepatitis, cirrhosis, or NASH, making HCC a highly attractive target for GPC3-based therapies. This is of paramount importance, given that primary liver cancer is the sixth most common malignancy worldwide and the second leading cause of cancer-related death globally (Torre et al., Globalcancer statistics, 2012. CA Cancer J Clin. 2015 Mar;65(2):87-108).

[0006] To date, several anti-GPC3 antibodies have been described, such as hYP7, hYP9.1b (Zhang et al., Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma. Sci Rep. 2016 Sep 26;6:33878), VHH antibody HN3 (WO2012 / 145469; Feng et al., Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma. Proc Natl AcadSci US A. 2013 Mar 19;110(12)), and fully human antibodies 2-F7, 4-11G, 7-5B, 7-8B, and 17-4D (Yu et al., Generation of fully human anti-GPC3 antibodies with high-affinity recognition of GPC3 positive tumors. Invest New Drugs. 2021). Jun;39(3):615-626), or mouse anti-human GPC3 antibody aGPC3 (WO2023 / 056474; Labadie et al., Glypican-3-Targeted 227 Th α-TherapyReduces Tumor Burden in an Orthotopic Xenograft Murine Model of Hepatocellular Carcinoma. J Nucl Med. 2022 Jul;63(7):1033-1038). However, to date, none of these anti-GPC3 antibodies have entered the clinical stage.

[0007] Currently, the only clinically validated antibody targeting GPC3 is codrituzumab, a humanized version of the murine monoclonal antibody GC33. It binds specifically to the juxtamembrane domain of GPC3 with high affinity (Ishiguro et al., Anti-glypican 3 antibody as a potential antitumor agent for human liver cancer. Cancer Res. 2008 Dec 1;68(23):9832-8; Nakano et al., Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells. Biochem Biophys ResCommun. 2009 Jan 9;378(2):279-84). Codrituzumab induces antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), inhibiting tumor growth. Codrituzumab was well-tolerated in a phase I trial and showed antitumor effects in HCC patients. However, in a randomized phase II study, codrituzumab did not show any benefit over placebo (Abou-Alfa et al., Randomized phase II placebo-controlled study of codrituzumab in previously treated patients with advanced hepatocellular carcinoma. J Hepatol. 2016 Aug;65(2):289-95). Some subsequent clinical studies have shown that codrituzumab has prognostic value for HCC patients with high sensitivity and high expression of GPC3 associated with ADCC (Chen et al., Combining expression of GPC3 in tumors and CD16 on NK cells from peripheral blood to identify patients responding to codrituzumab. Oncotarget. 2018 Jan 2;9(12):10436-10444). Currently, a phase I clinical trial testing codrituzumab in children and adolescents with solid tumors is recruiting participants (ClinicalTrials.gov identifier:NCT04928677).

[0008] However, the ADCC / CDC activity of antibodies targeting GPC3 appears insufficient to effectively treat diseases in which GPC3 expression plays a role, thus necessitating the development of novel, GPC3-specific therapeutics. GPC3 is an oncoemulsifiable protein primarily expressed in specific cancer types, and not expressed or only minimally expressed in normal tissues, making it a suitable target for targeted radionuclide therapy. Targeted radionuclide therapy is a promising and developing field with the potential to deliver highly cytotoxic radiation specifically to disease-related cell types. Currently approved radiopharmaceuticals for human use most commonly employ radionuclides that emit beta and / or gamma rays. However, due to the stronger cytotoxic potential of alpha-ray-emitting nuclides, their application is receiving increasing attention, particularly in cancer treatment.

[0009] The unique mechanism of action of targeted alpha therapy (TAT) lies in the specific delivery of radioactive alpha particles, such as actinium-225, to tumors through the use of tumor antigen-targeting components, such as small molecules, peptides, or monoclonal antibodies, thereby inducing clusters of irreparable DNA double-strand breaks. Radioactive alpha particles are highly cytotoxic, thus requiring specific biological targeting antibodies to avoid unacceptable side effects. The biological targeting efficacy of isotopes is highly dependent on the stability of the chelating agent, the robustness of the antibody to the chelation process, and the antibody's targeting properties to its specific target in a given biological environment.

[0010] Typical alpha emitters have a radiation range of less than 100 micrometers in a physiological environment, equivalent to only a few cell diameters. This makes these radiation sources highly suitable for treating tumors, including micrometastases, because they have a radiation range covering adjacent cells within the tumor; however, when well-targeted, very little radiation energy penetrates beyond the target cells. Therefore, targeted delivery to every cell is unnecessary, while minimizing damage to surrounding healthy tissue (see Feindegen et al., Radiat. Res. 148:195-201 (1997)). In contrast, beta particles have a radiation range of 1 mm or more in water (see Wilbur DS, Antibody Immunoconj Radiopharm. 4: 85-97 (1991)).

[0011] Compared to the energy carried by beta particles, gamma rays, and X-rays, alpha particle radiation has a much higher energy, typically 5–8 MeV, or 5 to 10 times the energy of beta particles and 20 times or more the energy of gamma rays. This deposition of a large amount of energy over an extremely short distance results in alpha radiation exhibiting a very high linear energy transfer (LET), a high relative biological effect (RBE), and a low oxygen enhancement ratio (OER) compared to gamma and beta radiation (see Hall, *Radiobiology for the radiologist*, 5th ed., Lippincott Williams & Wilkins, Philadelphia PA, USA, 2000). This explains the unusual cytotoxicity of alpha-radioactive isotopes and also places stringent demands on the biological targeting of such isotopes and the control and research levels necessary to avoid unacceptable side effects.

[0012] To date, attention regarding the application of alpha-emitting radionuclides in radioimmunotherapy has primarily focused on... 211 At、 213 Bihe 225 Ac, these three radionuclides have been explored in clinical immunotherapy trials. However, the compounds currently in use, typically employing the macrocyclic chelator DOTA (2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid), lack stability, leading to the dissociation of the radionuclides from the chelating macrocycle. This results in reduced selectivity and activity for target tissues and increased toxicity to non-target tissues. Serum stability studies have shown that the [225Ac]Ac-DOTA complex dissociates over time (Ramogida et al., Evaluation of Polydentate Picolinic Acid Chelating Ligands and an α-Melanocyte-Stimulating Hormone Derivative for Targeted Alpha Therapy Using ISOL-Produced 225Ac. EJNMMI Radiopharm. Chem. 2019, 4, 21), resulting in 225Ac deposition in the liver and femur after administration of the DOTA-based conjugate (Deal et al., Improved in Vivo Stability of Actinium-225 Macrocyclic Complexes. J. Med. Chem. 1999, 42, 2988-2992.).

[0013] Further research is currently underway to identify and develop methods suitable for large ion Ac. 3+ Chelating agents (Thiele et al., Actinium-225 for Targeted α Therapy: Coordination Chemistry and Current Chelation Approaches. Cancer Biother. Radiopharm. 2018, 33, 336-348). Acyclic chelating agents (such as ethylenediaminetetraacetic acid (H4EDTA), diethylenetriaminepentaacetic acid (H5DTPA), etc.) have the problem of poor stability of the complexes formed (Davis et al., Comparison of 225Actinium Chelates: Tissue Distribution and Radiotoxicity. Nucl. Med. Biol. 1999, 26, 581-589). Macrocyclic chelating agents (such as tetraazacyclododecane-1,4,7,10-tetraacetic acid (H4DOTA), etc.) exhibit higher inertness. However, radiolabeling H4DOTA with 225Ac requires either high-temperature conditions (which are incompatible with temperature-sensitive biomolecules such as antibodies) or a long incubation time (which results in relatively low specific activity and purity of the obtained construct).

[0014] To date, only a few examples of radionuclide conjugates targeting GPC3 have been reported. WO2022115778 describes a targeting component for cancer therapy comprising a macrocyclic peptide conjugated to a metal chelator (e.g., DOTA), which can complex with a radioisotope (e.g., Lu177 or Ac225). As relatively small molecules, these peptides exhibit much faster tumor clearance rates than larger molecules (e.g., antibodies), with relatively low tumor uptake, as exemplified by RAYZ-8009 reported by Li et al., which comprises a macrocyclic peptide binding to GPC3, a linker, and DOTA (Li et al., Novelpeptide binder to Glypican-3 for targeted radiopharmaceutical therapy of hepatocellular carcinoma. Journal of Clinical Oncology 2023, 41:16_suppl,e16131-e16131).

[0015] Labadie et al. reported a 227Th-labeled antibody-drug conjugates targeting GPC3 ( 227 Th-octapa-αGPC3), used for the treatment of HCC in orthotopic mouse models (Labadie et al., Glypican-3-Targeted 227 Th α-Therapy Reduces Tumor Burden in an Orthotopic Xenograft Murine Model of Hepatocellular Carcinoma. J Nucl Med. 2022 Jul;63(7):1033-1038; WO2023 / 056474), in which the conjugate was observed to have reduced tumour burden in an orthotopic Xenograft Murine Model of Hepatocellular Carcinoma after 2 hours at room temperature. 227 The labeling yield of Th was as low as 70%. Furthermore, the radiopharmaceutical used... 227 The half-life of Th radionuclides is relatively long (18.7 days), and 227 The first generation of daughter nuclides of Th 223 Ra also has a longer half-life, thus achieving a greater efficiency compared to... 225 Ac is a slower delivery of radiation dose ( 225 Ac's progeny nuclides have shorter half-lives, enabling faster delivery of radiation doses.

[0016] Bell et al. reported codrituzumab-based actinide conjugates. 225 Ac]Ac–Macropa–GC33 (Bell et al., Glypican-3-Targeted Alpha Particle Therapy for Hepatocellular Carcinoma. Molecules. 2020 Dec 22;26(1):4), Kadassery et al. compared [ 225 Ac]Ac-GC33-BZmacropa and similar [ 225Ac]Ac-GC33-macropa conjugates (Kadassery et al., H2BZmacropa-NCS: A Bifunctional Chelator for Actinium-225 Targeted Alpha Therapy. Bioconjug Chem. 2022 Jun15;33(6):1222-1231). Although these conjugates employ the potent alpha-emitting radionuclide actinium-225, they are all based on the GPC3-binding antibody codrituzumab (GC33), which contains four lysine residues in its CDR. Similarly, the αGPC3 disclosed in WO2023 / 056474 (see above) contains 4 lysine residues in its CDR. In addition, the fully human antibodies 2-F7, 4-11G, 7-5B, 7-8B and 17-4D reported by Yu et al. (Yu et al., Generation of fully human anti-GPC3 antibodies with high-affinity recognition of GPC3 positive tumors. Invest New Drugs. 2021 Jun;39(3):615-626) also contain one or more lysine residues in their CDR (2-F7: 3 lysine residues; 4-11G, 7-5B, 7-8B and 17-4D: 1 lysine residue).

[0017] Conjugation of chelating agents (e.g., macropa) typically occurs via lysine residues of the antibody, but can also occur within the target-binding CDR region of the antibody. This labeling impairs the antibody's ability to bind to its target. Therefore, the presence of lysine residues in the CDR implies an increased risk of unbound fractions in the antibody conjugate, leading to, for example, decreased internalization or toxic side effects. At approximately 10 chelating agent labeling levels per antibody, Bell et al. (2020) (see above) observed their codrituzumab-based actinide conjugates [ 225 Ac]Ac–Macropa–GC33 showed significant nonspecific accumulation in the liver. This finding is most likely due to increased unbound fractions of the conjugate by labeling lysine in the GC33 CDR. Another drawback of using the humanized anti-GPC3 antibody codrituzumab in targeting α-conjugates is its relatively high potential for immunogenicity, which could trigger immunogenicity-related side effects.

[0018] Purpose of the invention In view of the prior art, the object of the present invention is to provide novel therapeutic anti-GPC3 antibodies and their antigen-binding fragments, said antibodies and fragments being particularly suitable as conjugates for targeted radionuclide therapy, overcoming the disadvantages of existing anti-GPC3 antibodies and GPC3-targeting radionuclide conjugates. Specifically, the object of the present invention is to provide (i) novel anti-GPC3 antibodies and their antigen-binding fragments, said antibodies and fragments having high target affinity, cross-reactivity with cynomolgus monkey antigens, low immunogenicity, high thermal stability, and good manufacturability through recombinant production, while having as low a number of lysine residues and other sequence defects in the CDR as possible; and (ii) radionuclide conjugates targeting radioactive alpha particles, preferably targeting actinium conjugates (TACs), comprising these anti-GPC3 antibodies and their antigen-binding fragments, which still have high binding affinity, high stability, low chelator-antigen ratio (CAR), and can chelate in high yield under mild conditions.

[0019] These novel anti-GPC3 antibodies and their anti-GPC3-TACs will offer significant advantages in the treatment of hyperproliferative diseases such as cancers (e.g., hepatocellular carcinoma (HCC)).

[0020] In addition, these novel antibodies can also be used as imaging agents, for example, when labeled with positron-emitting electrons by a suitable chelating agent, for PET imaging. Summary of the Invention

[0021] The foregoing and other objectives are achieved through the teachings of this invention. This invention is based on the discovery of novel antibodies targeting GPC3, which are particularly suitable for targeting radionuclide therapeutic conjugates and delivering therapeutic benefits to subjects.

[0022] Codrituzumab, a humanized version of the murine monoclonal antibody GC33, is the only clinically validated antibody targeting GPC3. However, this antibody suffers from drawbacks such as a high immunogenicity score, numerous germline deviations, the presence of lysine residues in the CDR that hinder chelating agent binding via amino acid coupling, and a potential aspartic acid isomerization site in the CDR. To optimize these characteristics, codrituzumab underwent sequence germination, and further modifications were introduced, such as removing lysine residues or potential aspartic acid isomerization sites within the CDR. Nakano et al. have described the humanization and optimization of GC33 to obtain Codrituzumab. They showed that even a single amino acid substitution can lead to a complete loss of binding ability (e.g., N33D in CDR-L1). Therefore, it is expected that little further optimization can be performed without completely sacrificing beneficial properties. Furthermore, due to the high number of deviations from human lineages (32), the sequence space is too large to be systematically evaluated as a whole, further significantly reducing the potential for immunogenicity inherent in antibodies derived from mouse immunity.

[0023] Surprisingly, despite the extremely high number of mutations in both VH and VL compared to Codrituzumab, the applicant was still able to identify antibody variants with the following characteristics: a) still producible via recombinant methods; b) exhibiting comparable or superior affinity for recombinant or cellular human GPC3; c) still exhibiting binding to cynomolgus GPC3; and d) still exhibiting stability of the Fab fraction Tm above 65°C. However, the number of germline biases was drastically reduced, which is also reflected in significantly lower in-silico immunogenicity scores (IPAD, Epibase) and in vitro immunogenicity assessments (Epibase). The IPAD immunogenicity score is even more promising than the scores of the aforementioned fully human antibodies 2-F7, 4-11G, 7-5B, 7-8B, and 17-4D.

[0024] Surprisingly, by combining germlineation with CDR sequence alteration, the applicant identified novel anti-GPC3 antibodies with promising binding properties and reduced immunogenicity potential, which do not contain any lysine residues in their CDRs, making them particularly suitable as the targeting moiety of radiopharmaceuticals or other antibody-drug conjugates coupled via lysine residues.

[0025] Furthermore, the chelating agent Macropa-NCS used in the targeted actinide conjugate (TAC) of the present invention can achieve a high immunoreactivity fraction (IRF) at a low chelator-antigen (CAR) ratio and has high monomer purity, resulting in a high proportion of radionuclide-labeled compounds with GPC3 affinity. In addition, the GPC3-TAC of the present invention exhibits favorable biodistribution in vivo and low accumulation in the liver, indicating reduced dissociation of the radionuclide from the chelate and thus increased stability. The antibody chelating agent conjugate of the present invention can be radionuclide-labeled under very mild conditions and at a low temperature (i.e., room temperature), thereby reducing antibody denaturation and the amount of non-GPC3-binding fraction in the final product. Therefore, the TAC of the present invention exhibits an improved tumor-to-liver ratio, thus enabling more effective and selective treatment of diseases, maintaining high potency against cancer cells in target tissues while reducing damage to non-target tissues.

[0026] In addition to being suitable as conjugates for targeted radionuclide therapy, the antibodies or fragments thereof of the present invention can be used for a variety of other purposes, such as imaging, antibody-drug conjugates, or as therapeutic agents without a payload. For example, when conjugated with a chelating agent DFO* (e.g., as described in WO2021051168) and used in combination... 89 When Zr-labeled, the antibodies or fragments thereof of the present invention can be used for PET imaging purposes. Detailed Implementation

[0027] The invention can be more readily understood by referring to the following detailed description of the invention and its embodiments.

[0028] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. However, the following references provide general definitions of many terms used in this invention for those skilled in the art, and these references may be cited and used provided that such definitions are consistent with their common understanding in the art. Such references include, but are not limited to: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); Lackie et al., The Dictionary of Cell & Molecular Biology (3rd ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, WB Saunders Company. Any other technical resources available to those skilled in the art provide definitions of terms used herein with meanings commonly understood in the art. For the purposes of this invention, the following terms are further defined. Other terms are defined elsewhere in the specification. When used herein and in the appended claims, the singular forms “a(a)” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a gene” refers to one or more genes and includes their equivalents known to those skilled in the art, etc.

[0029] In the context of this invention, the term "comprising" or "including" means "including, but not limited to." This term is open-ended and is used to define the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout this application and particularly in the claims may be replaced by the term "consisting of."

[0030] In this document, the terms “about” or “approximately” mean between 80% and 120% of a given value or range, or between 90% and 110%, including 95% and 105%.

[0031] If any item in this document is stated as “as mentioned herein,” it means that it can be mentioned anywhere in this document.

[0032] In the context of this invention, the term "GPC3" refers to phosphatidylinositol proteoglycan 3. GPC3 is a carcinoembryonic acid heparan sulfate glycoprotein anchored to the cell membrane via glycosylphosphatidylinositol (Filmus J, Capurro M. Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma. FEBS J. May 2013;280(10):2471-6). GPC3 contains 580 amino acids and has a molecular weight of 70 kDa. Two HS chains are attached near the C-terminal portion. Single-chain GPC3 is processed by furin at the Arg358-Cys359 bond to produce mature GPC3, which consists of a 40-kDa N-terminal subunit and a 30-kDa C-terminal subunit linked by disulfide bonds (De CatB, Muyldermans SY, Coomans C, Degeest G, Vanderschueren B, Creemers J, BiemarF, Peers B, David G. Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements. J Cell Biol. 2003 Nov 10;163(3):625-35). Specific binding fragments such as Fab, Fab', F(ab')2, and single-chain specific binding antibodies are typical examples.

[0033] The human GPC3 protein is encoded by the GPC3 gene (NCBI gene ID 2719). Other names for GPC3 include SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, and GTR2-2. GPC3 proteins include homologs in humans, mice, cynomolgus monkeys, and other mammals and non-mammals.

[0034] Human GPC3 (SEQ ID NO: 321): QPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINV DDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILS LEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGM KNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH The terms “anti-GPC3 antibody” or “anti-phosphatidylinositol proteoglycan 3 antibody” and “antibody binding phosphatidylinositol proteoglycan 3” or “antibody binding GPC3” are used synonymously herein to refer to an antibody capable of binding GPC3, preferably having an affinity sufficient to enable the antibody to be used as a diagnostic and / or therapeutic agent targeting GPC3.

[0035] Codrituzumab (CAS Registry No. 1365267-33-9) is a humanized monoclonal anti-GPC3 antibody. Its preparation, isolation, and purification have been described, for example, in US 7,919,086. Codrituzumab (also referred to herein as "TPP-14890") comprises the heavy chain protein sequence according to SEQ ID NO. 57 and the light chain protein sequence according to SEQ ID NO. 58. Furthermore, Codrituzumab comprises at least three heavy chain CDR sequences according to SEQ ID NO. 42, SEQ ID NO. 43, and SEQ ID NO. 44, and three light chain CDR sequences according to SEQ ID NO. 46, SEQ ID NO. 47, and SEQ ID NO. 48. Codrituzumab CDR heavy chain sequence SEQ ID NO. 42: DYEMH SEQ ID NO. 43: ALDPKTGDTAYSQKFKG SEQ ID NO. 44: FYSYTY Codrituzumab CDR light chain sequence SEQ ID NO. 46: RSSQSLVHSNRNTYLH SEQ ID NO. 47: KVSNRFS SEQ ID NO. 48: SQNTHVPPT The terms “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0036] In this article, amino acids can be referred to by their well-known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides can be referred to by their generally accepted single-letter codes.

[0037] As used herein, the term "antibody" is intended to refer to immunoglobulin molecules, including but not limited to full-length antibodies and monovalent antibodies. A "full-length antibody" preferably comprises four polypeptide chains, typically two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated VH herein) and a heavy chain constant region. The heavy chain constant region may contain, for example, three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated VL herein) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and at most four FRs, arranged from the amino terminus to the carboxyl terminus in, for example, the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. As used herein, a “monovalent antibody” preferably comprises three polypeptide chains: two heavy (H) chains and one light (L) chain, typically linked by disulfide bonds. One heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region may contain, for example, three domains: CH1, CH2, and CH3. The other heavy chain consists solely of the heavy chain constant region. The light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and at most four FRs, arranged from the amino terminus to the carboxyl terminus in, for example, the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0038] As used herein, the term “complementarity-determining region” (CDR; e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain that are essential for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity-determining region may contain amino acid residues from what Kabat defines as “complementarity-determining regions” (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from “hypervariant rings” (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; (Chothia and Lesk; J Mol Biol)). 196: 901-917 (1987)). In some cases, the complementarity-determining region may include amino acids from the CDR region as defined by Kabat and amino acids from the hypervariable ring.

[0039] Intact antibodies can be classified into different “classes” based on the amino acid sequence of the constant domain of their heavy chains. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into “subclasses” (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. A preferred class of immunoglobulins used in this invention is IgG. In a particular embodiment, the antibody of this invention is an IgG1, IgG2, IgG3, or IgG4 antibody, more specifically an IgG1 antibody. Different isotypes may have different effector functions. Human light chains are divided into Kappa (κ) and lambda (λ) light chains. In both the light and heavy chains, variable and constant regions are linked by a “J” region of about 12 or more amino acids, and the heavy chain also contains a “D” region of about 10 more amino acids. General reference: Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). The heavy chain constant domains corresponding to different classes of antibodies are designated as [α], [δ], [ε], [γ], and [μ]. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known. As used herein, antibodies are conventionally known antibodies and their functional fragments.

[0040] The “functional fragment” or “antigen-binding antibody fragment” of an antibody / immunoglobulin is thus defined as a fragment of an antibody / immunoglobulin that retains an antigen-binding region (e.g., the variable region of IgG). The “antigen-binding region” of an antibody is typically found in one or more hypervariable regions of the antibody, such as CDR1, -2, and / or -3 regions; however, variable “framework” regions can also play an important role in antigen binding, for example, by providing a scaffold for the CDR. Preferably, the “antigen-binding region” comprises at least amino acid residues 4-103 of the variable light (VL) chain and amino acid residues 5-109 of the variable heavy (VH) chain, more preferably amino acid residues 3-107 of the VL and amino acid residues 4-111 of the VH, and particularly preferably intact VL and VH chains (amino acid sites 1-109 of the VL and amino acid sites 1-113 of the VH; according to WO 97 / 08320).

[0041] Non-limiting examples of “functional fragments” or “antigen-binding antibody fragments” include Fab, Fab', F(ab')2, Fv fragments, domain-specific antibodies (dAb), complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), single-chain antibody fragments, biantibodies, triantibodies, tetraantibodies, microantibodies, linear antibodies (Zapata et al., Protein Eng., 8 (10): 1057-1062 (1995)); chelated recombinant antibodies, tribodies or bibodies, intracellular antibodies, nanobodies, small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, camelized antibodies, antibodies containing VHH, or mutant proteins or derivatives thereof, and peptides containing at least a portion of an immunoglobulin (sufficient to confer specific antigen-binding ability) such as a CDR sequence (provided the antibody retains the desired biological activity); and multispecific antibodies formed from antibody fragments, such as bispecific and trispecific antibodies (CA K Borrebaeck, editor (1995) AntibodyEngineering). (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). Antibodies other than “bispecific” or “bifunctional” antibodies are understood to have identical binding sites. F(ab’)2 or Fab can be modified to minimize or completely eliminate intermolecular disulfide interactions occurring between the CH1 and CL domains. Antibody digestion with papain produces two identical antigen-binding fragments called “Fab” (each with a single antigen-binding site) and a residual “Fc” fragment (whose name reflects its tendency to crystallize). Pepsin treatment produces an F(ab’)2 fragment containing two “Fv” fragments. The “Fv” fragment is the smallest antibody fragment containing both complete antigen recognition and binding sites. This region consists of a tightly non-covalently associated dimer of a heavy chain variable domain and a light chain variable domain. In this configuration, the three CDRs of each variable domain interact to define antigen-binding sites on the surface of the VH-VL dimer. The six CDRs collectively confer antibody antigen-binding specificity. However, even a single variable domain (or half an Fv containing only three CDRs specific to a particular antigen) can recognize and bind to an antigen.

[0042] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain the VH and VL domains of the antibody, where these domains are present in a single polypeptide chain.

[0043] Preferably, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables Fv to form the structure required for antigen binding. A review of Fv can be found in Pluckthun's The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0044] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that it has several residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name used in this paper to refer to the Fab' fragment in which the cysteine ​​residues of the constant domain have a free thiol group. The F(ab')2 antibody fragment was initially formed as paired Fab' fragments with hinge cysteine ​​residues between them.

[0045] The terms "mutant protein" and "variant" are used interchangeably to refer to an antibody or antigen-binding fragment containing at least one amino acid substitution, deletion, or insertion in a variable region or an equivalent region, provided that the mutant protein or variant retains the desired binding affinity or biological activity. The antibody or antigen-binding antibody fragment variants contemplated in this invention are molecules that retain the binding activity of both antibody and antigen-binding antibody fragments.

[0046] "Humanized antibody" is generally defined as: (I) an antibody derived from a non-human source (e.g., a transgenic mouse carrying a heterologous immune system) and based on a human lineage sequence; or (II) a CDR transplantation antibody, wherein the CDR of the variable region is derived from a non-human source, and one or more frame regions and / or portions of the CDR sequence of the variable region are derived from a human source, and the constant region (if any) is derived from a human source.

[0047] Therefore, the term "humanized antibody" includes antibodies modified in such a way that at least partially replaces the variable region of the heavy or light chain, or both, of a human antibody with one or more CDRs from a non-human antibody with known specificity, and may also include partial frame region substitutions and sequence alterations if necessary. In other words, antibodies in which one or more "donor" CDRs from a non-human antibody with known specificity (such as mouse, rat, rabbit, or non-human primate antibodies) are transplanted into the frame region of a human heavy or light chain are referred to herein as "humanized antibodies." Replacing all CDRs with intact CDRs of the donor variable domain to transfer the antigen-binding capacity of one variable domain to another may not be necessary. Instead, it may only be necessary to transfer those residues essential to maintaining the activity of the target binding site.

[0048] The frame regions (FRs) within the variable regions of the heavy or light chain, or both, of humanized antibodies may contain only human residues; in this case, these frame regions of the humanized antibody are referred to as "fully human frame regions." Human frame regions containing a mixture of human and donor frame residues are referred to herein as "partially human frame regions." Furthermore, humanized antibodies may contain residues not present in the recipient or donor antibody. These modifications are made to further optimize antibody performance (e.g., to achieve the desired affinity).

[0049] Generally, humanized antibodies will therefore contain at least one, typically two, variable regions, wherein all or part of the CDR corresponds to the CDR of a non-human immunoglobulin, and all or substantially all of the FR corresponds to the FR of the corresponding human immunoglobulin sequence. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically the corresponding portion of a human immunoglobulin. For further details, see Jones et al., Nature 331:522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); and Presta, Curr. Opin. Struct. Biol. 2:593-96 (1992), each incorporated herein by reference.

[0050] "Human antibody" or "fully human antibody" includes a human CDR, i.e., a CDR of human origin. In this document, a "human" antibody is defined as an antibody that is not chimeric or "humanized" and is (wholly or partially) not derived from a non-human species. Human antibodies or functional antibody fragments may be derived from humans or may be synthetic human antibodies. A "synthetic human antibody" is defined herein as an antibody having a sequence wholly or partially derived from a synthetic sequence through in-silico computer simulation, said synthetic sequence being based on the analysis of known human antibody sequences. The computer simulation design of human antibody sequences or fragments thereof can be achieved, for example, by analyzing a database of human antibody or antibody fragment sequences and designing amino acid sequences using the data obtained therefrom. Another example of a human antibody or functional antibody fragment is the antibody encoded by a nucleic acid isolated from a human antibody sequence library (i.e., such a library is based on antibodies obtained from naturally occurring human sources).

[0051] As used herein, the term “monoclonal antibody” refers to an antibody derived from a substantially homogeneous group of antibodies, i.e., the individual antibodies constituting the group are identical except for the possibility of mutations (e.g., naturally occurring mutations) that may be present in very small amounts. Thus, the term “monoclonal” indicates that the antibody is characterized as not being a mixture of discrete antibodies. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. In addition to their specificity, monoclonal antibody formulations have the advantage that they are generally not contaminated by other immunoglobulins. The term “monoclonal” should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used may be prepared by the hybridoma method (first described in Kohler et al., Nature, 256: 495

[1975] ) or by the recombinant DNA method (see, for example, US Patent No. 4,816,567). “Monoclonal antibody” may also be, for example, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, a Human Engineered™ antibody, or an antibody fragment.

[0052] "Isolated" antibodies are antibodies that have been identified and separated from the components of the cells that express them. Cellular contamination components are substances that can interfere with the diagnostic or therapeutic use of antibodies and can include enzymes, hormones, and other protein- or non-protein solutes.

[0053] "Isolated" nucleic acids are nucleic acids that have been identified and isolated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0054] As used herein, an antibody that "specifically binds to," "specifically targets," or "specifically recognizes" a target antigen (such as GPC3) is an antibody that binds to an antigen target with sufficient affinity so that the antibody can be used as a therapeutic agent targeting cells or tissues expressing that antigen; or it refers to an antibody that binds to an antigen-binding peptide target with sufficient affinity so that the antibody can be used as a reversal agent to neutralize the therapeutic activity of that antigen-binding peptide (such as an antigen-binding antibody), and the antibody does not exhibit significant cross-reactivity with other proteins, or with proteins other than orthologs and variants (such as mutant forms, splice variants, or proteolytic truncated forms) of the aforementioned target. The terms "specifically recognizes," "specifically binds to," or "specifically targets" an epitope on a specific peptide or specific peptide target, as used herein, can be determined, for example, by the monovalent dissociation constant (K) of the antigen. D (Below approximately 10) -4 M, or less than approximately 10 -5 M, or less than approximately 10 -6 M, or less than approximately 10 -7 M, or less than approximately 10 -8 M, or less than approximately 10 -9 M, or less than approximately 10 -10 M, or less than approximately 10 -11 M, or less than approximately 10 -12 M, or lower, of the antibody or its antigen-binding fragment is used. If an antibody can distinguish between an antigen and one or more reference antigens, then such an antibody "specifically binds to," "specifically targets," or "specifically recognizes" the said antigen. In its most general form, "specifically binds," "specifically binds to," "specifically targets," or "selectively recognizes" refers to the ability of an antibody, as determined by, for example, one of the following methods: surface plasmon resonance (SPR), Western blotting, ELISA assays, RIA assays, ECL assays, IRMA assays, and peptide scanning. For example, a standard ELISA assay can be performed. Scoring can be performed by standard colorimetric methods (e.g., horseradish peroxidase-conjugated secondary antibody and hydrogen peroxide with tetramethylbenzidine). The reaction in some wells is scored by, for example, optical density at 450 nm. Typical background (= negative reaction) can be 0.1 OD; typical positive reaction can be 1 OD. This means that the difference between positive and negative can be greater than 5, 10, 50, preferably greater than 100. Typically, binding specificity is determined not by using a single reference antigen, but by a group of about three to five unrelated antigens (such as milk powder, BSA, transferrin, etc.).

[0055] “Binding affinity” or “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise stated, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of an antibody or fragment thereof for a target can be determined using techniques known in the art, such as by ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), flow cytometry, or fluorescence polarization assay. The dissociation constant “K” is also mentioned. D "This term is commonly used to describe the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), that is, how tightly the ligand binds to a specific protein. Ligand-protein affinity is influenced by non-covalent intermolecular interactions between the two molecules. Affinity can be measured by conventional methods known in the art, including those described herein. Preferably, affinity is expressed as a dissociation constant K." D The affinity is provided in the form of an EC50 value, or, in one embodiment, the "K" of the invention. D "or "K D The value was measured using surface plasmon resonance determination with a Biacore T200 instrument (Cytiva). Other suitable instruments include the Biacore X100, Biacore S200, Biacore 8K, Biacore 8K+, Biacore 4000 SPR (Cytiva), Alto Digital SPR (Nicoya Lifesciences Inc.), or SIERRA SPR-32 PRO (Bruker Corporation).

[0056] The "half-maximal effective concentration" (EC50) refers to the concentration of a drug, modulator, antibody, fragment, conjugate, or molecule that produces half the response between baseline and maximum after a specific incubation time. Therefore, in the context of affinity, EC50 reflects the concentration of the conjugate (such as an antibody) required to achieve half-maximal binding. EC50 can be determined if the inflection point can be determined through mathematical modeling (e.g., nonlinear regression) of the dose-response curve describing the relationship between the applied drug, antibody, fragment, conjugate, or molecule concentration and the signal. For example, if the dose-response curve follows an S-shaped curve, EC50 can be determined. When the response is inhibitory, EC50 is called the "half-maximal inhibitory concentration" (IC50). 50 ).

[0057] As used herein, the term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins or T-cell receptors. Epitope determinants typically consist of chemically active surface groups of a molecule (such as amino acid or sugar side chains, or combinations thereof) and usually possess specific three-dimensional structural features and specific charge properties.

[0058] The terms "matured antibody" or "matured antigen-binding fragment," such as matured Fab variants or "optimized" variants, refer to antibodies or antibody fragment derivatives that exhibit stronger binding (i.e., enhanced affinity binding) to the extracellular domains of a given antigen, such as a target protein. Maturation is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment that result in this increased affinity. The maturation process is a combination of molecular biological methods that introduce mutations into the antibody and screen for the identification of improved bindings.

[0059] The term "antibody conjugate" refers to a conjugate comprising at least one antibody moiety and one or more other molecules or moietyes. These other molecules or moietyes may be selected from, but are not limited to, pharmaceuticals, chelating agents, radioactive elements, cytotoxic agents, other antibody or antigen-binding fragments.

[0060] The term “antibody-drug conjugate” (“ADC”) refers to an antibody-drug conjugate that contains at least one drug moiety.

[0061] The term "germination" refers to replacing residues in the variable domain of an antibody with residues present in a pre-mutated germline gene to reduce the likelihood of immunogenicity.

[0062] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fcγ receptors (FcγRs) present on certain cytotoxic cells, such as NK cells, neutrophils, and macrophages, thereby enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells, for example, with cytotoxins. To assess the ADCC activity of a target antibody, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337, or U.S. Patent No. 6,737,056 (Presta). Useful effector cells for such assays include PBMCs and NK cells.

[0063] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component (C1q) of the complement system to an antibody (an antibody of an appropriate subclass) that binds to its homologous antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). The binding of peptide variants with altered Fc region amino acid sequences (peptides with a variant Fc region) and increased or decreased C1q binding is described, for example, in U.S. Patents 6,194,551 B1 and WO 1999 / 51642.

[0064] The "percentage of sequence identity (%)" relative to a reference polynucleotide or polypeptide sequence is defined, respectively, as the percentage of nucleic acid or amino acid residues in the candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after (if necessary) alignment and the introduction of gaps to achieve the maximum percentage of sequence identity. Conservative substitutions are not considered part of sequence identity. Gap-free alignment is preferred. Alignments aimed at determining the percentage of amino acid sequence identity can be performed in a variety of ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MegAlign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences to be compared.

[0065] Substantial sequence identity / similarity can be understood as having at least 80% sequence similarity / identity with the complete sequence, and / or at least 90% sequence similarity / identity with specific binding regions (such as CDR regions). Preferred sequence similarity or more preferred identity may be at least 92%, 95%, 97%, 98%, or 99%. Sequence similarity and / or identity can be determined using the "BestFit" program in the Genetics Computer Group Version 10 software package from the University of Wisconsin. This program uses Smith and Waterman's local HAD algorithm with the following default values: gap generation penalty = 8, gap extension penalty = 2, average match = 2.912, and average mismatch = 2.003.

[0066] As used interchangeably herein, the terms "polynucleotide" or "nucleic acid" refer to a nucleotide chain of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerases. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs.

[0067] "Sequence homology" refers to the percentage of identical or conserved amino acid substitutions.

[0068] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which they have been introduced. Some vectors are capable of guiding the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0069] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells into which at least one exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” “transfectants” and “transfected cells,” and “transduced cells,” which include primary transformed / transfected / transduced cells and their derived progeny, regardless of passage number. The nucleic acid composition of the progeny may not be identical to that of the parent cells and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0070] As used in this article, the phrase “therapeutic effective amount” means the amount of therapeutic or preventive antibody that, when administered according to the required treatment regimen, is suitable to elicit the desired therapeutic or preventive effect or response (including relief of some or all of these symptoms of the disease or reduction of susceptibility to the disease).

[0071] The term "pharmaceutical formulation" / "pharmaceutical composition" refers to a formulation in a form that allows the bioactivity of the active ingredient contained therein to be effective, and that does not contain any additives that would have unacceptable toxicity to a subject who would administer the formulation.

[0072] A "radioactive nuclide" (also known as a "radioactive isotope" or "radioactive isotope") is an atom that undergoes radioactive decay. In a non-limiting sense, a radioactive nuclide can be, for example, a radioactive nuclide that emits beta particles (beta emitters), a radioactive nuclide that emits alpha particles (alpha emitters), or a radioactive nuclide that emits Auger electrons (Auger electron emitters).

[0073] "Radioactive nuclide that emits beta particles" or "beta emitter" is a radioactive nuclide that emits beta particles. Examples of beta emitters include, but are not limited to, copper-67 (…). 67 Cu), Strontium-89 ( 89 Sr), Yttrium-90 ( 90 Y), Rhodium-105 ( 105 Rh), Iodine-131 ( 131 I), promethium-149 ( 149 Pm), Holmium-166 ( 166 Ho), Lutetium-177 ( 177 Lu), Rhenium-186 ( 186 Re), Rhenium-188 ( 188 Re), Jin-198 ( 198 Au) and Gold-199 ( 199 Au. Jongho reviewed various techniques for chelating the individual isotopes of these radioisotopes (Jeon, Jongho. "Review of therapeutic applications of radiolabeled functional nanomaterials." International journal of molecular sciences 20.9 (2019):2323.).

[0074] A “radionium-emitting nuclide” or “Auger electron emitting radionuclide” is a radionuclide that emits Auger electrons. Examples of Auger electron emitting radionuclides include, but are not limited to, bromine-77, indium-111, iodine-123, and iodine-125.

[0075] A “radioactive nuclide that emits alpha particles” or “alpha radionuclide” is a radioactive nuclide that emits alpha particles, specifically a +2 charged 4He nucleus. Non-limiting examples of alpha radions include bismuth-213 (…). 213 Bi), characterized by a half-life of 45.6 minutes; Actinide-225 ( 225 Ac), characterized by a half-life of 9.9 days; Astatine-211 ( 211 At), characterized by a half-life of 7.2 hours; Radium-223 ( 223 Ra), characterized by a half-life of 11.4 days; Radium-224 ( 224 Ra), characterized by a half-life of 3.7 days; Thorium-227 ( 227 Th), characterized by a half-life of 18.7 days.

[0076] In the context of this invention, the term "Ac" refers to the ion of at least one actinium isotope that emits α. Preferably, the actinium isotope that emits α is... 225Ac. Preferably, the ion of at least one alpha-emitting actinium isotope is a triple positively charged ion. 225 Ac, that is 225 AC 3+ Therefore, in the context of this invention, the term "Ac" preferably, but not exclusively, refers to 225 AC 3+ .

[0077] Radionuclides can be obtained in ways known in the art. For example, as described by Poty, Sophie, et al. 211 At can be produced by a cyclotron, that is, by bombarding natural bismuth with a beam of medium-energy alpha particles. 209 Bi(α,2n)211At reaction ("Poty, Sophie et al. α-Emitters for radiotherapy: from basic radiochemistry toclinical studies—part 1 / 2." Journal of Nuclear Medicine 59.6 / 59.7 (2018):878–884 / 1020-1027). 227 Th and 223 Ra can all originate from their common parent body. 227 Acetate was obtained through isolation. Clinical preparation. 223 Ra uses based on 227 AC / 227 Th generator. The parent isotope is loaded onto an actinide chromatography resin, eluted with 1M HCl or HNO3, followed by cation exchange column chromatography, evaporation, and dissolution in physiological saline solution to obtain... 223 Ra chloride solution.

[0078] "Chelation" refers to the formation or presence of two or more independent coordinate bonds between a ligand and a single central metal atom. Ligands capable of forming these coordinate bonds are called "chelating agents," "chelating reagents," or "sequestering agents."

[0079] "Chelating fraction" is a chelating agent capable of chelating a given radionuclide or a group of radionuclides (e.g., but not limited to alpha, beta, or Auger electron chemiluminescent materials). Various chelating agents are known in the art and can be used according to the present invention, such as Price, Eric W., and Chris Orvig, "Matching chelators to radiometals for radiopharmaceuticals." Chemical Society Reviews 43.1 (2014): 260-290, which is cited in full and incorporated herein by reference.

[0080] In the context of this invention, the chelating portion includes, for example, an 18-membered chelating portion, such as the macrocyclic chelator “macropa” (N,N′-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6). Macrocyclic chelators such as macropa have been described, for example, in Thiele et al., Angew. Chem. Int. Ed. 2017, 56(46), 14712 and references therein. Macropa chelating agents and their derivatives, such as "Macropa-NCS" (6-[[16-[(6-carboxy-2-pyridyl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl]methyl]-4-[2-(4-isothiocyanate-phenyl)ethoxy]pyridine-2-carboxylic acid), have also been described in WO2018 / 183906 and WO2020 / 106886 and their references.

[0081] The chelating moiety used for complexing actinium (or "chelating moiety of complexed actinium") may be terminated with an isothiocyanate or carboxylic acid moiety to facilitate its coupling with the target moiety (e.g., the anti-GPC3 antibody or its antigen-binding fragment according to the invention). When using macropa chelating agents, one of the substituents on the pyridine ring of the macropa chelating agent is typically substituted to allow it to couple with the target moiety. This can be a simple functional group (such as a carboxyl-COOH) or a more complex chemical structure, as long as it can couple with the target moiety (e.g., the anti-GPC3 antibody or its antigen-binding fragment according to the invention). For example, the substituent on the pyridine ring of the macropa chelating agent may be terminated with an isothiocyanate or carboxylic acid moiety.

[0082] As used herein, a “targeting portion” is any chemical structure that binds to a biological target, such as GPC3 or a cell expressing GPC3. The targeting portion positions itself, for example, as part of a larger structure, to a specific site where its presence is required to exert a desired effect (e.g., in the case of TAC, delivery of radioactive decay). Thus, the role of a tissue-targeting group or portion is to better localize a molecule or conjugate to at least one desired site in a subject's body compared to the concentration of an equivalent complex that does not contain a targeting portion. The targeting portion can be, for example, selected from, but not limited to, the group consisting of nucleotides, DNA and RNA fragments, aptamers, peptides, proteins, antibodies or fragments thereof, nanoparticles or small molecules, or any combination thereof. According to the invention, the targeting portion is preferably an antibody or an antigen-binding fragment thereof (i.e., the anti-GPC3 antibody or a fragment thereof of the present invention).

[0083] "Targeted actinium conjugate" (also known as "TAC") is a conjugate comprising a targeting portion (i.e., the anti-GPC3 antibody or a fragment thereof of the present invention) and at least one chelating agent or chelating group (e.g., macropa, preferably Macropa-NCS) for complexing actinium and optionally containing actinium.

[0084] The term "DOTA" refers to 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid or 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid and its derivatives, which can chelate radionuclides. DOTA is used to complex radionuclides (e.g., radioactive alpha particles) with radioactive nuclides. 212 Bi、 213 Bi、 225 Ac、 227 Chelating agents for Th. Chelating agents such as DOTA form stable complexes with chelated As-225 in the +3 oxidation state. To achieve adequate labeling of DOTA-conjugated antibodies, the complexation step should preferably be performed in two steps, or directly at elevated temperatures.

[0085] In the context of this invention, the term CAR refers to the ratio of chelating agent to antigen, which is a measure of the specific activity of a radiolabeled compound (e.g., the targeted actinium conjugate of this invention).

[0086] In the context of this invention, the term IRF refers to the immunoreactive fraction, which is the fraction of labeled products (i.e., the targeted actinium conjugate of this invention) capable of binding to the target (i.e., GPC3). The Lindmo assay (Lindmo T et al., (1984) "Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess." J. Immunol. Methods. 72, 77–89) is the most commonly used method for determining the immunoreactive fraction.

[0087] The anti-GPC3 antibody according to the present invention According to a first aspect, the present invention relates to isolated antibodies or antigen-binding fragments thereof capable of binding to GPC3.

[0088] The antibody may be, for example, an IgG antibody, such as human IgG1, IgG2, IgG3, or IgG4, or mouse IgG1, IgG2a, IgG2b, or IgG2c. In some highly preferred embodiments, the antibody isolated according to this aspect or its antigen-binding fragment is a humanized IgG1 antibody. In some further preferred embodiments, the antibody according to this aspect or its antigen-binding fragment is a Fab', F(ab')2, Fab, Fv, rlgG, or scFv fragment.

[0089] In some preferred embodiments, the antibody or antigen-binding fragment thereof according to this aspect binds to human GPC3. In some such preferred embodiments, the antibody or antigen-binding fragment thereof according to this aspect cross-reacts with GPC3 from other species, such as monkey, long-tailed macaque (Macaca fascicularis), common macaque (Macacamulatta), rodent, mouse, rat, horse, cattle, pig, dog, cat and / or camel GPC3, preferably cross-reacting with cynomolgus monkey GPC3.

[0090] According to some preferred embodiments, the antibody or its antigen-binding fragment has a binding affinity K for human GPC3 of SEQ ID NO:321. D Below 2E-07 M, preferably below 1E-08 M, 1E-09 M, 10E-10 M, or 1E-10 M. According to some embodiments that may be the same or different, the binding affinity K of the antibody or antigen-binding fragment to the cynomolgus monkey GPC3 of SEQ ID 323 is... DBelow 2E-07 M, preferably below 1E-08 M, 1E-09 M, 10E-10 M or 1E-10 M.

[0091] Preferably, the antibody or its antigen-binding fragment is in the same order of magnitude K D Combining human GPC3 and cynomolgus monkey GPC3, for example, with K values ​​below 2E-07 M, preferably below 1E-08 M, 1E-09 M, 10E-10 M or 1E-10 M. D Combine.

[0092] In a further preferred embodiment, the antibody or its antigen-binding fragment binds to human GPC3 of SEQ ID 321 at an EC50 of less than 1E-07 M, preferably less than 10E-08 M, 1E-08 M, 10E-9 M, or 1E-09 M. According to some embodiments that may be the same or different, the antibody or antigen-binding fragment has an EC50 of less than 2E-07 M, preferably less than 10E-08 M, 1E-08 M, 10E-9 M, or 1E-09 M for cynomolgus monkey GPC3 of SEQ ID 323.

[0093] Preferably, the antibody or antigen-binding fragment binds to human GPC3 and cynomolgus monkey GPC3 at the same order of magnitude of EC50, for example, at an EC50 of less than 2E-07 M, preferably less than 10E-08 M, 1E-08 M, 10E-9 M or 1E-09 M.

[0094] In some preferred embodiments, the number of germline deviations between the antibody or its antigen-binding fragment according to this aspect and the closest germline light chain IGKV2-28-IGKJ2 (SEQ ID NO:325) and the closest germline heavy chain IGHV1-46-IGHJ4 (SEQ-ID NO: 324) other than CDR-H3 is low, which is less than 32, preferably less than 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 5, 4, 3, 2 or 1.

[0095] In a further preferred embodiment, the immunogenicity risk of the antibody or its antigen-binding fragment according to this aspect is low, as evidenced by an IPAD score (as predicted by the v4.03 algorithm described in Example 6) of less than 1000, preferably less than 900, 800, 700, 600 or 550.

[0096] In a further preferred embodiment, the antibody or antigen-binding fragment of this aspect contains a low number of lysine residues in its CDR. Preferably, the number of lysine residues in the six CDRs of the antibody or antigen-binding fragment is less than 4, more preferably less than 3, even more preferably less than 2, and most preferably less than 1. According to a highly preferred embodiment, the antibody or antigen-binding fragment of this aspect does not contain lysine residues in its six CDRs.

[0097] The isolated antibody or antigen-binding fragments according to the present invention can exhibit any combination of the above-described characteristics.

[0098] In some preferred embodiments, the isolated antibody or antigen-binding fragment according to the present invention comprises: i) Containing a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 82, H-CDR2 of SEQ ID NO: 83, and H-CDR3 of SEQ ID NO: 84, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 86, L-CDR2 of SEQ ID NO: 87, and L-CDR3 of SEQ ID NO: 88 (TPP-29312); or ii) Containing a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 102, H-CDR2 of SEQ ID NO: 103, and H-CDR3 of SEQ ID NO: 104, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 106, L-CDR2 of SEQ ID NO: 107, and L-CDR3 of SEQ ID NO: 108 (TPP-29537); or iii) Containing a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 122, H-CDR2 of SEQ ID NO: 123, and H-CDR3 of SEQ ID NO: 124, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 126, L-CDR2 of SEQ ID NO: 127, and L-CDR3 of SEQ ID NO: 128 (TPP-29538); or iv) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 142), H-CDR2 (SEQ ID NO: 143), and H-CDR3 (SEQ ID NO: 144), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 146), L-CDR2 (SEQ ID NO: 147), and L-CDR3 (SEQ ID NO: 148) (TPP-29636); or v) Contains a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 162, H-CDR2 of SEQ ID NO: 163, and H-CDR3 of SEQ ID NO: 164, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 166, L-CDR2 of SEQ ID NO: 167, and L-CDR3 of SEQ ID NO: 168 (TPP-29638); or vi) Contains a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 182, H-CDR2 of SEQ ID NO: 183, and H-CDR3 of SEQ ID NO: 184, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 186, L-CDR2 of SEQ ID NO: 187, and L-CDR3 of SEQ ID NO: 188 (TPP-29642).

[0099] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises: i) Containing a heavy chain antigen-binding region comprising H-CDR1 of SEQ ID NO: 102, H-CDR2 of SEQ ID NO: 103, and H-CDR3 of SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 of SEQ ID NO: 106, L-CDR2 of SEQ ID NO: 107, and L-CDR3 of SEQ ID NO: 108; or ii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 122), H-CDR2 (SEQ ID NO: 123), and H-CDR3 (SEQ ID NO: 124), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 126), L-CDR2 (SEQ ID NO: 127), and L-CDR3 (SEQ ID NO: 128); or iii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 142), H-CDR2 (SEQ ID NO: 143), and H-CDR3 (SEQ ID NO: 144), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 146), L-CDR2 (SEQ ID NO: 147), and L-CDR3 (SEQ ID NO: 148); or iv) A heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 162, H-CDR2 containing SEQ ID NO: 163, and H-CDR3 containing SEQ ID NO: 164, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 166, L-CDR2 containing SEQ ID NO: 167, and L-CDR3 containing SEQ ID NO: 168.

[0100] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 102, H-CDR2 of SEQ ID NO: 103, and H-CDR3 of SEQ ID NO: 104, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 106, L-CDR2 of SEQ ID NO: 107, and L-CDR3 of SEQ ID NO: 108 (TPP-29537).

[0101] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 122, H-CDR2 containing SEQ ID NO: 123, and H-CDR3 containing SEQ ID NO: 124, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 126, L-CDR2 containing SEQ ID NO: 127, and L-CDR3 containing SEQ ID NO: 128 (TPP-29538).

[0102] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 142, H-CDR2 containing SEQ ID NO: 143, and H-CDR3 containing SEQ ID NO: 144, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 146, L-CDR2 containing SEQ ID NO: 147, and L-CDR3 containing SEQ ID NO: 148 (TPP-29636).

[0103] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 162, H-CDR2 of SEQ ID NO: 163, and H-CDR3 of SEQ ID NO: 164, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 166, L-CDR2 of SEQ ID NO: 167, and L-CDR3 of SEQ ID NO: 168 (TPP-29638).

[0104] In a further preferred embodiment, the isolated antibody or antigen-binding fragment according to the present invention comprises: i) a variable heavy chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 81 and / or a variable light chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 85; or ii) a variable heavy chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 101 and / or a variable light chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105; or iii) a variable heavy chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 121 and / or a variable light chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 125; or iv) A variable heavy chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 141 and / or a variable light chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 145; or v) A variable heavy chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 161 and / or a variable light chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 165; or vi) A variable heavy chain domain having at least 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 181 and / or a variable light chain domain having at least 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 185.

[0105] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises: i) a variable heavy chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 101 and / or a variable light chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105; or ii) a variable heavy chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 121 and / or a variable light chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 125; or iii) a variable heavy chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 141 and / or a variable light chain domain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 145; or iv) A variable heavy chain domain having at least 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 161 and / or a variable light chain domain having at least 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 165.

[0106] In a further preferred embodiment, the isolated antibody or antigen-binding fragment according to the present invention comprises: i) Containing a variable heavy chain domain of SEQ ID NO: 81 and a variable light chain domain of SEQ ID NO: 85; or ii) Containing a variable heavy chain domain of SEQ ID NO: 101 and a variable light chain domain of SEQ ID NO: 105; or iii) Containing a variable heavy chain domain of SEQ ID NO: 121 and a variable light chain domain of SEQ ID NO: 125; or iv) Containing a variable heavy chain domain of SEQ ID NO: 141 and a variable light chain domain of SEQ ID NO: 145; or v) Containing a variable heavy chain domain of SEQ ID NO: 161 and a variable light chain domain of SEQ ID NO: 165; or vi) Contains a variable heavy chain domain of SEQ ID NO: 181 and a variable light chain domain of SEQ ID NO: 185.

[0107] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises: i) Containing a variable heavy chain domain of SEQ ID NO: 101 and a variable light chain domain of SEQ ID NO: 105; or ii) Containing a variable heavy chain domain of SEQ ID NO: 121 and a variable light chain domain of SEQ ID NO: 125; or iii) Containing a variable heavy chain domain of SEQ ID NO: 141 and a variable light chain domain of SEQ ID NO: 145; or iv) Containing a variable heavy chain domain of SEQ ID NO: 161 and a variable light chain domain of SEQ ID NO: 165; or In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises a variable heavy chain domain containing SEQ ID NO: 101 and a variable light chain domain containing SEQ ID NO: 105.

[0108] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises a variable heavy chain domain containing SEQ ID NO: 121 and a variable light chain domain containing SEQ ID NO: 125.

[0109] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises a variable heavy chain domain containing SEQ ID NO: 141 and a variable light chain domain containing SEQ ID NO: 145.

[0110] In some highly preferred embodiments, the isolated antibody or antigen-binding fragment according to the invention comprises a variable heavy chain domain containing SEQ ID NO: 161 and a variable light chain domain containing SEQ ID NO: 165.

[0111] In a particular embodiment, the antibody isolated according to the present invention is an IgG1, IgG2, IgG3, or IgG4 antibody. Most particularly, the antibody isolated according to the present invention is an IgG1 antibody.

[0112] In a further preferred embodiment, the isolated antibody or antigen-binding fragment according to the present invention comprises: i) a heavy chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 97 and / or a light chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 98; or ii) a heavy chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 117 and / or a light chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 118; or iii) a heavy chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 137 and / or a light chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 138; or iv) a heavy chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 157 and / or a light chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 158; or v) a heavy chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 177 and / or a light chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 178; or vi) Heavy chains having at least 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 197 and / or light chains having at least 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 198.

[0113] In some highly preferred embodiments, the isolated antibody according to the invention comprises: i) a heavy chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 117 and / or a light chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 118; or ii) a heavy chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 137 and / or a light chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 138; or iii) a heavy chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 157 and / or a light chain having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 158; or iv) Heavy chains having at least 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 177 and / or light chains having at least 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 178.

[0114] In some preferred embodiments, the isolated antibody according to the present invention comprises: i) A heavy chain containing SEQ ID NO: 97 and a light chain containing SEQ ID NO: 98; or ii) a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118; or iii) A heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138; or iv) a heavy chain containing SEQ ID NO: 157 and a light chain containing SEQ ID NO: 158; or v) A heavy chain containing SEQ ID NO: 177 and a light chain containing SEQ ID NO: 178; or vi) The heavy chain containing SEQ ID NO: 197 and the light chain containing SEQ ID NO: 198.

[0115] In some highly preferred embodiments, the isolated antibody according to the invention comprises: i) a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118; or ii) a heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138; or iii) A heavy chain containing SEQ ID NO: 157 and a light chain containing SEQ ID NO: 158; or iv) The heavy chain containing SEQ ID NO: 177 and the light chain containing SEQ ID NO: 178; or In some highly preferred embodiments, the antibody isolated according to the invention comprises a heavy chain containing SEQ ID NO: 117 and a light chain (TPP-29537) containing SEQ ID NO: 118.

[0116] In some highly preferred embodiments, the antibody isolated according to the invention comprises a heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138 (TPP-29538).

[0117] In some highly preferred embodiments, the antibody isolated according to the invention comprises a heavy chain containing SEQ ID NO:157 and a light chain containing SEQ ID NO:158 (TPP-29636).

[0118] In some highly preferred embodiments, the antibody isolated according to the invention comprises a heavy chain containing SEQ ID NO:177 and a light chain (TPP-29638) containing SEQ ID NO:178.

[0119] In a particular embodiment, the antigen-binding fragment according to the invention is scFv, Fab, Fab' fragment or F(ab')2 fragment.

[0120] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is a monoclonal antibody or antigen-binding fragment.

[0121] In particular embodiments, the isolated antibody or antigen-binding fragment according to the invention is a human, humanized, or chimeric antibody or antigen-binding fragment, more particularly a humanized antibody or antigen-binding fragment.

[0122] In a particular embodiment, the isolated antibody or antigen-binding fragment according to the invention is a monospecific antibody. In other particular embodiments, the isolated antibody or antigen-binding fragment according to the invention is a multispecific antibody that binds to GPC3 and at least one other antigen, such as a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0123] The preferred amino acid sequences of the antibodies according to the present invention are listed in Table 1.

[0124] Table 1: Amino acid sequences of preferred antibodies according to the present invention The preferred nucleic acid sequences of the antibodies of this invention are listed in Table 2.

[0125] Table 2: Nucleic acid sequences of preferred antibodies according to the present invention peptide variants The antibody or antigen-binding fragments of this invention are not limited to the specific peptide sequences provided herein. Rather, this invention also includes variants of these peptides. Based on this disclosure and conventionally available techniques and references, those skilled in the art can prepare, test, and utilize functional variants of the antibodies disclosed herein, while understanding that these variants having the ability to bind GPC3 fall within the scope of this invention.

[0126] Variants may include, for example, antibodies having at least one altered complementarity-determining region (CDR) (hypervariate region) and / or frame (FR) (variable region) domain / position compared to the peptide sequence disclosed herein.

[0127] By altering one or more amino acid residues in the CDR or FR regions, technicians can routinely generate mutated or diversified antibody sequences, which can be screened against antigens to, for example, obtain new or improved properties.

[0128] A further preferred embodiment of the invention involves selecting antibody or antigen-binding fragments with VH and VL sequences as shown in Table 1. Those skilled in the art can use the data in Table 1 to design peptide variants within the scope of the invention. Variants are preferably constructed by altering amino acids within one or more CDR regions; variants may also have one or more altered frame regions. Alterations can also be made within the frame regions. For example, the FR domain of the peptide can be altered where there is a deviation in residues compared to the germline sequence.

[0129] Alternatively, a technician may perform the same analysis by comparing the amino acid sequences disclosed herein with known sequences of similar antibodies of the same class, using, for example, the method described in Knappik A. et al., JMB 2000, 296:57-86.

[0130] Furthermore, variants can be obtained by using an antibody as a starting point for further optimization, by diversifying one or more amino acid residues in the antibody (preferably one or more amino acid residues in the CDR), and by screening the resulting set of antibody variants to obtain variants with improved performance. Diversification of one or more amino acid residues in the CDR3 of VL and / or VH is particularly preferred. Diversification can be carried out, for example, by synthesizing a set of DNA molecules using trinucleotide mutagenesis (TRIM) technology (Virnekäs B. et al., Nucl. Acids Res. 1994, 22: 5600). Antibodies or their antigen-binding fragments comprise molecules with modifications / changes, including but not limited to modifications that result in alterations such as half-life (e.g., modification of the Fc portion or attachment of other molecules such as PEG), alterations in binding affinity, or alterations in ADCC or CDC activity.

[0131] Conserved amino acid variants Peptide variants that retain the overall molecular structure of the antibody peptide sequence described herein can be prepared. Considering the characteristics of individual amino acids, those skilled in the art will recognize some reasonable substitutions. Amino acid substitutions, or "conservative substitutions," can be made, for example, based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues involved.

[0132] For example, (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitution can generally occur within groups (a)–(d). Furthermore, based on their ability to disrupt the α-helix, glycine and proline can substitute for each other. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine, and lysine, are more common in the α-helix, while valine, isoleucine, phenylalanine, tyrosine, tryptophan, and threonine are more common in the β-sheet. Glycine, serine, aspartic acid, asparagine, and proline are common in the turn. Some preferred substitutions can be made in the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L, and I. Given the known genetic code and recombination and synthetic DNA techniques, skilled scientists can readily construct DNA encoding conserved amino acid variants.

[0133] Glycosylation variants When an antibody contains an Fc region, the carbohydrates attached to it can be altered. Naturally occurring antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are usually attached to Asn297 (using Kabat EU number) of the CH2 domain of the Fc region via N-linkage; see, for example, Wright et al. Trends Biotechnol. 15: 26-32 (1997).

[0134] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibodies are glycosylated. Adding or deleting glycosylation sites in the antibody can be conveniently achieved by altering the expression system (e.g., host cell) and / or by changing the amino acid sequence to generate or remove one or more glycosylation sites.

[0135] In one embodiment of the invention, a glycosylated antibody or antibody derivative with reduced effector function is prepared by expression in a prokaryotic host. Suitable prokaryotic hosts include, but are not limited to, various species of *Escherichia coli*, *Bacillus subtilis*, *Salmonella typhimurium*, and *Pseudomonas*, *Streptomyces*, and *Staphylococcus*.

[0136] In one embodiment, an antibody variant with reduced effector function is provided, characterized by modification at a conserved N-linker site in the CH2 domain of the Fc portion of the antibody. In one embodiment of the invention, the modification comprises a mutation at a heavy chain glycosylation site to prevent glycosylation at that site. Thus, in a preferred embodiment of the invention, a glycosylated antibody or antibody derivative is prepared by mutation at the heavy chain glycosylation site (i.e., using the mutation with Kabat EU number N297) and expressed in a suitable host cell.

[0137] In another embodiment of the invention, the glycosylated antibody or antibody derivative has reduced effector function, wherein the modification at the conserved N-linking site in the CH2 domain of the Fc portion of the antibody or antibody derivative comprises the removal of the CH2 domain glycan, i.e., deglycosylation. These glycosylated antibodies can be generated by conventional methods and then enzymatically deglycosylated. Methods for enzymatic deglycosylation of antibodies are well known in the art (e.g., Winkelhake and Nicolson (1976), J Biol Chem. 251(4):1074-80).

[0138] In another embodiment of the invention, deglycosylation can be achieved using the glycosylation inhibitor tunicamycin (Nose and Wigzell (1983), Proc Natl Acad Sci USA, 80(21):6632-6). That is, the modification is to prevent glycosylation at the conserved N-linkage site in the CH2 domain of the Fc portion of the antibody.

[0139] In one embodiment, an antibody variant is provided having a sugar structure lacking (directly or indirectly) fucose attached to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU number of Fc region residues) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants can possess improved ADCC function.

[0140] Examples of published literature on “defucosylated” or “fucosylated” antibody variants include: Okazaki et al., J Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).

[0141] Examples of cell lines capable of producing defucosylated antibodies include Lec13CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); and WO 2004 / 056312) and knockout cell lines, such as CHO cells with the α-1,6-fucosyltransferase gene FUT8 knocked out (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)).

[0142] Antibody variants with bimeric oligosaccharides are also provided, for example, wherein a biantennary oligosaccharide attached to the Fc region of the antibody is bimeric by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; US Patent No. 6,602,684; and US 2005 / 0123546.

[0143] Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have enhanced CDC function. Such antibody variants are described, for example, in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764.

[0144] FC region variant In some implementations, one or more amino acid modifications (e.g., substitutions) may be introduced into the Fc region of the antibody provided herein (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) to generate an Fc region variant.

[0145] In some embodiments, the invention envisions an antibody variant possessing some, but not all, effector functions, making it a desirable candidate for applications where in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding capacity. In some embodiments, alterations are made in the Fc region that result in altered (i.e., increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC).

[0146] In some embodiments, the present invention envisions antibody variants having increased or decreased half-lives. Antibodies having increased half-lives and improved binding to the neonatal Fc receptor (FcRn) (which is responsible for transferring maternal IgG to the fetus (Guyer et al., J Immunol. 117:587 (1976) and Kim et al., J Immunol. 24:249 (1994))) are described in US2005 / 0014934 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions that improve the binding of the Fc region to the FcRn.

[0147] DNA molecule of the present invention This invention also relates to isolated nucleic acid sequences encoding antibodies or antigen-binding fragments thereof of the present invention. Isolated nucleic acid sequences encoding antibodies or antigen-binding fragments thereof of the present invention can be generated, for example, by techniques or chemical synthesis (e.g., techniques described in Oligonucleotide Synthesis (1984, Gait, ed., IRLPress, Oxford)) as described in Sambrook et al., 1989 and Ausubel et al., 1989. DNA sequences for expression of antibodies are listed in Table 2. These sequences are optimized for mammalian expression in certain cases. The DNA molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the present invention can be described by reference to their physical properties in hybridization. Those skilled in the art will recognize that nucleic acid hybridization techniques can be used to identify complementary sequences and their equivalents or homologs of DNA (because DNA is double-stranded). It should also be recognized that hybridization can be performed with less than 100% complementarity. However, if appropriate conditions are chosen, hybridization techniques can be used to distinguish between DNA sequences based on the structural relevance of the DNA sequence to a specific probe. For guidance on such conditions, see Sambrook et al., 1989 (see above) and Ausubel et al., 1995 (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, and Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

[0148] The structural similarity between two polynucleotide sequences can be expressed as a function of the “strictness” of the conditions under which the two sequences hybridize. As used herein, the term “strictness” refers to the degree to which conditions are unfavorable to hybridization. Strict conditions strongly discourage hybridization, and under such conditions only molecules that are structurally most related can hybridize. Conversely, non-strict conditions favor hybridization of molecules that exhibit a lower degree of structural relevance. Therefore, hybridization strictness is directly related to the structural relationship between two nucleic acid sequences.

[0149] Hybridization strictness varies with many factors, including total DNA concentration, ionic strength, temperature, probe size, and the presence of reagents that disrupt hydrogen bonds. Factors that promote hybridization include high DNA concentration, high ionic strength, low temperature, long probe size, and the absence of reagents that disrupt hydrogen bonds. Hybridization typically occurs in two phases: a "binding" phase and a "washing" phase.

[0150] Functionally equivalent DNA variants Another class of DNA variants within the scope of this invention can be described with reference to the products they encode. These functionally equivalent polynucleotides are characterized in that, due to the degeneracy of the genetic code, they encode the same peptide sequence.

[0151] It should be recognized that variants of the DNA molecules presented herein can be constructed in several different ways. For example, they can be constructed as fully synthetic DNA. Methods for the efficient synthesis of oligonucleotides are widely available. See Ausubel et al., Section 2.11, Supplement 21 (1993). Overlapping oligonucleotides can be synthesized and assembled in a manner first reported by Khorana et al., J. Mol. Biol. 72:209217 (1971); also see Ausubel et al., see above, Section 8.2. Preferably, the synthesized DNA is designed with readily engineered restriction sites at the 5' and 3' ends of the gene to facilitate cloning into appropriate vectors.

[0152] As shown, methods for generating variants begin with one of the DNAs disclosed herein, followed by site-directed mutagenesis. See Ausubel et al., above, Chapter 8, Supplement 37 (1997). In a typical approach, the target DNA is cloned into a single-stranded DNA phage vector. The single-stranded DNA is isolated and hybridized with an oligonucleotide containing one or more desired nucleotide variations. A complementary strand is synthesized, and the double-stranded phage is introduced into a host. Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing. Furthermore, various methods are available to increase the probability of progeny phages containing the desired mutant. These methods are well known to those skilled in the art, and kits for generating such mutants are commercially available.

[0153] Recombinant DNA constructs and expression The present invention further provides recombinant DNA constructs comprising one or more of the nucleotide sequences of the present invention. The recombinant constructs of the present invention can be used in conjunction with vectors (such as plasmids, phage particles, bacteriophages, or viral vectors) to insert DNA molecules encoding antibodies of the present invention or their antigen-binding fragments or variants thereof into said vectors.

[0154] Therefore, on the one hand, the present invention relates to vectors containing the nucleic acid sequences of the present invention.

[0155] The antibodies, antigen-binding moieties, or variants thereof provided herein can be prepared by recombinantly expressing nucleic acid sequences encoding light and heavy chains or portions thereof in host cells. To recombinantly express antibodies, antigen-binding moieties, or variants thereof, host cells can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding light and / or heavy chains or portions thereof, thereby expressing the light and heavy chains in the host cells. Nucleic acids encoding heavy and light chains are prepared and / or obtained using standard recombinant DNA methods, these nucleic acids are inserted into a recombinant expression vector, and the vector is introduced into a host cell, such as those described in the following literature: Sambrook, Fritsch, and Maniatis (eds.), *Molecular Cloning: A Laboratory Manual*, Second Edition, Cold Spring Harbor, NY, (1989); Ausubel, FM et al. (eds.) *Current Protocols in Molecular Biology*, Greene Publishing Associates, (1989); and Boss et al., US Pat. No. 4,816,397.

[0156] Furthermore, nucleic acid sequences encoding variable regions of the heavy and / or light chains can be converted into nucleic acid sequences encoding, for example, full-length antibody chains, Fab fragments, or scFv. DNA fragments encoding VL or VH can be operatively linked to another DNA fragment encoding, for example, antibody constant regions or flexible linkers (such that the amino acid sequences encoded by the two DNA fragments are within the same reading frame). Sequences of the human heavy and light chain constant regions are known in the art (see, for example, Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification.

[0157] To generate a polynucleotide sequence encoding scFv, the nucleic acids encoding VH and VL can be operatively linked to another segment encoding a flexible linker, so that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VL and VH regions are linked by a flexible linker (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

[0158] To express antibodies, their antigen-binding fragments, or variants thereof, standard recombinant DNA expression methods can be used (see, for example, Goeddel, Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For instance, DNA encoding the desired polypeptide can be inserted into an expression vector and then transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples of prokaryotic host cells include bacteria, and examples of eukaryotic host cells include yeast, insects and insect cells, plants and plant cells, transgenic animal or mammalian cells. Recombinant constructs can be introduced into host cells using standard techniques such as calcium phosphate transfection, DEAE-mediated transfection, electroporation, transduction, or phage infection.

[0159] In some implementations, the DNA encoding the heavy and light chains is inserted into different vectors. In other implementations, the DNA encoding the heavy and light chains is inserted into the same vector. It should be understood that the design of the expression vector (including the selection of the regulatory sequence) is influenced by factors such as the choice of host cell, the desired protein expression level, and whether the expression is constitutive or inducible.

[0160] Therefore, on the other hand, the present invention relates to isolated cells expressing antibodies or antigen-binding fragments of the present invention and / or containing nucleic acids of the present invention or vectors of the present invention.

[0161] The isolated cells can be almost any cell with an available expression vector. The isolated cells can be, for example, higher eukaryotic host cells such as mammalian cells, lower eukaryotic host cells such as yeast cells, and prokaryotic cells such as bacterial cells.

[0162] On the other hand, the present invention relates to a method for producing the isolated antibody or antigen-binding fragment of the present invention, comprising culturing the cells of the present invention. In a particular embodiment, the cells of the present invention are cultured under conditions suitable for antibody expression, and the antibody or antigen-binding fragment is recovered. In a particular embodiment, the antibody or antigen-binding fragment is purified, particularly to a homogeneity of at least 95% by weight.

[0163] Bacterial expression Efficient bacterial expression vectors are constructed by inserting the DNA sequence encoding the desired protein, along with appropriate translation initiation and termination signals, into an operable reading phase within a functional promoter. The vector will contain one or more phenotypic selection markers and a replication origin to ensure the maintenance of the vector, and as such If necessary, amplification can be provided within the host. Suitable prokaryotic hosts for transformation include, but are not limited to, various species of *Escherichia coli*, *Bacillus subtilis*, *Salmonella typhimurium*, *Pseudomonas*, *Streptomyces*, and *Staphylococcus*.

[0164] Bacterial vectors can be, for example, based on bacteriophages, plasmids, or phage particles. These vectors may contain selection markers and bacterial origins of replication derived from commercially available plasmids, typically containing elements of the well-known cloning vector pBR322 (ATCC 37017). After transformation of a suitable host strain and allowing the host strain to grow to an appropriate cell density, the repression / induction of the selected promoter is lifted by appropriate methods (e.g., temperature change or chemical induction), and the cells are cultured for an additional time. Cells are typically harvested by centrifugation, lysed by physical or chemical methods, and the resulting crude extract is retained for further purification.

[0165] In bacterial systems, a variety of expression vectors can be advantageously selected depending on the intended use of the expressed protein. For example, when producing large quantities of such proteins, such as for antibody production or for screening peptide libraries, a vector that directs the high-level expression of an easily purified fusion protein product may be required.

[0166] Therefore, one embodiment of the present invention is an expression vector comprising a nucleic acid sequence encoding the novel antibody of the present invention.

[0167] The antibodies or antigen-binding fragments or variants thereof of the present invention include naturally purified products, products of chemical synthesis methods, and products generated from a prokaryotic host by recombinant technology, said prokaryotic host including, for example, *Escherichia coli*. E. coli Bacillus subtilis ( Bacillus subtilis Salmonella typhimurium ( Salmonella typhimurium Various species of Pseudomonas, Streptomyces and Staphylococcus, with Escherichia coli cells being preferred.

[0168] mammalian expression Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level expression of proteins in mammalian cells, such as those derived from promoters and / or enhancers of: cytomegalovirus (CMV) (such as CMV promoters / enhancers), simian virus 40 (SV40) (such as SV40 promoters / enhancers), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyomaviruses. Antibody expression can be constitutive or regulated (e.g., induced by the addition or removal of small molecule inducers, such as tetracycline bound to the Tet system). Further descriptions of viral regulatory elements and their sequences can be found, for example, in Stinski's US 5,168,062, Bell et al.'s US 4,510,245, and Schaffner et al.'s US 4,968,615. Recombinant expression vectors may also include origins of replication and selection markers (see, for example, US 4,399,216, 4,634,665, and US 5,179,017). Suitable selection markers include genes conferring resistance to drugs (such as G418, puromycin, hygromycin, blastomycin, Zeocin / bleomycin, or methotrexate) on host cells already introduced with the vector, or the use of auxotrophic selection markers such as glutamine synthase (Bebbington et al., Biotechnology (NY). 1992 Feb;10(2):169-75). For example, the dihydrofolate reductase (DHFR) gene confers methotrexate resistance, the neo gene confers G418 resistance, the bsd gene from Aspergillus terreus confers blastomycin resistance, puromycin N-acetyltransferase confers puromycin resistance, the Shble gene product confers Zeocin resistance, and hygromycin resistance is conferred by the Escherichia coli hygromycin resistance gene (hyg or hph). Selection markers such as DHFR or glutamine synthase can also be used in amplification techniques combined with MTX and MSX.

[0169] Transfection of expression vectors into host cells can be performed using standard techniques such as electroporation, nuclear transfection, calcium phosphate precipitation, liposome transfection, and polycation-based transfections such as polyethyleneimine (PEI)-based transfection and DEAE-dextran transfection.

[0170] Suitable mammalian host cells for expressing the antibodies, their antigen-binding fragments, or variants thereof provided herein include Chinese hamster ovary (CHO cells) such as CHO-K1, CHO-S, CHO-K1SV [including dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun;33(2):405-12, which are used with, for example, DHFR selection markers described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621; and Fan et al., Biotechnol Bioeng. 2012] Other knockout cells listed in Apr;109(4):1007-15] include NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells, and SP2 cells.

[0171] In expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7, or CAP-T cells, expression may also be transient or semi-stable (e.g., Durocher et al., Nucleic Acids Res. 2002 Jan 15;30(2):E9).

[0172] In some implementations, the expression vector is designed such that the expressed protein is secreted into the culture medium in which the host cells grow. The antibody, its antigen-binding fragment, or a variant thereof can be recovered from the culture medium using standard protein purification methods.

[0173] purification The antibodies or antigen-binding fragments or variants thereof of the present invention can be recovered and purified from recombinant cell cultures by well-known methods, including but not limited to ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography, protein G chromatography, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (“HPLC”) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each incorporated herein by reference in its entirety.

[0174] The antibodies of the present invention, or their antigen-binding fragments or variants thereof, include naturally purified products, products of chemical synthesis, and products generated from eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells, via recombinant technology. Depending on the host used in the recombinant production method, the antibodies of the present invention may be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, see above, Sections 17.37–17.42; Ausubel, see above, Chapters 10, 12, 13, 16, 18, and 20.

[0175] In a preferred embodiment, the antibody is purified (1) to a weight greater than 95%, and in a further preferred embodiment, to a weight greater than 99%, as determined, for example, by the Lowry method, UV-Vis spectroscopy, or by SDS capillary gel electrophoresis (e.g., on a Caliper LabChip® GXII, GX 90, or Bio-Rad bioanalyzer), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) to homogenization by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated naturally occurring antibodies include recombinant intracellular in situ antibodies, since at least one component of the antibody's native environment will be absent. However, typically, isolated antibodies can be prepared by at least one purification step.

[0176] Antibody conjugates In addition to naked antibodies or their antigen-binding fragments, various antibody- or antibody-based conjugates can be designed using the antibodies or antigen-binding fragments disclosed herein.

[0177] In one aspect, the present invention relates to antibody-drug conjugates targeting GPC3, comprising the isolated antibody or antigen-binding fragment of the present invention. These conjugates can be for diagnostic, therapeutic, research applications, and various other purposes. The antibody or its antigen-binding fragment can be conjugated, for example, to radionuclides, cytotoxic agents, organic compounds, protein toxins, immunomodulators (such as cytokines), fluorescent fractions, cells, other antibodies or their antigen-binding fragments.

[0178] In some other embodiments, the conjugate contains a cytotoxic agent, for example, to form an antibody-drug conjugate (ADC). For example, the cytotoxic agent may be selected from auristatin, maytansinoid, kinin-spindle protein (KSP) inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, or pyrrolobenzodiazepine derivatives.

[0179] The cytotoxic agents and / or cell growth inhibitors of ADCs can be any agent known to inhibit cell growth and / or replication and / or kill cells. Numerous agents with cytotoxic and / or cell growth inhibitory properties are known in the literature. Non-limiting examples of the aforementioned classes of cytotoxic agents and / or cell growth inhibitors include, but are not limited to, cell cycle regulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, alkylating agents, DNA cross-linking agents, intercalating agents, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA / DNA antimetabolites, and antimitotic agents.

[0180] The connector that links the cytotoxic agent and / or cell growth inhibitor to the target portion of the ADC can be inherently long, short, flexible, rigid, hydrophilic, or hydrophobic, or can comprise segments with different characteristics, such as flexible segments, rigid segments, etc. The connector can be chemically stable to the extracellular environment, for example, chemically stable in bloodstream, or can include unstable connections that release the cytotoxic agent and / or cell growth inhibitor into the extracellular environment. Various connectors for linking drugs to antigen-binding portions (e.g., antibodies) in the context of ADCs are known in the art.

[0181] The number of cytotoxic agents and / or cell growth inhibitors (drug-to-antibody ratio: DAR) attached to the target portion of the anti-GPC3 ADC can vary and is limited only by the number of available binding sites on the target portion and the number of reagents attached to a single adapter. Typically, one adapter attaches one cytotoxic agent and / or cell growth inhibitor to the target portion of the ADC. In embodiments of ADCs that include more than one cytotoxic agent and / or cell growth inhibitor, each reagent can be the same or different.

[0182] In some other embodiments, the conjugate is a chimeric antigen receptor conjugate engineered for GPC3 targeting. Recently, CAR T cells have gained attention due to their clinical success and accelerated FDA approval (see WO2020 / 102240, the full text of which is incorporated herein by reference). In the CAR T cell approach, T cells are engineered to express a CAR that is specific to an antigen present on tumor cells. These engineered T cells are then re-administered to the same patient. After injection, the CAR T cells recognize the targeting antigen on the target cells to induce target cell death. Thus, T cells expressing chimeric antigen receptors (CAR T cells) constitute a novel form of therapy for medical uses, such as cancer treatment. A chimeric antigen receptor (CAR) is a genetically engineered receptor designed to target a specific antigen, such as a tumor antigen. This targeting can lead to cytotoxicity against tumors, for example, enabling CAR T cells expressing CARs to target and kill tumors via specific tumor antigens.

[0183] According to the present invention, antibodies or antigen-binding fragments provided for GPC3 can be used to engineer CAR T cells to specifically recognize cells expressing GPC3.

[0184] Preferably, the CAR according to the invention comprises a co-stimulatory domain (e.g., CD137, CD28, or CD134) to achieve prolonged activation of T cells in vivo. The addition of the co-stimulatory domain enhances the in vivo proliferation and survival of CAR-containing T cells, and initial clinical data show that such constructs are promising therapeutic agents for treating diseases such as cancer. According to the invention, the CAR T cells can be used to treat any disease with localized or systemic abnormalities in the expression of GPC3.

[0185] In some other embodiments, the conjugate is or comprises a bispecific antibody or a multispecific antibody. In some preferred embodiments, the bispecific antibody comprises at least one Fc domain.

[0186] In some preferred embodiments, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment according to the invention, and the second binding portion of the bispecific antibody is the same or a different antibody or antigen-binding fragment according to the invention.

[0187] In some further embodiments, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment according to the invention, and the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment that binds to a cell surface protein (such as a cell type-specific antigen). In some of these embodiments, the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment that targets a checkpoint protein, such as an anti-PD1 antibody or an anti-PD-L1 antibody. Suitable checkpoint protein targeting antibodies include nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cimiplimab, dostarlimab, or ipilimumab. In other implementations of this type, the second binding portion of the bispecific antibody is a HER2-targeting antibody, such as trastuzumab, pertuzumab, and / or margetuximab.

[0188] Techniques for preparing the bispecific or multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and chemical conjugation of two different monoclonal antibodies (see Staerz et al. (1985) Nature 314 (6012): 628-31). The multispecific antibodies can also be prepared by crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)), by using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol, 148(5): 1547-1553 (1992)), by using bispecific antibody technology to prepare bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), by using single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol, 152:5368 (1994)), by preparing trispecific antibodies as described in Tutt et al., J. Immunol. 147: 60 (1991), and according to Labrijn AF et al., Proc. Natl Acad Sci USA 2013; 110:5145-50 Controlled Fab Arm Exchange (cFAE).

[0189] In some other embodiments, the conjugate includes a detectable portion. Examples of the detectable portion include various enzymes, cofactors, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-emitting metals, non-radioactive paramagnetic metal ions, and reactive portions.

[0190] Detectable substances can be directly or indirectly linked to or coupled to antibodies or fragments thereof using techniques known in the art, such as via connectors or other parts known in the art. Examples of enzyme labeling include luciferases (e.g., firefly luciferase and bacterial luciferase; US4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, acetylcholinesterase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and so on.

[0191] Examples of suitable cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; examples of luminescent substances include luminol; examples of bioluminescent substances include luciferase, luciferin, and amylopectin; examples of suitable radioactive substances include 125I, 131I, 111In, or 99mTc.

[0192] In some embodiments, the coupling material may contain a radionuclide, such as a beta particle, alpha particle, gamma particle, or Auger electron emitter. For example, the radionuclide may be selected from... 43 Sc、 44 Sc、 47 Sc、 89 Zr、 90 Y、 111 In、 149 Tb, 152 Tb, 155 Tb, 161 Tb, 166 Ho、 177 Lu、 186 Re、 188 Re、 212 Bi、 213 Bi、 225 Ac、 227 Th and 232 Th. Suitable β-radiators of the present invention are, for example, 67 Cu、 89 Sr、 89 Zr、 90 Y、 105 Rh、 131 I, 149 Pm, 166 Ho、 177 Lu、 186 Re、 188 Re、198 Au. In some preferred embodiments of the invention, the radionuclide is... 89 Zr. Suitable radionuclides that emit Auger electrons, for example, are... 67 Ga、 71 Ge 77 Br、 99 mTc, 103 Pd, 111 In、 123 I, 125 I, 140 Nd, 178 Ta、 193 Pt, 195 mPt, 197 Hg. Suitable alpha emitters of the present invention are, for example, 211 At、 212 Pb, 213 Bi、 223 Ra、 224 Ra、 225 Ac or 227 Th.

[0193] In some preferred embodiments, the radionuclide is a radionuclide that emits alpha particles, for example... 225 Ac.

[0194] In some particularly preferred embodiments, the radionuclide is 225 Ac.

[0195] In some preferred embodiments, the conjugate further comprises a chelating agent for immobilizing radionuclides, such as those described elsewhere herein.

[0196] Targeted radionuclide conjugates according to the present invention According to another aspect, the present invention relates to a targeted radionuclide conjugate comprising the anti-GPC3 antibody of the present invention or its antigen-binding fragment and a chelating portion for chelating a radionuclide.

[0197] In some embodiments of this aspect of the invention, the chelating portion is capable of complexing α-radioactive nuclides, β-radioactive nuclides, γ-radioactive nuclides, or Auger electron radiators.

[0198] In some preferred embodiments of this aspect of the invention, the chelating portion is capable of complexing with a radionuclide that emits alpha radiation. In some embodiments, the radionuclide that emits alpha radiation may be, for example, but not limited to, actinium (A). 225 Ac 3+ ),radium( 233 Ra 2+ ),bismuth( 213 Bi3+ , 212 Bi 3+ or 211 Bi 3+ ),lead( 212 Pb 2+ or 212 Pb 4+ ),thorium( 227 Th 4+ or 226 Th 4+ ),dysprosium( 152 Dy 3+ ) or astatine ( 211 At + or 217 At + In some highly preferred embodiments of this aspect of the invention, the radionuclide that is complexed with the chelate portion of the radioactive alpha is actinium (A). 225 Ac), preferably 225 Ac 3+ .

[0199] In some preferred embodiments of this aspect of the invention, the chelating portion capable of complexing actinium comprises an 18-membered chelating portion. In some further preferred embodiments of this aspect of the invention, the chelating portion comprises the macrocyclic macropa chelating agent N,N′-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6. In some highly preferred embodiments of the invention, the chelating portion comprises the chelating agent Macropa-NCS (6-[[16-[(6-carboxy-2-pyridyl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl]methyl]-4-[2-(4-isothiocyanate-phenyl)ethoxy]pyridine-2-carboxylic acid) (abbreviated as “Macropa” in the examples), for example as described in WO2020 / 106886.

[0200] In some preferred embodiments of the invention, the chelating moiety capable of complexing actinium is terminated by an isothiocyanate or carboxylic acid moiety to facilitate its conjugation to the antibody or antigen-binding fragment of the invention. When using a macropa chelating agent, one of the substituents on the pyridine ring of the macropa chelating agent is typically substituted to allow it to conjugate to the anti-GPC3 antibody or antigen-binding fragment of the invention. This can be a simple functional group (such as a carboxyl-COOH) or a more complex chemical structure, as long as it can conjugate to the anti-GPC3 antibody or antigen-binding fragment of the invention. In a particular embodiment of the invention, the substituent on the pyridine ring of the macropa chelating agent is preferably terminated by an isothiocyanate or carboxylic acid moiety.

[0201] This aspect of the invention relates to a targeted actinide conjugate (TAC) of formula (I). [Ab] refers to a monoclonal antibody or its antigen-binding fragment that can bind to GPC3.

[0202] In a further embodiment, the present invention relates to a targeted actinium conjugate (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding to GPC3, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0203] In a further embodiment, the present invention relates to a targeted actinium conjugate (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, selected from antibodies or antigen-binding fragments isolated according to the first aspect of the present invention, comprising: i) Contains a heavy chain antigen-binding region comprising H-CDR1 of SEQ ID NO: 82, H-CDR2 of SEQ ID NO: 83, and H-CDR3 of SEQ ID NO: 84, and a light chain antigen-binding region comprising L-CDR1 of SEQ ID NO: 86, L-CDR2 of SEQ ID NO: 87, and L-CDR3 of SEQ ID NO: 88; or ii) Contains a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 102, H-CDR2 containing SEQ ID NO: 103, and H-CDR3 containing SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 106, L-CDR2 containing SEQ ID NO: 107, and L-CDR3 containing SEQ ID NO: 108; or iii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 122), H-CDR2 (SEQ ID NO: 123), and H-CDR3 (SEQ ID NO: 124), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 126), L-CDR2 (SEQ ID NO: 127), and L-CDR3 (SEQ ID NO: 128); or iv) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 142), H-CDR2 (SEQ ID NO: 143), and H-CDR3 (SEQ ID NO: 144), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 146), L-CDR2 (SEQ ID NO: 147), and L-CDR3 (SEQ ID NO: 148); or v) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 162), H-CDR2 (SEQ ID NO: 163), and H-CDR3 (SEQ ID NO: 164), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 166), L-CDR2 (SEQ ID NO: 167), and L-CDR3 (SEQ ID NO: 168); or vi) Contains a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 182, H-CDR2 of SEQ ID NO: 183, and H-CDR3 of SEQ ID NO: 184, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 186, L-CDR2 of SEQ ID NO: 187, and L-CDR3 of SEQ ID NO: 188.

[0204] In a further embodiment, the present invention relates to a targeted actinium conjugate (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, selected from antibodies or antigen-binding fragments isolated according to the first aspect of the present invention, comprising: i) Contains a heavy chain antigen-binding region comprising H-CDR1 of SEQ ID NO: 82, H-CDR2 of SEQ ID NO: 83, and H-CDR3 of SEQ ID NO: 84, and a light chain antigen-binding region comprising L-CDR1 of SEQ ID NO: 86, L-CDR2 of SEQ ID NO: 87, and L-CDR3 of SEQ ID NO: 88; or ii) Contains a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 102, H-CDR2 containing SEQ ID NO: 103, and H-CDR3 containing SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 106, L-CDR2 containing SEQ ID NO: 107, and L-CDR3 containing SEQ ID NO: 108; or iii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 122), H-CDR2 (SEQ ID NO: 123), and H-CDR3 (SEQ ID NO: 124), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 126), L-CDR2 (SEQ ID NO: 127), and L-CDR3 (SEQ ID NO: 128); or iv) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 142), H-CDR2 (SEQ ID NO: 143), and H-CDR3 (SEQ ID NO: 144), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 146), L-CDR2 (SEQ ID NO: 147), and L-CDR3 (SEQ ID NO: 148); or v) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 162), H-CDR2 (SEQ ID NO: 163), and H-CDR3 (SEQ ID NO: 164), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 166), L-CDR2 (SEQ ID NO: 167), and L-CDR3 (SEQ ID NO: 168); or vi) Contains a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 182, H-CDR2 of SEQ ID NO: 183, and H-CDR3 of SEQ ID NO: 184, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 186, L-CDR2 of SEQ ID NO: 187, and L-CDR3 of SEQ ID NO: 188.

[0205] [Ab] is bound to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0206] In some preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) Containing a heavy chain antigen-binding region comprising H-CDR1 of SEQ ID NO: 102, H-CDR2 of SEQ ID NO: 103, and H-CDR3 of SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 of SEQ ID NO: 106, L-CDR2 of SEQ ID NO: 107, and L-CDR3 of SEQ ID NO: 108; or ii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 122), H-CDR2 (SEQ ID NO: 123), and H-CDR3 (SEQ ID NO: 124), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 126), L-CDR2 (SEQ ID NO: 127), and L-CDR3 (SEQ ID NO: 128); or iii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 142), H-CDR2 (SEQ ID NO: 143), and H-CDR3 (SEQ ID NO: 144), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 146), L-CDR2 (SEQ ID NO: 147), and L-CDR3 (SEQ ID NO: 148); or iv) A heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 162, H-CDR2 containing SEQ ID NO: 163, and H-CDR3 containing SEQ ID NO: 164, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 166, L-CDR2 containing SEQ ID NO: 167, and L-CDR3 containing SEQ ID NO: 168.

[0207] In some preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) Containing a heavy chain antigen-binding region comprising H-CDR1 of SEQ ID NO: 102, H-CDR2 of SEQ ID NO: 103, and H-CDR3 of SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 of SEQ ID NO: 106, L-CDR2 of SEQ ID NO: 107, and L-CDR3 of SEQ ID NO: 108; or ii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 122), H-CDR2 (SEQ ID NO: 123), and H-CDR3 (SEQ ID NO: 124), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 126), L-CDR2 (SEQ ID NO: 127), and L-CDR3 (SEQ ID NO: 128); or iii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 142), H-CDR2 (SEQ ID NO: 143), and H-CDR3 (SEQ ID NO: 144), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 146), L-CDR2 (SEQ ID NO: 147), and L-CDR3 (SEQ ID NO: 148); or iv) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 162), H-CDR2 (SEQ ID NO: 163), and H-CDR3 (SEQ ID NO: 164), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 166), L-CDR2 (SEQ ID NO: 167), and L-CDR3 (SEQ ID NO: 168). [Ab] is bound to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0208] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 102, H-CDR2 containing SEQ ID NO: 103, and H-CDR3 containing SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 106, L-CDR2 containing SEQ ID NO: 107, and L-CDR3 containing SEQ ID NO: 108.

[0209] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 122, H-CDR2 containing SEQ ID NO: 123, and H-CDR3 containing SEQ ID NO: 124, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 126, L-CDR2 containing SEQ ID NO: 127, and L-CDR3 containing SEQ ID NO: 128.

[0210] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 142, H-CDR2 containing SEQ ID NO: 143, and H-CDR3 containing SEQ ID NO: 144, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 146, L-CDR2 containing SEQ ID NO: 147, and L-CDR3 containing SEQ ID NO: 148.

[0211] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 162, H-CDR2 containing SEQ ID NO: 163, and H-CDR3 containing SEQ ID NO: 164, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 166, L-CDR2 containing SEQ ID NO: 167, and L-CDR3 containing SEQ ID NO: 168.

[0212] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 102, H-CDR2 containing SEQ ID NO: 103, and H-CDR3 containing SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 106, L-CDR2 containing SEQ ID NO: 107, and L-CDR3 containing SEQ ID NO: 108, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0213] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a heavy chain antigen-binding region comprising H-CDR1 of SEQ ID NO: 122, H-CDR2 of SEQ ID NO: 123, and H-CDR3 of SEQ ID NO: 124, and a light chain antigen-binding region comprising L-CDR1 of SEQ ID NO: 126, L-CDR2 of SEQ ID NO: 127, and L-CDR3 of SEQ ID NO: 128, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0214] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 142, H-CDR2 containing SEQ ID NO: 143, and H-CDR3 containing SEQ ID NO: 144, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 146, L-CDR2 containing SEQ ID NO: 147, and L-CDR3 containing SEQ ID NO: 148, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0215] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a heavy chain antigen-binding region comprising H-CDR1 containing SEQ ID NO: 162, H-CDR2 containing SEQ ID NO: 163, and H-CDR3 containing SEQ ID NO: 164, and a light chain antigen-binding region comprising L-CDR1 containing SEQ ID NO: 166, L-CDR2 containing SEQ ID NO: 167, and L-CDR3 containing SEQ ID NO: 168, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0216] In a further preferred embodiment, the present invention relates to a targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding to GPC3, selected from isolated antibodies or antigen-binding fragments, including: i) Containing a variable heavy chain domain of SEQ ID NO: 81 and a variable light chain domain of SEQ ID NO: 85; or ii) Containing a variable heavy chain domain of SEQ ID NO: 101 and a variable light chain domain of SEQ ID NO: 105; or iii) Containing a variable heavy chain domain of SEQ ID NO: 121 and a variable light chain domain of SEQ ID NO: 125; or iv) Containing a variable heavy chain domain of SEQ ID NO: 141 and a variable light chain domain of SEQ ID NO: 145; or v) Containing a variable heavy chain domain of SEQ ID NO: 161 and a variable light chain domain of SEQ ID NO: 165; or vi) Contains a variable heavy chain domain of SEQ ID NO: 181 and a variable light chain domain of SEQ ID NO: 185.

[0217] In a further preferred embodiment, the present invention relates to a targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) Containing a variable heavy chain domain of SEQ ID NO: 81 and a variable light chain domain of SEQ ID NO: 85; or ii) Containing a variable heavy chain domain of SEQ ID NO: 101 and a variable light chain domain of SEQ ID NO: 105; or iii) Containing a variable heavy chain domain of SEQ ID NO: 121 and a variable light chain domain of SEQ ID NO: 125; or iv) Containing a variable heavy chain domain of SEQ ID NO: 141 and a variable light chain domain of SEQ ID NO: 145; or v) Containing a variable heavy chain domain of SEQ ID NO: 161 and a variable light chain domain of SEQ ID NO: 165; or vi) Contains a variable heavy chain domain of SEQ ID NO: 181 and a variable light chain domain of SEQ ID NO: 185. [Ab] is bound to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0218] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) Containing a variable heavy chain domain of SEQ ID NO: 101 and a variable light chain domain of SEQ ID NO: 105; or ii) Containing a variable heavy chain domain of SEQ ID NO: 121 and a variable light chain domain of SEQ ID NO: 125; or iii) Containing a variable heavy chain domain of SEQ ID NO: 141 and a variable light chain domain of SEQ ID NO: 145; or iv) Contains a variable heavy chain domain of SEQ ID NO: 161 and a variable light chain domain of SEQ ID NO: 165.

[0219] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) Containing a variable heavy chain domain of SEQ ID NO: 101 and a variable light chain domain of SEQ ID NO: 105; or ii) Containing a variable heavy chain domain of SEQ ID NO: 121 and a variable light chain domain of SEQ ID NO: 125; or iii) Containing a variable heavy chain domain of SEQ ID NO: 141 and a variable light chain domain of SEQ ID NO: 145; or iv) Contains a variable heavy chain domain of SEQ ID NO: 161 and a variable light chain domain of SEQ ID NO: 165. [Ab] is bound to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0220] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a variable heavy chain domain containing SEQ ID NO: 101 and a variable light chain domain containing SEQ ID NO: 105.

[0221] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a variable heavy chain domain containing SEQ ID NO: 121 and a variable light chain domain containing SEQ ID NO: 125.

[0222] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a variable heavy chain domain containing SEQ ID NO: 141 and a variable light chain domain containing SEQ ID NO: 145.

[0223] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a variable heavy chain domain containing SEQ ID NO: 161 and a variable light chain domain containing SEQ ID NO: 165.

[0224] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a variable heavy chain domain containing SEQ ID NO: 101 and a variable light chain domain containing SEQ ID NO: 105, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0225] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a variable heavy chain domain containing SEQ ID NO: 121 and a variable light chain domain containing SEQ ID NO: 125, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0226] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a variable heavy chain domain containing SEQ ID NO: 141 and a variable light chain domain containing SEQ ID NO: 145, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0227] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a variable heavy chain domain containing SEQ ID NO: 161 and a variable light chain domain containing SEQ ID NO: 165, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0228] In a particular embodiment, the isolated monoclonal antibody contained in the targeted actinide conjugate according to the invention is an IgG1, IgG2, IgG3, or IgG4 antibody. Most particularly, the isolated monoclonal antibody according to the invention is an IgG1 antibody.

[0229] In a further preferred embodiment, the present invention relates to a targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) A heavy chain containing SEQ ID NO: 97 and a light chain containing SEQ ID NO: 98; or ii) a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118; or iii) A heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138; or iv) a heavy chain containing SEQ ID NO: 157 and a light chain containing SEQ ID NO: 158; or v) A heavy chain containing SEQ ID NO: 177 and a light chain containing SEQ ID NO: 178; or vi) The heavy chain containing SEQ ID NO: 197 and the light chain containing SEQ ID NO: 198.

[0230] In a further preferred embodiment, the present invention relates to a targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) A heavy chain containing SEQ ID NO: 97 and a light chain containing SEQ ID NO: 98; or ii) a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118; or iii) A heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138; or iv) a heavy chain containing SEQ ID NO: 157 and a light chain containing SEQ ID NO: 158; or v) A heavy chain containing SEQ ID NO: 177 and a light chain containing SEQ ID NO: 178; or vi) The heavy chain containing SEQ ID NO: 197 and the light chain containing SEQ ID NO: 198, [Ab] is bound to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0231] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118; or ii) a heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138; or iii) A heavy chain containing SEQ ID NO: 157 and a light chain containing SEQ ID NO: 158; or iv) The heavy chain containing SEQ ID NO: 177 and the light chain containing SEQ ID NO: 178.

[0232] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). Wherein [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, selected from isolated antibodies or antigen-binding fragments, comprising: i) a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118; or ii) a heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138; or iii) A heavy chain containing SEQ ID NO: 157 and a light chain containing SEQ ID NO: 158; or iv) Heavy chains containing SEQ ID NO: 177 and light chains containing SEQ ID NO: 178 [Ab] is bound to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or its antigen-binding fragment.

[0233] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding to GPC3, comprising: a heavy chain containing SEQ ID NO: 117 and a light chain containing SEQ ID NO: 118 (TPP-29537).

[0234] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment that can bind to GPC3, comprising: a heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138 (TPP-29538).

[0235] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment that can bind to GPC3, comprising: a heavy chain containing SEQ ID NO: 157 and a light chain containing SEQ ID NO: 158 (TPP-29636).

[0236] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or its antigen-binding fragment that can bind to GPC3, comprising: a heavy chain containing SEQ ID NO: 177 and a light chain containing SEQ ID NO: 178 (TPP-29638).

[0237] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a heavy chain containing SEQ ID NO: 117 and a light chain (TPP-29537) containing SEQ ID NO: 118, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0238] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a heavy chain containing SEQ ID NO: 137 and a light chain (TPP-29538) containing SEQ ID NO: 138, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0239] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a heavy chain containing SEQ ID NO: 157 and a light chain (TPP-29636) containing SEQ ID NO: 158, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0240] In some highly preferred embodiments, the present invention relates to targeted actinide (TAC) of formula (I). [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3, comprising: a heavy chain containing SEQ ID NO: 177 and a light chain (TPP-29638) containing SEQ ID NO: 178, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of the lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0241] The targeted actinium conjugate (TAC) of formula (I) of the present invention comprises a chelated portion containing a chelated actinium atom (Ac).

[0242] In the targeted actinium coupling (TAC) of formula (I) of the present invention, the actinium (Ac) atom is shown to be complexed with four oxygen atoms and two nitrogen atoms of the macrocycle and with two carboxyl groups. Although this is a hypothetical complexation mode of the actinium atom, the description includes all possible and conceivable cases in which one or more bonds are absent, such as where the actinium atom is not bonded to any heteroatoms of the macrocycle, or not bonded to the carboxyl groups, etc.

[0243] The anti-GPC3 antibody or its antigen-binding fragment is preferably coupled to the remainder of the molecule forming the targeted actinium conjugate of the present invention via a lysine residue of the antibody. Therefore, the chelating portion of the complexing actinium and the anti-GPC3 antibody or its antigen-binding fragment can be coupled via an amide or thiourea portion. When the preferred macrocyclic chelating agent macropa is used, the terminal isothiocyanate or carboxylic acid portion is coupled to the terminal amino group of the lysine residue in the anti-GPC3 antibody or its antigen-binding fragment, resulting in the formation of a thiourea or amide group, respectively. Therefore, the chelating portion of the preferred complexing actinium containing the macropa chelating agent is attached to the anti-GPC3 antibody or its antigen-binding fragment via a thiourea or amide bond derived from the terminal amino group of the lysine residue in the anti-GPC3 antibody or its antigen-binding fragment.

[0244] Synthesis of targeted actinide conjugates according to the present invention In another aspect, the present invention relates to a method for preparing a targeted actinium conjugate (TAC) of formula (I). [Ab] refers to a monoclonal antibody or its antigen-binding fragment that binds to GPC3. The method includes: (i) The chelation part of formula (II) Conjugated to monoclonal antibodies or their antigen-binding fragments capable of binding to GPC3, and (ii) The resulting product is contacted with an aqueous solution containing ions of at least one actinium isotope that emits α radiation.

[0245] In another embodiment, the present invention relates to a method for preparing a targeted actinium conjugate (TAC) of formula (I). [Ab] refers to a monoclonal antibody or its antigen-binding fragment that can bind to GPC3. The method includes: (i) The chelation part of formula (II) Coupled with a monoclonal antibody or its antigen-binding fragment capable of binding to GPC3 via a lysine residue of the monoclonal antibody or its antigen-binding fragment, and (ii) The resulting product is contacted with an aqueous solution containing ions of at least one actinium isotope that emits α radiation.

[0246] In another embodiment, the present invention relates to a method for preparing a targeted actinium conjugate (TAC) of formula (I). Wherein [Ab] is an anti-GPC3 antibody or its antigen-binding fragment selected from TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638 and TPP-29642. The method includes: (i) The chelation part of formula (II) Conjugated to anti-GPC3 antibodies or antigen-binding fragments selected from TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638 and TPP-29642 via lysine residues of the anti-GPC3 antibody or antigen-binding fragment, and (ii) The resulting product is contacted with an aqueous solution containing ions of at least one actinium isotope that emits α radiation.

[0247] In another embodiment, the present invention relates to a method for preparing a targeted actinium conjugate (TAC) of formula (I). [Ab] is an anti-GPC3 antibody selected from TPP-29537, TPP-29538, TPP-29636, or TPP-29638, or its antigen-binding fragment. The method includes: (i) The chelation part of formula (II) Conjugated to an anti-GPC3 antibody or its antigen-binding fragment selected from TPP-29537, TPP-29538, TPP-29636 or TPP-29638 via a lysine residue of a monoclonal antibody or its antigen-binding fragment, and (ii) The resulting product is contacted with an aqueous solution containing ions of at least one actinium isotope that emits α radiation.

[0248] In step (i), the chelation portion of formula (II) The anti-GPC3 antibody is conjugated to a monoclonal antibody capable of binding to GPC3 or its antigen-binding fragment. Preferably, the anti-GPC3 antibody is selected from TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 or their antigen-binding fragments. More preferably, the anti-GPC3 antibody is selected from TPP-29537, TPP-29538, TPP-29636, and TPP-29638 or their antigen-binding fragments. Most preferably, the anti-GPC3 antibody is TPP-29537.

[0249] Preferably, the chelating portion of formula (II) is coupled to an anti-GPC3 antibody or its antigen-binding fragment via a lysine residue of a monoclonal antibody, thereby forming a thiourea portion that links the chelating portion to the anti-GPC3 antibody or its antigen-binding fragment.

[0250] Preferably, the coupling in step (i) is carried out at an alkaline pH, i.e., at a pH higher than 7. More preferably, the coupling in step (i) is carried out at a pH of 8 to 10, and most preferably at a pH of 9.

[0251] Following step (i), an antibody-drug conjugate (ADC) of formula (III) is generated. Therefore, in another embodiment, the present invention relates to an antibody-drug conjugate of formula (III). [Ab] refers to a monoclonal antibody or its antigen-binding fragment that can bind to GPC3.

[0252] Preferably, the anti-GPC3 antibody or its antigen-binding fragment is selected from TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 or their antigen-binding fragments. More preferably, the anti-GPC3 antibody is selected from TPP-29537, TPP-29538, TPP-29636, and TPP-29638 or their antigen-binding fragments. Most preferably, the anti-GPC3 antibody is TPP-29537.

[0253] In step (ii), the product generated from the coupling in step (i) is contacted with an aqueous solution containing ions of at least one actinium isotope that emits α. Preferably, the actinium isotope that emits α is... 225 Ac. Preferably, the ion of at least one alpha-emitting actinium isotope is a triple positively charged ion. 225 Ac, that is 225 Ac 3+The present invention allows step (ii) to be carried out at room temperature, i.e., at a temperature lower than that required for the preparation of the corresponding DOTA conjugate (which requires a higher temperature (60°C)).

[0254] Treatment The treatment method involves administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof or a variant thereof and / or a targeted actinide conjugate (TAC) conjugate to a subject in need of treatment.

[0255] use On the other hand, the antibodies and / or targeted actinide conjugates (TACs) of the present invention can be used to treat diseases in human or non-human animal subjects. In particular, the antibodies and / or targeted actinide conjugates (TACs) of the present invention can be used to treat proliferative and / or neoplastic diseases, such as cancer. In one embodiment, the antibodies and / or targeted actinide conjugates (TACs) of the present invention have specificity and / or binding affinity for GPC3.

[0256] As described herein, various aspects of the invention relate to the treatment of diseases, particularly selective targeted therapy to diseased tissues, and to complexes, conjugates, pharmaceuticals, formulations, kits, etc., that can be used in such methods. In all aspects, the diseased tissue may be located in a single site within the body (e.g., in the case of a localized solid tumor) or may be located in multiple sites (e.g., in arthritis where several joints are affected or in the case of distributed or metastatic cancerous diseases).

[0257] The antibodies and / or targeted actinide conjugates (TACs) of the present invention preferably have binding affinity for phosphatidylinositol proteoglycan-3 (GPC3). The expression of GPC3 in tumor cells has been observed in different types of diseases, particularly hyperproliferative diseases such as cancer (Nishida T, Kataoka H. Glypican 3-Targeted Therapy in Hepatocellular Carcinoma. Cancers (Basel). 2019 Sep 10;11(9):1339). Examples of diseases and / or tumors suitable for treatment with the preferred antibodies and / or targeted actinide conjugates (TACs) of this invention include: hepatocellular carcinoma (HCC), lung cancer (including squamous non-small cell lung cancer), neuroendocrine prostate cancer, Merkel cell carcinoma, pediatric indications (such as pediatric solid embryonal tumors (including most hepatoblastomas), Wilms' tumor, rhabdoid tumors, certain germ cell tumor subtypes (such as yolk sac tumors + choriocarcinomas and some rhabdomyosarcomas), melanoma, ovarian cancer, colorectal cancer, esophageal cancer, cervical cancer, head and neck cancer, renal cell carcinoma, breast cancer (including triple-negative breast cancer), gastric cancer, sarcoma, and bladder cancer.

[0258] In some embodiments, the present invention includes methods for treating diseases, more preferably hyperproliferative diseases, using the antibodies and / or targeted actinide conjugates (TACs) of the present invention, and even more particularly, wherein the hyperproliferative disease is cancer, and even more particularly, wherein the cancer is hepatocellular carcinoma (HCC). In some embodiments, the present invention includes methods for treating diseases, preferably hyperproliferative diseases, more preferably cancer, using the antibodies and / or targeted actinide conjugates (TACs) of the present invention, characterized by ectopic expression of GPC3 in tumor cells.

[0259] In some embodiments, the present invention includes a method for treating hyperproliferative diseases, more particularly cancers, and more particularly hepatocellular carcinoma, said method comprising administering to a subject in need an effective amount of at least one antibody and / or targeting actinide conjugate (TAC) of the present invention. In some embodiments, the present invention includes a method for treating hyperproliferative diseases characterized by ectopic expression of GPC3 in tumor cells, more particularly wherein the hyperproliferative disease is cancer, more particularly hepatocellular carcinoma, said method comprising administering to a subject in need an effective amount of at least one antibody and / or targeting actinide conjugate (TAC) of the present invention and other agents.

[0260] In some embodiments, the present invention provides the antibodies and / or targeted actinide conjugates (TACs) described above for the treatment and / or prevention of diseases, particularly hyperproliferative disorders, more particularly cancers, and even more particularly hepatocellular carcinoma. In some embodiments, the present invention includes the antibodies and / or targeted actinide conjugates (TACs) described above for the treatment and / or prevention of hyperproliferative diseases characterized by ectopic expression of GPC3 in tumor cells.

[0261] In some embodiments, the present invention includes the antibody and / or targeted actinide conjugate (TAC) of the present invention, in a method for inhibiting cell proliferation and / or inducing apoptosis, said method comprising contacting cells with the antibody and / or targeted actinide conjugate (TAC). In some embodiments, the present invention includes the antibody and / or targeted actinide conjugate (TAC) of the present invention, in a method for inhibiting tumor cell proliferation and / or inducing tumor cell apoptosis, said method comprising contacting tumor cells with the antibody and / or targeted actinide conjugate (TAC), wherein GPC3 is overexpressed in the tumor cells.

[0262] In some embodiments, the present invention includes antibodies and / or targeted actinide conjugates (TACs) for methods of treating hyperproliferative diseases, more particularly wherein the hyperproliferative disease is cancer, and even more particularly wherein the cancer is hepatocellular carcinoma. In some embodiments, the present invention includes antibodies and / or targeted actinide conjugates (TACs) for methods of treating hyperproliferative diseases characterized by ectopic expression of GPC3 in tumor cells.

[0263] In some embodiments, the present invention includes the use of antibodies and / or targeted actinide conjugates (TACs) for the treatment and / or prevention of hyperproliferative diseases. In some embodiments, the present invention includes the use of antibodies and / or targeted actinide conjugates (TACs) for the manufacture of medicaments for the treatment and / or prevention of hyperproliferative diseases characterized by ectopic expression of GPC3 in tumor cells.

[0264] In some embodiments, the present invention includes the use of antibodies and / or targeted actinide (TACs) for the manufacture of medicaments for the treatment of hyperproliferative diseases, particularly cancers and more particularly hepatocellular carcinoma.

[0265] The aforementioned conditions have been well characterized in humans, but also exist in other animals (including mammals) with similar causes, and can be treated by applying the pharmaceutical compositions of the present invention.

[0266] The antibodies or antigen-binding fragments thereof of the present invention, or the targeted actinide conjugates (TACs) or variants thereof of the present invention, can be co-administered with known drugs, and in some cases, the antibodies or antigen-binding fragments thereof may be modified themselves. For example, antibodies or antigen-binding fragments thereof or variants thereof, or TACs, can be conjugated to drugs or other peptides or proteins to potentially further enhance therapeutic efficacy.

[0267] The antibody or antigen-binding fragment thereof according to the invention, or the targeted actinide conjugate (TAC) according to the invention, may be administered as a single agent or in combination with one or more other therapeutic agents, wherein the combination does not cause unacceptable adverse reactions.

[0268] Therefore, on the other hand, the present invention relates to antibodies or antigen-binding fragments thereof according to the invention, or targeted actinide conjugates according to the invention, or pharmaceutical compositions according to the invention, for use simultaneously, separately or sequentially in combination with one or more other bioactive therapeutically active compounds, the bioactivity preferably targeting cancer, especially hepatocellular carcinoma.

[0269] The following lists non-limiting examples of therapeutically active compounds for use in conjunction with the antibodies or antigen-binding fragments thereof of the present invention or the targeted actinide conjugates (TACs) of the present invention. These compounds are defined by their categories, thereby enabling those skilled in the art to identify such compounds belonging to said categories.

[0270] In the context of this invention, TK inhibitors refer to compounds that are pharmacological inhibitors of tyrosine kinases. Tyrosine kinases are receptors responsible for activating many proteins through signal transduction cascades. Examples include, but are not limited to, sorafenib, lenvatinib, regorafenib, or cabozantinib.

[0271] In the context of this invention, VEGFR inhibitors refer to compounds that act as pharmacological inhibitors of vascular endothelial growth factor receptor (VEGFR) signaling, which plays a role in regulating tumor-induced angiogenesis. Examples include, but are not limited to, bevacizumab and ramucirumab.

[0272] In the context of this invention, a chemotherapeutic agent refers to a compound suitable for chemotherapy of GPC3-positive cancers, particularly HCC. Examples include, but are not limited to, doxorubicin, epirubicin, or cisplatin.

[0273] In the context of this invention, examples of early pipeline drugs that can be used in combination with antibodies or antigen-binding fragments thereof according to the invention, or targeted actinide conjugates (TACs) according to the invention, include, but are not limited to: tivozanib (AVEOOncology), namodenoson (Can-Fite BioPharma), GT90001 (Kintor Pharmaceutical), ARTEMIS T-cell therapy (Eureka Therapeutics), MTL-CEBPA (MiNA Therapeutics), fostroxacitabine bralpamide / fostrox (Medivir), or various TIGIT inhibitors.

[0274] In the context of this invention, transarterial chemoembolization refers to a compound suitable for intra-arterial administration to an artery that specifically supplies blood to the site of intrahepatic HCC lesions. Examples include, but are not limited to, doxorubicin, epirubicin, or cisplatin, drug-eluting beads that release doxorubicin, iodized oil, yttrium-90 (β-radioactive) labeled glass or resin microspheres, or embolizing agents such as gelfoam or biodegradable starch microspheres.

[0275] In the context of this invention, external beam radiation therapy refers to radiation therapy in which an external source of ionizing radiation is directed and / or applied to a specific part of the body of a subject.

[0276] In the context of this invention, the term "combination" refers not only to a dosage form (also known as a fixed-dose combination) containing all components (i.e., the anti-GPC3 antibody or fragment thereof of the present invention and / or the anti-GPC3-TAC of the present invention) and other pharmaceutical agents, and to a combination package containing components that are separate from each other, but also to components that are administered simultaneously or sequentially, provided that these components are used to prevent or treat the same disease.

[0277] The amount of active ingredient administered can vary widely depending on factors such as the specific compound and dosage unit used, the route and timing of administration, the treatment cycle, the age, sex and general condition of the patient being treated, the nature and extent of the disease being treated, the rate of drug metabolism and excretion, potential drug combinations and drug-drug interactions, etc.

[0278] Pharmaceutical composition and administration On the other hand, this invention relates to pharmaceutical compositions comprising the isolated antibody or antigen-binding fragment of the present invention or the targeted actinide conjugate of the present invention. For the treatment of any of the aforementioned conditions, the pharmaceutical compositions used according to the present invention can be formulated in any conventional manner using one or more physiologically acceptable carriers, excipients, or adjuvants. Further details regarding formulation and administration techniques can be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co., Easton, Pa.).

[0279] The isolated antibody or antigen-binding fragment according to the invention, or the targeted actinide conjugate according to the invention, can be administered in any suitable manner, which may vary depending on the type of disease being treated. Possible routes of administration include oral, parenteral, or local administration. Parenteral delivery methods include intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal, or intranasal administration. Furthermore, the isolated antibody or antigen-binding fragment according to the invention, or the targeted actinide conjugate according to the invention, can be administered via pulsatile infusion, for example, using a decreasing dose. Administration by injection is preferred, and intravenous injection is most preferred. The dosage will depend on various factors, such as clinical symptoms, individual weight, and whether other medications are being administered. Those skilled in the art should recognize that the route of administration may be varied depending on the condition or disease to be treated.

[0280] Pharmaceutical compositions according to the invention may contain, alone, an antibody or antigen-binding fragment according to the invention or a targeted actinide conjugate according to the invention, or in combination with at least one other agent (such as a stabilizer). The antibody or antigen-binding fragment according to the invention or the targeted actinide conjugate according to the invention may be administered in any sterile, biocompatible pharmaceutical carrier (including, but not limited to, saline, buffered saline, dextran, and water). In a particular embodiment, the pharmaceutical composition according to the invention may contain one or more other pharmaceutically active compounds, particularly one or more other pharmaceutically active compounds suitable for treating GPC3-related conditions and / or conditions associated with ischemic events resulting from partial or complete vascular occlusion. Any of these agents may be administered to a patient alone or in combination with other agents or drugs in the form of a pharmaceutical composition (mixed with one or more excipients or pharmaceutically acceptable carriers). In a particular embodiment, the pharmaceutically acceptable carrier is pharmaceutically inert.

[0281] Orally administered pharmaceutical compositions may be formulated in dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, pastes, suspensions, etc., for oral administration to patients.

[0282] Orally administered pharmaceutical formulations can be obtained by combining an active compound with a solid excipient, optionally grinding the resulting mixture, adding suitable adjuvants as needed, and then processing the granular mixture to obtain tablets or sugar-coated pellet cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starches from corn, wheat, rice, potatoes, or other plants; celluloses such as methylcellulose, hydroxypropyl methylcellulose, or sodium carboxymethylcellulose; gums, including gum arabic and tragacanth; and proteins such as gelatin and collagen. Disintegrants or solubilizers, such as cross-linked polyvinylpyrrolidone, agar, alginate, or their salts, such as sodium alginate, may be added as needed.

[0283] The sugar-coated core may be provided with a suitable coating, such as a concentrated sugar solution, and may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solution, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the coating of the tablets or sugar-coated cores for product identification or characterization of the amount, i.e., dosage, of the active compound.

[0284] Orally administered pharmaceutical formulations include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a coating (such as glycerin or sorbitol). Push-fit capsules may contain the active ingredient mixed with a filler or binder (such as lactose or starch), a lubricant (such as talc or magnesium stearate), and optionally a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol, with or without a stabilizer.

[0285] Parenteral drug formulations comprise aqueous solutions of the active compound. For injection, the pharmaceutical compositions of the present invention can be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Furthermore, suspensions of the active compound can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or excipients include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or reagents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions.

[0286] For local or nasal administration, a penetrant suitable for the specific barrier to be penetrated can be incorporated into the formulation. Such penetrants are well known in the art.

[0287] The pharmaceutical compositions of the present invention can be prepared in a manner known in the art, for example by conventional methods of mixing, dissolving, granulation, sugar-coated pelleting, grinding, emulsification, encapsulation, embedding, or lyophilization.

[0288] The pharmaceutical composition may be provided in the form of a salt and may form with an acid, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. Salts tend to be more soluble in aqueous or other protic solvents than their corresponding free base forms. In other cases, preferred formulations may be liquid formulations or lyophilized powders in their histidine or phosphate or Tris buffer, sucrose, and / or mannitol, with a pH range of 4.5-7.5, optionally containing additional substances such as polysorbate, which are bound to a buffer prior to use.

[0289] After preparing a pharmaceutical composition containing the compounds of the present invention formulated in an acceptable carrier, it can be placed in a suitable container and labeled for the treatment of the indicated disease. For administration of anti-GPC3 antibodies or their antigen-binding fragments or anti-GPC3-TACs, such labels will include the amount, frequency, and method of administration.

[0290] Therapeutic effective dose Pharmaceutical compositions applicable to the present invention include compositions containing an effective amount of the active ingredient to achieve an intended purpose (e.g., treating a specific disease state characterized by ectopic expression of GPC3).

[0291] Determining the effective dosage is within the capabilities of those skilled in the art. The determination of the therapeutically effective amount of the novel antibody of the present invention, its antigen-binding fragment, or a variant thereof depends primarily on the specific patient characteristics, route of administration, and nature of the disease being treated. General guidance is described, for example, in publications of the International Conference on Harmonization and REMINGTON'S PHARMACEUTICAL SCIENCES, Chapters 27 and 28, pp. 484-528 (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). More specifically, determining the therapeutically effective amount will depend on factors such as the toxicity and efficacy of the drug. Toxicity can be determined using methods well-known in the art and found in the foregoing references.

[0292] For any compound, the therapeutically effective dose can be determined in cell culture or initially assessed in animal models, typically mice, rabbits, dogs, pigs, or monkeys. Animal models can also be used to obtain the desired concentration range and route of administration. This information can then be used to determine the effective dose and route of administration in humans.

[0293] Therapeutic effective doses refer to the amount of antibody or its antigen-binding fragment, or antibody-drug conjugate, that improves symptoms or disease. The efficacy and toxicity of these compounds can be determined through standard pharmaceutical procedures performed in cell cultures or laboratory animals, such as ED (Effective Drug Reduction). 50 (The effective dose in 50% of the population) and LD50 50 (The 50% lethal dose for a population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio ED. 50 / LD 50 Pharmaceutical compositions exhibiting a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies were used to formulate a range of human dosages. The dosages of these compounds are preferably within a range of circulating concentrations, which includes ED (extracorporeal active ingredient) concentrations with little or no toxicity. 50 The dosage may vary within this range depending on the dosage form used, patient sensitivity, and route of administration.

[0294] The exact dosage is determined by an individual physician based on the patient being treated. Dosage and administration are adjusted to provide an adequate level of the active component or to maintain the desired effect. Other factors that may be considered include the severity of the disease state, the patient's age, weight, and sex; diet, timing and frequency of administration, concomitant medications, sensitivity to response, and tolerance / response to treatment. Depending on the specific formulation's half-life and clearance rate, long-acting drug compositions may be administered, for example, every 3 to 4 days, weekly, every two weeks, or every three weeks.

[0295] Depending on the route of administration, the normal dose can vary from 0.1 to 100,000 micrograms, with a maximum total dose of about 10 g. Specific dosage and delivery instructions are provided in the literature. See US Pat. Nos. 4,657,760, 5,206,344, or 5,225,212.

[0296] Reagent test kit On the other hand, the present invention relates to kits comprising isolated antibodies or antigen-binding fragments according to the invention, or targeted actinide conjugates according to the invention, and instructions for use. In a particular embodiment, the kit comprises one or more containers containing one or more components of the aforementioned compositions of the invention. Such containers may be accompanied by a statement in the format prescribed by a government agency regulating the production, use, or sale of pharmaceuticals or biological products, reflecting that agency's approval for the production, use, or sale of products for human use.

[0297] Diagnostic methods According to another aspect, antibodies or antigen-binding fragments according to the invention or antibody conjugates according to the invention can be used in their original or combined forms for research and diagnosis, or as analytical reference standards, etc.

[0298] The anti-GPC3 antibody or its antigen-binding fragment or antibody-conjugate according to the present invention can be used to detect the presence of GPC3. Therefore, in another aspect, the present invention relates to isolated antibodies or antigen-binding fragments according to the present invention, or antibody-conjugates according to the present invention, which are used as diagnostic agents.

[0299] The detection of GPC3 expression typically involves contacting a biological sample (an individual's tumor, cells, tissue, or body fluid) with one or more antibodies or fragments according to the invention (optionally conjugated with a detectable portion) and detecting whether the sample is GPC3 positive or whether the expression in the sample has changed (e.g., decreased or increased) compared to a control sample.

[0300] In some embodiments, the provided conjugates comprise one or more detectable moieties, i.e., are “labeled” with one or more such moieties. In some such embodiments, the conjugates of this disclosure can be used for diagnostic or imaging applications, such as cancer diagnosis or imaging. Any of a variety of detectable moieties can be used in the labeled antibody conjugates described herein. Suitable detectable moieties include, but are not limited to: various ligands; radionuclides; fluorescent dyes (such as fluorescence resonance energy dyes (FRET), such as Cy5, Cy7); chemiluminescent agents (such as acridinium esters, stabilized dioxane, etc.); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (such as gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (such as dyes, colloidal gold, etc.); biotin; digoxigenin; haptens; and proteins from which antiserum or monoclonal antibodies can be obtained.

[0301] According to certain preferred embodiments of the invention, the detectable portion may be a fluorescent dye, such as Alexa Flour dye (Invitrogen), or a fluorescence resonance energy dye (FRET), such as the pH-sensitive dye CypHer5E-NHS-Ester (Cytiva) that can be used to directly label antibodies or antigen-binding fragments via lysine residues.

[0302] PET imaging According to another aspect, the antibody or antigen-binding fragment and / or its conjugate of the present invention can be used as an imaging agent, preferably as a PET imaging agent.

[0303] Positron emission tomography (PET) is a medical imaging technique that detects gamma photons produced by positron emitters and reconstructs these signals to visualize the location of the positron emitters. When combined with computed tomography (CT), PET / CT can precisely locate positron emitters within the anatomical framework of the body. Several positron emitters are routinely used clinically, such as C-11, F-18, Ga-68, and Zr-89. Preferably, Zr-89 is used as a positron emitter in conjunction with an antibody or antigen-binding fragment according to the invention or an antibody-drug conjugate according to the invention, due to its long half-life (78.4 hours) and efficient labeling reaction under mild conditions.

[0304] Suitable chelating agents for Zr-89 in PET imaging applications include, but are not limited to, DFO (deferoxamine), N-suc-deferoxamine-tetrafluorophenol ester (N-suc-DFO-TFP ester), benzyl-deferoxamine p-isothiocyanate (DFO-Bz-NCS, also known as DFO-Ph-NCS), deferoxamine-maleimide (DFO-maleimide), deferoxamine-squamamide (DFO-sq), or DFO*. All of these chelating agents can be conjugated to antibodies or antibody fragments to provide a means of targeting the imaging agent to the tumor to be imaged. According to a preferred embodiment of the invention, the chelating agent is conjugated to the remainder of the molecule via the terminal amino group of a lysine residue of a monoclonal antibody or its antigen-binding fragment.

[0305] DFO* is an extended version of DFO. 89 Zr-DFO* complexes exhibit increased in vitro stability and improved in vivo properties (WO2021051168), and are therefore preferred chelating agents for Zr-89 when conjugated with the antibodies or antibody fragments of the present invention for PET imaging.

[0306] DFO* refers to 5,11,16,22-tetraazahexacosanodiamide, N 1 -[5-(acetylhydroxyamino)pentyl]-N 26 5,16-Trihydroxy-N 26 -[5-[[[(4-isothiocyanate phenyl)amino]thiomethyl]amino]pentyl]-4,12,15,23-tetraoxo (CAS no. 1810009-29-0): Furthermore, according to certain embodiments of the present invention, it can be conjugated to an antibody or its antigen-binding fragment [Ab], preferably to a monoclonal antibody that binds to GPC3 or its antigen-binding fragment. Preferably, the anti-GPC3 antibody is selected from TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638 and TPP-29642 or their antigen-binding fragments. More preferably, the anti-GPC3 antibody is selected from TPP-29537, TPP-29538, TPP-29636 and TPP-29638 or their antigen-binding fragments.

[0307] According to certain highly preferred embodiments of the present invention, DFO* is coupled to the anti-GPC3 antibody or its antigen-binding fragment of the present invention via a lysine residue of a monoclonal antibody, thereby forming a thiourea moiety that links the chelating portion to the anti-GPC3 antibody or its antigen-binding fragment.

[0308] Those skilled in the art can utilize the foregoing information to the fullest extent possible. Attached Figure Description

[0309] Figure 1 Anti-GPC3 antibodies TPP-14890 and TPP-29537 bind to the FACS of human GPC3 transiently transfected into CHO cells. TPP-5657 is an isotype control antibody.

[0310] Figure 2 Anti-GPC3 antibodies TPP-14890 and TPP-29537 bound to the FACS of cynomolgus monkey GPC3 transiently transfected into CHO cells. TPP-5657 was an isotype control antibody.

[0311] Figure 3 Anti-GPC3 antibodies TPP-14890 and TPP-29537 bind to the FACS of mouse GPC3 transiently transfected into CHO cells. TPP-5657 is an isotype control antibody.

[0312] Figure 4 Anti-GPC3 antibodies TPP-14890 and TPP-29537 bound to FACS of transiently mimicked CHO cells. TPP-5657 was an isotype control antibody.

[0313] Figure 5 Vesicle area per cell was calculated 5 hours after the addition of the directly labeled antibody to HepG2 cells. TPP-5657 was an isotype control antibody.

[0314] Figure 6 Vesicle area per cell was calculated 5 hours after adding Fabfluor-labeled antibody to HepG2 cells. TPP-5657 was an isotype control antibody.

[0315] Figure 7 Antibody binding capacity on cell surfaces was measured by flow cytometry using Alexa 488-labeled TPP-14890 in cell lines HFF-1, SU-DHL-10, PLC / PRF / 5, HepG2, Hep3B2, and primary fibroblasts HCF and HPF.

[0316] Figure 8 Antibody binding capacity on cell surfaces was measured by flow cytometry using Alexa 488-labeled TPP-29537 in cell lines HFF-1, SU-DHL-10, PLC / PRF / 5, HepG2, Hep3B2, and primary fibroblasts HCF and HPF.

[0317] Figure 9The binding of Ac-225-Macropa-TPP-29537 and Ac-225-Macropa-TPP-14890 to peptide-coated GPC3 magnetic beads. Total binding was defined as [(activity in beads - activity in supernatant) / (total activity)] × 100.

[0318] Figure 10 In vitro cytotoxicity data of Ac-225-Macropa-TPP-29537 (CAR 0.5 and CAR 11), Ac-225-Macropa-TPP-14890 (CAR 0.7 and CAR 10) and Ac-225-Macropa-isotype-control (CAR 0.7) in HepG2 cells (A) and HEP-3B cells (B).

[0319] Figure 11 Biodistribution of Ac-225-Macropa-TPP-29537 (CAR 0.7) and Ac-225-Macropa-Allotype-Control (CAR 0.7) in different tissues of the human hepatocellular carcinoma xenograft model Huh-7. Animals were administered a single intravenous (iv) dose of 0.75 mg / kg total antibody, 500 kBq / kg. Cumulative Ac-225 is expressed as % (%ID / g) of the injected dose.

[0320] Figure 12 A) In vivo efficacy of Ac-225-Macropa-TPP-29537 (CAR 0.7) and Ac-225-Macropa-allotype-control (CAR 0.7) in the human hepatocellular carcinoma xenograft model Huh-7. Tumors were measured using digital calipers, and results are expressed as tumor area (mm²). 2 () indicates that the calculation is the longest diameter of the tumor multiplied by the shortest diameter. B) % survival rate in in vivo efficacy studies in the Huh-7 tumor model.

[0321] Figure 13 The binding of Zr-89-DFO*-TPP-29537 to GPC3 peptide-coated magnetic beads. Total binding was defined as [(activity in beads - activity in supernatant) / (total activity)] × 100.

[0322] Figure 14 Coronal PET / CTMIP images of mice carrying HEP-3B tumors, imaged using Zr-89-DFO*-TPP-29537. 2h n=1, 4h n=1, 24h n=3, 72h n=3, and 168h n=2.

[0323] Figure 15: Ex vivo and in vivo PET / CT quantification of Zr-89-DFO*-TPP-29537 in the HEP-3B tumor model, and ex vivo biodistribution of Ac-225-Macropa-TPP-29537. Ex vivo biodistribution data: n=3 at each time point. Imaging-based analysis: 2h n=1, 4h n=1, 24h n=3, 72h n=3, and 168h n=2. Data are expressed as mean + SD.

[0324] Experimental Section Chemical names were generated using Dassault Systèmes' BIOVIA Draw 2019. In some cases, generally accepted names of commercially available reagents were used instead of those generated by BIOVIA Draw.

[0325] The following table lists the abbreviations used in this paragraph and the Examples section (unless otherwise explained in the text). Other abbreviations have their usual meaning to those skilled in the art.

[0326] Table 3: Abbreviations The following table lists the abbreviations used in this article.

[0327] 225 AcAc-225 Ac-225 Actinide-225 ACC antibody-chelating agent conjugate CAR chelating agent-antibody ratio CT computed tomography scan DCTA1,2-Diaminocyclohexanetetraacetic acid DMAN, N-dimethylacetamide DMSO dimethyl sulfoxide EdU5-ethynyl-2'-deoxyuridine ESI Electrospray Ionization EtOH (ethanol) FPLC (Fast Protein Liquid Chromatography) FSC forward scattering GPC3 Phosphatidylinositol Proteoglycan-3 HPGe high purity germanium HPLC (High Performance Liquid Chromatography) iTLC instant thin-layer chromatography IRF immune response score mAb monoclonal antibody min minutes MS mass spectrometry NaCl sodium chloride nm nanometer nmol nanomolar PBS phosphate buffered saline PET Positron Emission Tomography RAC radioactivity concentration RCP radiochemical purity RPMI Roswell Park Memorial Institute 1640 culture medium SEC size exclusion chromatography SCC lateral scattering TAC-targeted actinide (Ac-225-tagged ACC) TFA (trifluoroacetic acid) Time of Flight (TOF) UPLC (Ultra-High Performance Liquid Chromatography) UV ultraviolet rays All reagents not described in the experimental section are commercially available compounds, or known compounds, or compounds that can be formed from known compounds by a person skilled in the art using known methods.

[0328] The various aspects of the invention described in this application are illustrated by the following embodiments, which are not intended to limit the invention in any way.

[0329] The exemplary test experiments described herein are for illustrative purposes only, and the invention is not limited to the embodiments given.

[0330] Anti-GPC3 antibodies and their targeted radionuclide conjugates Example 1: Generation of anti-GPC3 antibody and reference compound All antibodies were expressed in HEK293 cells using a standard transient transfection procedure and purified from cell culture supernatant by Protein-A and size exclusion chromatography.

[0331] In some embodiments, the sequences of existing antibodies, such as those of TPP-14890 (Codrituzumab), are randomly altered. Only a few of the resulting antibodies exhibit improved or nearly unchanged behavior. This document includes only examples with improved or nearly unchanged properties.

[0332] Furthermore, existing antibody sequences, such as TPP-14890, were germinated and further modified, such as by removing lysine residues or potential aspartic acid isomerization sites from the CDR. The resulting antibodies (TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, TPP-29642) were tested for monovalent binding affinity (Kb) to human GPC3 and cynomolgus monkey GPC3 using surface plasmon resonance (SPR) assay as described in Example 2.D Cell binding (EC50) to CHO cells transfected with human GPC3 or cynomolgus monkey GPC3 was determined by flow cytometry as described in Example 3. Furthermore, the melting temperature (TmFab) of Fab was determined by differential scanning calorimetry (DSC) as described in Example 4. All these data are summarized in Table 4.

[0333] Table 4: Summary table comparing the properties of the antibody of the present invention with those of TPP-14890. Surprisingly, despite the extremely high number of mutations in both the VH and VL regions compared to TPP-14890, variants were identified that possess the following characteristics: a) are still producible via recombinant methods; b) exhibit comparable or superior affinity for recombinant or cellular human GPC3; c) still bind to cynomolgus monkey GPC3; d) still exhibit stability of the Fab fraction Tm above 65°C; and e) show expression rates above 145 mg / L in the standard HEK293E expression system (as listed in Table 4), indicating good producibility. However, the number of germline biases was significantly reduced, which is also reflected in the significantly lower computer-simulated immunogenicity scores (IPAD, Epibase) and in vitro immunogenicity assessments (Epibase). Furthermore, the number of lysine residues in the CDR was reduced from four lysine residues in the TPP-14890 CDR to one lysine residue or even none in the CDR of the antibody according to the invention (Table 4). Reducing the number of lysine residues within the CDR region lowers the risk of chelating agents conjugating to antibodies within the CDR region. Binding of chelating agents within the CDR can affect the binding of antibody conjugates to their target GPC3. For the variants TPP-29537, TPP-29538, TPP-29636, and TPP-29638, which do not contain any lysine residues in the CDR, this risk is completely avoided.

[0334] Example 2: SPR comparison of TPP-14890 and the antibody according to the present invention To evaluate the binding kinetics and affinity of the anti-GPC3 antibody and its cross-reactivity with cynomolgus GPC3, surface plasmon resonance (SPR) binding assays were performed. SPR-based experiments were conducted at 25°C on a Biacore T200 instrument (formerly GE Healthcare Inc., now Cytiva) using an anti-human Fc capture antibody covalently coupled to the CM5 sensor chip (Cytiva).

[0335] Anti-human Fc capture antibodies were covalently conjugated to approximately 5,000 RU using an amine conjugation kit (Cytiva) according to the manufacturer's instructions. Capture levels, RMax, and resulting % activity showed comparable behavior for all variants in SPR (data not shown). Analytes (human and cynomolgus monkey GPC3, R&D Systems, catalog numbers 2119-GP and 10509-GP, respectively) were injected in a multi-cycle kinetic series from 0.039 nM to 200 nM. Experiments were performed in HBS-EP+ (Cytiva). Association time was set to 300 s, dissociation time to 1,200 s, and flow rate to 30 μL / min. The resulting data were subjected to dual-reference processing (subtraction of the reference cell and the 0 nM concentration signal) and fitted to a 1:1 Langmuir binding isotherm using Biacore T200 evaluation software (RI set to constant). Results are shown in Table 5.

[0336] Table 5: Kinetic data of TPP-14890 and the antibody according to the present invention using SPR. The kinetic data show that TPP-29312, TPP-29537, TPP-29538, and TPP-29636 exhibit affinity (K0.05) comparable to that of TPP-14890. D Compared to TPP-14890 and other variants, TPP-29638 and TPP-29642 exhibit slightly higher affinity.

[0337] Example 3: Binding of GPC3 antibody to CHO cells transiently transfected with human, mouse, or cynomolgus monkey GPC3 To evaluate the binding of anti-GPC3 antibodies to human, cynomolgus monkey, or mouse GPC3, the binding of TPP-14890 and the antibody according to the present invention to CHO cells transiently transfected with human, cynomolgus monkey, or mouse GPC3 was assessed by FACS.

[0338] CHO cells were transiently transfected using a GPC3 expression construct. CHO-3E7 cells were cultured in 35 mL rotary tubes (TPP, #87050) under the following conditions: Freestyle F17 (fisherscientific, #10388033), 4 mM Glutamax, 0.1% Pluronic, 5% CO2, 37°C, and 75 rpm. Human GPC3 (SEQ ID NO:321), mouse GPC3 (SEQ ID NO:322), or long-tailed macaque (cynomolgus monkey) GPC3 (SEQ ID No:323) expression plasmids previously generated using recombinant DNA technology were cultured at a cell density of 3 × 10⁻⁶ cells / year.5 Cells were transfected into CHO-3E7 cells at a rate of 1 mL / h and then incubated at 37°C and 5% CO2 for three days.

[0339] The binding of the antibody to GPC3-transfected CHO cells was analyzed using FACS. Add 5 µL of the test antibody solution in 500 µg / mL PBS (pH 7.4) to the wells of a microplate (Eppendorf, #0030601300). Then, prepare a 1:5 dilution series, diluting down to 0.0064 µg / mL. Next, add 45 µL of the solution containing 2.5 × 10⁻⁶ PBS to the wells of a microplate. 4 A solution of GPC3-transfected CHO cells (pre-incubated on ice for 30 minutes) was added to each well. After incubating the microplate on ice for 45 minutes, the plate was centrifuged at 400 x g and 4 °C for 5 minutes. The supernatant was discarded, and the cells were resuspended in 110 µL of ice-cold FACS buffer (PBS, 3% FCS), centrifuged again, and resuspended in 110 µL of ice-cold FACS buffer for another wash. After a third centrifugation, the cells were resuspended in 50 µL of FACS buffer containing a 1:200 dilution of anti-human Fabdimer specific and Alexa Fluor 488 labeled secondary reagent (Dianova, #109-456-097). After incubating on ice for 40 minutes, the plate was centrifuged at 400 x g and 4 °C for 5 minutes. The supernatant was discarded, and the cells were resuspended in 110 µL of FACS buffer for the final centrifugation step. Finally, the cells were resuspended in FACS buffer containing propidium iodide (1 µg / mL), vortexed at 2500 RPM for 5 seconds, and FACS measurements were performed on an iQue3 instrument using a 4-second aspiration buffer as the instrument setting. Results for TPP-14980, TPP-29537, and the non-binding isotype control TPP-5657 are shown below. Figure 1 , Figure 2 , Figure 3 and Figure 4 .

[0340] EC50 values ​​were calculated from FACS binding curves using nonlinear regression analysis integrated in GraphPad Prism software. Results for all antibody variants described herein have been determined and are summarized in Table 6.

[0341] Table 6: EC50 values ​​of TPP-14890 and the antibody according to the present invention on CHO cells transiently transfected with human, cynomolgus monkey, or mouse GPC3. In summary, TPP-14890, TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 exhibited comparable FACS binding properties: all antibodies showed strong binding to human and cynomolgus monkey GPC3, while no binding to mouse GPC3 was detected. The isotype control antibody TPP-5657 did not show any binding to human, mouse, or cynomolgus monkey GPC3.

[0342] Example 4: Thermostability of Anti-GPC3 Antibody The thermostability of various anti-GPC3 antibodies was determined by differential scanning calorimetry (DSC) using a VP-Capillary DSC system (Malvern Instruments GmbH, formerly MicroCal Inc.) with a cell volume of 0.137 mL. All samples were diluted to a final concentration of 0.5 mg / mL with PBS buffer, and a protein-free buffer control was used as a reference. Samples were scanned from 20°C to 95°C at a scan rate of 120°C / h. Data were analyzed using Origin 7.0 Data Analysis (OriginLabCorp.). The results are shown in Table 6.

[0343] Table 6: Fab melting temperatures of various GPC3 antibodies. All antibodies of this invention exhibit Fab Tm values ​​well above 65°C, making them highly suitable for clinical development for human treatment.

[0344] Example 5: Germination of antibody sequences The existing antibody TPP-14890 carries several deviations from human lineage as described in the IMGT database (see http: / / www.imgt.org / ).

[0345] TPP-14890 carries 15 deviations from the closest phylogenetic light chain identified (IGKV2-28-IGKJ2, TPP-35038; sequential numbering; SEQ ID NO:325): I2V, L30V, G34R, Y35N, N36T, D39H, L55K, G56V, A60F, M94S, A96N, L97T, Q98H, T99V, Y101P.

[0346] TPP-14890 carries 17 deviations from the closest phylogenetic heavy chain identified except CDR-H3 (IGHV1-46-IGHJ4, TPP-35037; serial number; SEQ-ID NO: 324): S31D, Y33E, I50A, I51L, N52D, S54K, G55T, S57D, S59A, A61S, Q65K, M70L, R72A, T74K, V79A, R87T, A97T.

[0347] Compared to the closest human germline sequence, the lower germline deviation of the antibody reduces the murine component of the antibody or phage-derived human antibody without affecting antigen binding, thereby increasing the overall "humanity" and reducing the risk of immunogenicity caused by these molecules (Hwang et al.; Methods, Vol. 36, Issue 1, 2005, pp. 35-42, ISSN 1046-2023). See Table 7 for details.

[0348] Table 7: Number of germline deviations and their respective mutations in the light and heavy chains (excluding CDR-H3) compared to the human reference sequence of the TPP-14890 family. Compared to the TPP-14890 antibody, the antibody according to the present invention carries a reduced number of germline amino acid deviations. In fact, TPP-14890 has a total of 32 germline deviations from the human lineage. In contrast, the antibody according to the present invention has only 14-16 germline deviations from the human lineage, thereby reducing the risk of immunogenicity in human treatment.

[0349] In summary, the number of germline deviations of TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 is significantly lower than that of the existing humanized antibody TPP-14890, thereby further reducing the risk of immunogenic reactions when used for human treatment.

[0350] Example 6: Computer-simulated (in silico) immunogenicity assessment The likelihood of triggering an immunogenic response in human treatment was assessed using two independent computer-based prediction methods: iPad rating The IPAD v4.03 algorithm relies on the NetMHCIIpan algorithm (Nielsen et al., Quantitative predictions of peptide binding to any HLA-DR molecule of known sequence: NetMHCIIpan. PLoS Comput Biol. 2008 Jul 4;4(7):e1000107), which predicts the binding of peptides to HLA class II molecules. In short, VH and VL sequences are run through the algorithm, filtering out non-species peptides. HLA scores are calculated by multiplying the number of predicted HLA-binding peptides by the HLA frequency as a percentage and a scaling factor (0.19352). The IPAD score is then generated by summing all individual HLA scores. Using this method, IPAD scores between 600 and 1000 are comparable to those of marketed fully human and humanized antibodies, scores below 600 are more favorable, while scores above 1000 indicate a higher risk of immunogenicity.

[0351] Table 8: Comparison of IPAD scores between prior art antibodies and the antibodies of the present invention As shown in Table 8, TPP-14890 has an IPAD score of 1129, indicating a higher risk of immunogenicity compared to the antibodies TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 (which have much lower IPAD scores). TPP-29312, TPP-29537, TPP-29538, and TPP-29636 even have IPAD scores below 600. The fully human antibodies 2-F7, 4-11G, 7-5B, 7-8B, and 17-4D exhibit comparable or worse IPAD scores compared to the antibodies of the present invention. YP7, a mouse antibody, has the highest IPAD score. The humanized variant hYP7 shows significant improvement but still exhibits a worse IPAD score compared to the antibodies of the present invention. Similarly, hYP9.1b (also a humanized antibody) showed a worse IPAD score compared to the antibody of the present invention, and was in a similar range to hYP7.

[0352] Epibase Computer Simulation Immunogenicity Platform (Lonza) Epibase Computer Simulation (IS) is Lonza's T-cell epitope screening platform that analyzes and predicts the potential immunogenicity of proteins.

[0353] Immunoprofiling of the TPP-14890 and TPP-29537 sequences was performed using the “HLA class II – Global v4.0” setting on the Epibase platform, and the content of putative HLA class II restriction epitopes of the antibodies was compared. At the allotype level, profiling was performed on 43 DRB1 receptors, 8 DRB3 / 4 / 5 receptors, 22 DQ receptors, and 12 DP receptors, for a total of 86 HLA class II receptors. The DRB1 score (the sum of the products of key epitope counts and allele frequencies of affected allotypes) is shown in Table 9.

[0354] Table 9: DRB1 risk score for tested antibodies in the global population. TPP-29537 has a lower number of key DRB1 epitopes (25 epitopes), while TPP-14890 has a higher number (30 epitopes). Similarly, for the DRB3 / 4 / 5 family, TPP-29537 has the lowest epitope count (10 epitopes), while TPP-14890 has a higher count of 12 epitopes. Neither antibody, TPP-14890 nor TPP-29537, has any key DQ epitopes. For the DP family, both TPP-29537 and TPP-14890 have 3 DP epitopes.

[0355] As can be seen from Table 9, TPP-29537 has a low DRB1 risk score of only 785.9, while TPP-14890 has a DRB1 risk score of 924.7.

[0356] In summary, TPP-29537 has a DRB1 risk score comparable to that of fully human therapeutic antibodies, with epitope counts at the upper limit for human antibodies, while TPP-14890 has a DRB1 risk score comparable to that of humanized therapeutic antibodies, with epitope counts at the lower limit for humanized antibodies. Therefore, considering the overall key epitope counts and the population frequency of affected HLA allotypes, TPP-14890 poses a greater immunogenic risk than TPP-29537 in human therapeutic applications.

[0357] In summary, both independently used computer simulations using iPad and Epibase to elicit immunogenic responses in human treatments indicate that TPP-29537 is less likely to be immunogenic than TPP-14890. Therefore, from a patient safety perspective, TPP-29537 is more advantageous than TPP-14890.

[0358] Example 7: In vitro immunogenicity assessment To further confirm the computer-simulated immunogenicity predictions, the potential for inducing an immunogenic response in human treatment was assessed using in vitro immunogenicity assessment methods. For this purpose, antibody-chelating conjugates of TPP-14890 and TPP-29537 were prepared using the chelating agent Macropa-NCS (as described in Example 10), resulting in conjugates TPP-29537-Macropa and TPP-14890-Macropa, which were then subjected to the Epibase in vitro PBMC proliferation assay (Lonza).

[0359] This assay utilized Lonza's in vitro T-cell assay platform, assessing T-cell proliferation using human peripheral blood mononuclear cells (PBMCs) from healthy donors. T-cell responses induced by the test molecule were evaluated in PBMCs from 31 healthy human donors whose HLA-DRB1 frequencies were similar to those in the global population. The PBMC assay employed flow cytometry to detect the number of proliferating cells following stimulation by the test molecule.

[0360] PBMCs were seeded in 96-well plates. The test protein, diluted in assay medium, was added to the cells. Assay medium alone was used as a blank, and KLH (keyhole hemocyanin) was used as an initial positive control. PBMCs were incubated at 5% CO2 and 37°C for 7 days. On day 7, PBMCs were labeled with CD3 and CD4 markers, and DNA-incorporated 5-ethynyl-2'-deoxyuridine (EdU) was used as a proliferation marker.

[0361] KLH was used as a positive control and is a potent driver of CD4+ T cell proliferation, indicating that the assay was performed as expected: 31 out of 31 donors (100%) showed a significant T cell response to KLH (SI ≥ 2.00 and p < 0.05) compared to the blank condition (mean SI 28.94, p < 0.0001). The results are shown in Table 10.

[0362] Table 10: Donor response under each test condition compared to blank.

[0363] SI = Stimulation Index. The p-value relates to the single-sample t-test hypothesis, which states that the SI frequency distribution for a given antigen represents a distribution with a mean SI value of 1. Consistent with computer simulation predictions, even when testing two different batches, the number of donors responding to the TPP-14890 antibody chelate conjugate (5 donors, equivalent to 16%) was greater than the number responding to the TPP-29537 antibody chelate conjugate (2 donors, equivalent to 6%).

[0364] In summary, the TPP-14890 antibody chelate conjugate elicited a significant CD4+ T cell response in 16% of donors, indicating a moderate risk of immunogenicity. In contrast, the TPP-29537 antibody chelate conjugate only elicited a response in 6% of donors. Therefore, the TPP-29537 antibody chelate conjugate can be assessed as having only a low risk of immunogenicity (<10% donor response) for human treatment. Thus, from a patient safety perspective, TPP-29537 is more advantageous than TPP-14890.

[0365] Example 8: Internalization of anti-GPC3 antibody into HepG2 cells The following sections describe the characterization of different anti-GPC3 antibodies regarding their internalization profile.

[0366] Internalization is the process by which antibodies are taken up and enter tumor cells after binding to their specific tumor targets. Internalization is a necessary prerequisite for the effectiveness of antibody-drug conjugates (ADCs) because the toxins bound to the antibody are released only within the cell and exert their cytotoxic effects. Internalization is not a prerequisite for the effectiveness of tumor-associated drugs (TACs) because, even without internalization, radioactive radiation can kill target cells after TACs bind to their receptors. However, internalization can also be beneficial in this context, as it can help improve tumor retention.

[0367] Internalization of anti-GPC3 antibody directly labeled with CypHer5E In the first step, antibodies TPP-29537 and TPP-14890 were directly labeled at a concentration of 1 mg / mL with the pH-sensitive dye CypHer5E-NHS ester (Cytiva, PA 15401), which was conjugated to the lysine residues of the antibodies according to the manufacturer’s instructions.

[0368] After labeling, the estimated dye-to-protein ratio for TPP-29537 was 21.5, and for TPP-14890 it was 16.4.

[0369] HepG2 cells (20,000 cells / well) were seeded in Greiner µCLEAR 96-well microplates (Greiner, #655090) in 100 µL EMEM medium (ATCC, 30-2003) containing 10% FCS. After overnight incubation at 37°C and 5% CO2, a staining solution containing 10 µg Hoechst 33342 (Invitrogen, #H3570) in 10 µL of medium was added to each well, and the cells were incubated in the dark for 10 minutes. The medium was then removed and replaced with 90 µL of fresh medium and the corresponding target antibody (10 µL per well, final concentration 2 µg / mL). The cells were then incubated at 37°C and 5% CO2. Images were taken using an ImageXpress (Molecular Devices) high-content analysis system at 0.15, 1, 3, 5, and 24 hours. Images were analyzed using the Transfluor module in MetaXpress software (Molecular Devices) with settings "Vesicles 5_10_500" and "Nuclear stain 6_32_100". The vesicle area for each cell was calculated for each individual antibody, and the values ​​obtained at 5 hours are shown below. Figure 5 .

[0370] Internalization of Fabfluor-labeled antibodies HepG2 cells (20,000 cells / well) were seeded in Corning BioCoat µClear 96-well MTP (356640) plates in 100 µL EMEM medium (ATCC, 30-2003) containing 10% FCS and incubated overnight at 37°C and 5% CO2. The next day, 5 µL of Incucyte® Human Fabfluor-pH Antibody Labeling Reagent (Satorius, #4722) (a dye-labeled Fab fragment that binds to Fc-containing proteins) at a concentration of 10 µg / mL was mixed with 5 µL of each test antibody solution at a concentration of 20 µg / mL at room temperature for 20 minutes. Then, 90 µL of EMEM medium was added, and the mixture was incubated for an additional 5 minutes on a shaker. Additionally, a staining solution containing 10 µg Hoechst33342 (Invitrogen, #H3570) in 10 µL of medium was added to each well of the cell-containing plate, and the plates were incubated in the dark for 10 minutes. Next, the culture medium in the tissue culture plate was removed and replaced with 90 µL of antibody-Fabfluor mixture. Cells were incubated in an incubator at 37°C and 5% CO2. Images were taken using the ImageXpress (Molecular Devices) high-content analysis system at 0.15, 1, 3, 5, and 24 hours. Images were analyzed using the Transfluor module (set to "Vesicles 5_10_500" and "Nuclear stain 6_32_100") in MetaXpress (Molecular Devices) software. The vesicle area for each cell was calculated for each individual antibody, and the values ​​obtained at 5 hours are shown below. Figure 6 .

[0371] It is evident that when antibodies are directly labeled using CypHer5E via covalent labeling of lysine residues, TPP-14890 exhibits lower internalization compared to TPP-29537 (see [link to article]). Figure 5 In contrast, when using Fabfluor non-covalent labeling, both anti-GPC3 antibodies showed comparable internalization rates (see [link to relevant documentation]). Figure 6These differences in internalization rates can be explained by the fact that direct labeling with lysine-conjugated chemistry also results in labeling one or more of the four lysine residues in the CDR of the prior art antibody TPP-14890, thus affecting binding and resulting in a lower internalization rate. Since the antibody TPP-29537 of the present invention does not contain lysine residues in its CDR, binding and internalization rates are unaffected. In the case of Fabfluor labeling, both anti-GPC3 antibodies exhibit comparable internalization rates because the lysine labeling step is not used in this experimental design. This example demonstrates that the antibody of the present invention is more suitable for lysine-based chemical conjugation applications, such as ADCs or targeted actinide conjugates, compared to prior art antibodies (such as antibody TPP-14890) that contain lysine residues in their CDR.

[0372] Example 9: GPC3 antibody binds to the cell surface of different cell lines Cell surface binding of TPP-14890, TPP-29537, and the isotype control TPP-754 was investigated using different cell cultures by flow cytometry. Cell lines SU-DHL-1, HFF-1, HepG2, Hep3B2, and PLC / PRF / 5 were cultured in RPMI + 10% FCS, DMEM + 15% FCS, RPMI + 10% FCS, MEM + 10% FCS, and MEM + 10% FCS, respectively. Primary human cardiac fibroblasts (HCF) and primary human lung fibroblasts (HPF) were purchased from Promocell and cultured according to the manufacturer's instructions. Cells were detached using a non-enzymatic dissociation solution (Gibco #13151-014) for surface staining. Cells were washed twice with ice-cold FACS buffer (PBS - / - containing 3% FCS, from Sigma #F2442). All subsequent steps were performed on ice to prevent antibody internalization. 100 µL of 200,000 cells were transferred to each well of a V-shaped 96-well plate. Cells were stained with Alexa Fluor 488-labeled TPP-14890, Alexa Fluor 488-labeled anti-GPC3 antibody TPP-29537, or FITC-labeled isotype control antibody TPP-754. Antibodies were directly labeled with Alexa Fluor 488 (A10235 from Invitrogen), conjugated to the primary amino group present on the lysine residue. Labeling degree was determined according to the manufacturer's instructions. The labeling degree (DOL) for TPP-14890 was 6.07; for TPP-29537, DOL = 7.22; and for TPP-754, DOL = 3.4. All antibodies were diluted to a final concentration of 10 µg / mL in FACS buffer. Staining was performed by shaking on a Titramax 100 at 4°C in the dark for 30 minutes. Cells were washed with PBS and centrifuged at 800g for 2 minutes at 4°C. The supernatant was then carefully removed. Cells were resuspended in FACS buffer containing 1:1000 Sytot Blue (Invitrogen S34857). Cell measurements were performed using a BD Biosciences FACSCanto™ II Flow Cytometry System and analyzed using FlowJo_v10.6.1 software (BDBiosciences). Live single cells were gated using forward scattering (FSC), side scattering (SCC), and Pacific Blue gating. MESF beads (Quantum 488C, lot number: 14506) were measured along with the cells. The measured fluorescence signals were converted to ABC values ​​(antibody binding capacity) according to the manufacturer's instructions using the MESF beads.After calculating the ABC value, the ABC value of the isotype control was subtracted from the value of the target antibody. The results were plotted as ABC (antibody binding capacity on cell surface), see below. Figure 7 , Figure 8 See Table 11.

[0373] Table 11: ABC values ​​of various cell lines determined using TPP-14890 or TPP-29537 Clearly, in the cases of HepG2 and Hep3B2, the Alexa Fluor 488-labeled TPP-29537 showed an ABC value approximately 20 times higher than that of the Alexa Fluor 488-labeled TPP-14890. For PLC / PRF / 5, the Alexa Fluor 488-labeled TPP-29537 showed an ABC value of 14495.1, while the Alexa Fluor 488-labeled TPP-14890 failed to detect any GPC3 on this cell line with low GPC3 content on the cell surface. For cell lines with ABC=0, the calculated ABC value of the GPC3 antibody was lower than that of the isotype control antibody in the corresponding cell line. An ABC value of 0 indicates no or very low GPC3 protein expression. These differences in ABC values ​​can be explained by the fact that direct labeling of Alexa Fluor 488 using lysine conjugation chemistry also results in labeling one or more of the four lysine residues in the CDR of the prior art antibody TPP-14890, thus affecting binding to GPC3. Since the anti-GPC3 antibody TPP-29537 of the present invention does not contain lysine residues in its CDR, the binding and the resulting ABC value are unaffected.

[0374] Example 10: Preparation of Targeted Actinide Conjugate (TAC) Conjugation of Macropa with GPC3 antibody (ACC) (6-[[16-[(6-carboxy-2-pyridinyl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl]methyl]-4-[2-(4-isothiocyanate-phenyl)ethoxy]pyridine-2-carboxylic acid (Macropa-NCS; hereinafter abbreviated as "Macropa") was dissolved in DMA to a concentration of 10 mg / mL. Anti-GPC3 antibodies TPP-29537 and TPP-14890 were dissolved in PBS to a concentration of 10 mg / mL, and the pH was adjusted to 9 with 1M carbonate buffer. The chelating agent and antibody were mixed and incubated at 350 rpm for two hours at room temperature on a temperature-controlled shaker. The molar ratio of chelating agent to antibody was selected according to the desired chelating agent to antibody ratio (CAR). The product was purified by FPLC (column: HiLoad 16 / 600 Superdex 200 pg column; flow rate: 1 mL / min; at 0.1... In M acetate buffer (pH 5); detection: UV 280 / 254 nm). Concentration and monomer purity were determined by SEC-UV (as described below and shown in Table 12). CAR was determined by SEC-MS using positive electrospray ionization (as described below) and the following formula: CAR = [(A*An) / A]0, where A = the intensity of the maximum entropy envelope of all signals, and An = the signal of the maximum entropy envelope containing the chelating agent. CARs for different ACCs were determined and are shown in Table 12.

[0375] Table 12: Chelating agent to antibody ratio (CAR), concentration, and monomer purity of different antibody chelating agent conjugates ACC characterization was performed using size exclusion chromatography and mass spectrometry. The CAR of ACC was determined by the following method: using SEC-MS (Water Acquity HPLC coupled with Waters XEVOTOF; run buffer: 50 / 50 / 0.1 (v / v / v) ACN / water / TFA; flow rate: 0.06 mL / min, online desalting column: WatersAcquity BEH SEC, 1.7 µm, 2.1 x 150 mm; ESI+ mode), the percentage of the MS peak height of each component (mAb, mAb + 1 chelator, mAb + 2 chelators, mAb + 3 chelators, etc.) to the main peak height was used, and the formula CAR = Sum(n*An) / Sum An was used, where n is equal to the number of chelators and An is equal to the strength of the antibody conjugate containing n chelators. The purity and concentration of ACC were determined by SEC-UV (Agilent 1260 Infinity HPLC system, run buffer: 10% DMSO / PBS; flow rate: 0.3 mL / min; column: Waters Acquity BEH SEC, 1.7 µm, 4.6 x 300 mm; detection: UV 280 nm). This method separates higher aggregates, dimers, and fragments from ACC monomers.

[0376] Radiolabeling of targeted actinium-225 conjugates (TAC) Actinium-225 (Ac-225) was added to 0.1 M acetate buffer (pH 5) containing TPP-14890-Macropa and TPP-29537-Macropa, with a specific activity of 2 MBq / mg and a radioactivity concentration of 1–2.5 kBq / µL. The mixture was incubated at room temperature for one hour, and then the radiochemical purity (RCP) was determined by iTLC.

[0377] Instantaneous thin-layer chromatography (iTLC): iTLC was performed to measure the radiochemical purity (RCP) of the Ac-225-Macropa-GPC3 conjugate solution. Briefly, an iTLC.SG. plate was cut to obtain an 11 cm x 1 cm band. The spotting point (1 cm), cut point (4 cm), and run endpoint (10 cm) were marked, and 2 µL of TAC was spotted onto the iTLC band. The band was developed in a 3 mL mobile phase vial in an LSC vial. Free Ac-225 migrated along the band with the solvent, while complexed Ac-225 remained at the spotting point. When the liquid front reached the marked endpoint, the band was removed from the vial and dried on aluminum foil. The band was cut in half at 4 cm: a shorter “spotting” half (A) and a longer “front” half (F). The portions were carefully folded and placed at the bottom of a scintillation vial. After 6 hours, Actinium-225 and its daughter products reached long-term equilibrium. The spotting portion was counted for 60 seconds and the leading edge portion for 120 seconds using a high-purity germanium detector (Ortec). For all samples listed in Table 12, the radiochemical purity (% RCP) (defined as [(spotting segment radioactivity) / (total radioactivity on the strip)] x 100) was 99.9%.

[0378] Example 11: Comparison of GPC3-TAC in Immunoreactivity Reactivity (IRF) Assay The binding of each radiolabeled sample to GPC3-coated M-270 carboxyl Dynabeads (Thermo Fisher Scientific) was determined by immunoreactivity fraction (IRF) assay. IRF was assessed by measuring the fraction of radiolabeled antibody bound to the magnetic antigen-coated beads compared to the fraction of unbound antibody remaining in the supernatant. Binding was evaluated directly after radiolabeling.

[0379] In short, magnetic beads were placed in 2 mL Eppendorf tubes at decreasing volumes of 400 to 0.1 µg and resuspended in 50 µL of buffer (PBS / 3% BSA). The radiolabeled compound was diluted with buffer to 5 Bq / µL and added to each tube containing beads (50 Bq total). The tubes were incubated at room temperature on an Eppendorf shaker at 750 rpm for 60–90 minutes. After the binding step was complete, 40 µL of buffer was added to each tube, and the beads were separated from the supernatant using a magnet (DynaMag-2). After at least 6 hours (to allow the progeny nuclides to reach long-term equilibrium), the radioactivity in the supernatant and beads of each sample was measured using a high-purity germanium detector (Ortec). Total binding was defined as [(activity in beads - activity in supernatant) / (total activity)] x 100. Nonspecific binding was obtained by the same calculation for the closed samples, and specific binding was defined as [total binding - nonspecific binding]. Results are shown in... Figure 9 And Table 13.

[0380] Table 13: CAR and EC of Ac-225-Macropa-TPP-29537 and Ac-225-Macropa-TPP-14890 50 (µg beads) and maximum %IRF For Ac-225-Macropa-TPP-29537, both low-CAR and high-CAR conjugates yielded comparable EC values. 50 And maximum % IRF, while for the Ac-225-Macropa-TPP-14890 conjugate with high CAR containing the existing anti-GPC3 antibody TPP-14890, EC compared to the low CAR version 50 The value decreased significantly, indicating impaired binding efficacy with human GPC3 peptide.

[0381] Example 12: In vitro cytotoxicity The in vitro cytotoxicity of Ac-225-Macropa-TPP-14890 (TPP-14890-TAC) CAR 0.7, Ac-225-macropa-TPP-14890 CAR 10, Ac-225-Macropa-TPP-29537 (TPP-29537-TAC) CAR 0.5, and Ac-225-Macropa-TPP-29537 CAR 11 was determined in human hepatocellular carcinoma (HCC) cell lines expressing different levels of GPC3. HepG2 and Hep3B2.1-7 cells were seeded at appropriate cell densities in MEM + 10% FCS cell culture medium. HepG2 cells showed higher GPC3 expression on their cell surface than Hep3B2.1-7 cells. One day after seeding, compounds with a specific activity of 2 MBq / mg and a radioactive concentration of 5 kBq / mL were titrated in parallel with a radiolabeled isotype control. Cells were incubated for 6 days, followed by Hoechst and PI staining to determine cell viability, and analysis was performed using the Operetta CLS high-content analysis system (Perkin Elmer). The obtained IC50 values... 50 The values ​​are shown in Table 14. Figure 10 A and Figure 10 B. Table 15 shows the isotype / target ratio for different TACs (a higher isotype / target ratio indicates higher specific cytotoxicity).

[0382] Table 14: IC50 of TPP-14890-TAC and TPP-29537-TAC for low CAR and high CAR 50 value Table 15.1: IC values ​​of TPP-14890-TAC and TPP-29537-TAC for low and high CAR levels 50 Ratio (isotype / target) Strong and specific cytotoxicity was observed in HepG2 and Hep3B2.1-7 cell lines. TPP-29537-TAC CAR 0.5 showed the highest specific cytotoxicity, exhibiting the highest IC50. 50 The ratio. The high-CAR TPP-29537 conjugate exhibited similar IC50 values ​​to the low-CAR TPP-14890-TAC. 50 TPP-14890-TAC CAR 10 exhibited significantly lower specific cytotoxicity, particularly in the Hep3B2.1-7 cell line, indicating lower cytotoxic efficiency compared to other tested compounds. This suggests that the impaired binding of high-CAR TPP-14890-TAC is attributed to the tagging of one or more of the four lysine residues in the CDR of the prior art antibody TPP-14890.

[0383] Example 13: Biodistribution study of GPC3-TAC The biodistribution of Ac-225-Macropa-TPP-29537 (CAR 0.7) and Ac-225-Macropa-isotype-control (CAR 0.7) was investigated in the Huh-7 (ABC: 22,000) human hepatocyte xenograft model with low GPC3 expression. 2 × 10⁻⁶ cells were used. 6 Huh-7 cells were seeded into the right flank of female RJ:NMRI-Fox1nu / nu mice. Animals were pretreated with 200 µg / mouse of nonspecific IgG2a antibody 24 hours prior to hot administration. Animals were treated with 500 kBq / kg Ac-225-macropa-TPP-29537 and Ac-225-Macropa-isotype-control, respectively, with a total protein dose of 0.75 mg / kg. Animals were sacrificed at 72, 168, 336, and 504 hours post-treatment, and tumors and organs from Ac-225-Macropa-TPP-29537 were isolated at each time point; the last time point (504 hours) was excluded for the Ac-225-Macropa-isotype-control due to reaching the humanitarian endpoint. Accumulated Ac-225 in blood, tumors, liver, kidneys, spleen, and femur was measured using a germanium detector HPGe (GEM-F8250-LB-C, Ortec). The results are shown in Figure 11 A and Figure 11 B.

[0384] Data from animals treated with Ac-225-Macropa-TPP-29537 showed specific, strong, and persistent tumor accumulation, while accumulation in normal organs was typically low and transient, and cleared over time.

[0385] Example 14: In vivo efficacy study comparing TPP-29537-TAC and homologous-TAC The in vivo efficacy of Ac-225-Macropa-TPP-29537 was evaluated in the human hepatocellular carcinoma model Huh-7. Mice of the Huh-7 human hepatocellular carcinoma model with low GPC3 expression were xenografted and administered a single intravenous (IV) dose of 500 kBq / kg, with a total antibody dose of 0.75 mg / kg, using Ac-225-Macropa-TPP-29537 (CAR 0.7) and Ac-225-Macropa-isotype-control (CAR 0.7). Animals were pretreated with 200 µg / mouse of nonspecific IgG2a antibody 24 hours prior to administration. Mice surviving on day 25 after the initial administration received a repeat dose of 250 kBq / kg and a total protein dose of 0.75 mg / kg.

[0386] As shown in Fehler! Verweisquelle konnte nicht gefunden werden., Ac-225-Macropa-TPP-29537 has antitumor activity and significantly inhibits the growth of Huh-7 tumors compared to the Ac-225-Macropa-isotype-control. Figure 12 A). The survival rate of mice in the Ac-225-Macropa-TPP-29537 group was also significantly better than that of mice in the solvent group and the Ac-225-Macropa-isotype-control group. No weight loss was observed in the treatment group.

[0387] Example 15: Preparation and characterization of Zr-89-DFO*-TPP-29537 for imaging To perform in vivo positron emission tomography (PET) imaging, the anti-GPC3 antibody TPP-29537 was conjugated with DFO* and radiolabeled with zirconium-89.

[0388] Conjugation and purification of DFO*-TPP-29537: 5,11,16,22-Tetraazahexacosanediamide, N1-[5-(acetylhydroxyamino)pentyl]-N26,5,16-trihydroxy-N26-[5-[[[(4-isothiocyanate-phenyl)amino]thiomethyl]amino]pentyl]-4,12,15,23-tetraoxo-(p-Phe-NCS-DFO* (DFO*), ABX advanced chemical compounds, 7272) was dissolved in DMSO to a final concentration of 10 mg / mL. Anti-GPC3 antibody TPP-29537 was dissolved in WFI to a final concentration of 10 mg / mL. The pH of the antibody was adjusted to 9 using 1M carbonate buffer. p-Phe-NCS-DFO* was added to the antibody, and the mixture was incubated at 37°C for 1 hour with shaking on a constant temperature shaker. The conjugate was purified using a PD-10 column according to the manufacturer's instructions (Cytiva, 17085101). The monomer purity of the DFO*-TPP-29537 conjugate was measured by SEC-UV. CAR was determined by SEC-MS using positive electrospray ionization and the following formula: CAR = [(A*An) / A]0, where A = the intensity of the maximum entropy envelope of all signals, and An = the signal of the maximum entropy envelope containing the chelating agent. The monomer purity and CAR were determined to be 99.2% and 1.2%, respectively.

[0389] Radiolabeling of DFO*-TPP-29537 and zirconium-89 1M oxalic acid containing zirconium-89 was added to 1M HEPES, pH 7.3 DFO*-TPP-29537 for labeling, with a specific activity of 34 MBq / mg and a RAC of 103 MBq / mL. After incubation at 37°C for 30 minutes, the sample was purified using a PD-10 column according to the manufacturer's instructions (Cytiva, 17085101), and the buffer was replaced with formulation buffer (10 mM L-histidine, 130 mM glycine, 5% (m / v) sucrose aqueous solution for injection) and diluted to a RAC of 24 MBq / mL.

[0390] Immunoreactivity score assay Immunoreactivity fraction (IRF) assays were used to evaluate the binding properties of the radiolabeled immunoreactive conjugate monoclonal antibody (mAb) product Zr-89-DFO*-TPP-29537. Shortly after radiolabeling, the binding of Zr-89-DFO*-TPP-29537 to GPC3 peptide-coated M-270 carboxyl Dynabeads was measured. Briefly, 0.36–800 µg beads were placed in 90 µL of buffer (PBS / 3% BSA) in 2 mL Eppendorf tubes. The radiolabeled compound was diluted to 5 Bq / µL with buffer, and 10 µL was added to each tube containing beads. Samples were tripled. Binding was performed for 60 minutes at room temperature while the tubes were shaken at 750 rpm on an Eppendorf shaker. After the binding step was complete, the beads were separated from half of the supernatant using a magnet (DynaMag-2). The radioactivity in the supernatant and beads of each sample was determined using a high-purity germanium detector (Ortec). Total binding was defined as [(activity in beads - activity in supernatant) / (total activity)] × 100. Figure 13 The binding efficacy of radiolabeled Zr-89-DFO*-TPP-29537 to benzyl-coupled GPC3 peptides is shown.

[0391] The number of coated beads selected to provide an excess of epitope antigen for binding was achieved by coating beads with 50–100 µg of GPC3 peptide. In summary, the IRF% measured in single-point assays was approximately 90%.

[0392] Example 16: Biodistribution of Ac-225-Macropa-TPP-29537 and its imaging pair Zr-89-DFO*-TPP-29537 The ex vivo biodistribution of Ac-225-Macropa-TPP-29537 (CAR 0.7) and its imaging pair Zr-89-DFO*-TPP-29537 (CAR 5.3) was evaluated in the HEP-3B HCC tumor model. Furthermore, in vivo PET imaging quantification of Zr-89-DFO*-TPP-29537 was performed to investigate its potential as an imaging pair for early development of Ac-225-Macropa-TPP-29537.

[0393] 4.1×10 6 One HEP-3B cell was seeded into the right flank of a female RJ:NMRI-Fox1nu / nu mouse. The animals were pretreated with 200 µg / mouse of nonspecific IgG2a antibody 24 hours prior to hot administration.

[0394] As described in Table 16, Zr-89-DFO*-TPP-29537 was injected into the tail vein of the animals via a slow bolus injection. PET / CT imaging was performed at 2 h (n=1), 4 h (n=3), 24 h (n=3), 72 h (n=3), and 168 h (n=2) post-injection. Imaging was acquired using a MILabsVector6 PET / SPECT / CT scanner with a HE-UHR-RM collimator, 4 mouse beds, a total acquisition time of 60 minutes, spiral acquisition mode, and a fixed field of view established during the initial acquisition.

[0395] Table 16: Overview of each group in the in vivo and in vitro biodistribution studies of Zr-89-DFO*-TPP-29537 and the in vitro biodistribution study of Ac-225-Macropa-TPP-29537. Administer the medication in batches to avoid having more than one mouse in the scanner at the same time.

[0396] To assess in vivo biodistribution, image analysis was performed using PMOD software. CT volumes of interest (spherical VOIs) were applied to the heart, liver, and kidneys. Tumor outlines were delineated on CT images, and restrictive VOIs (10% threshold) were applied to PET images. Data were attenuated to the injection time point. Figure 14 Coronal PET / CT MIP images of mice carrying HEP-3B tumors, imaged with Zr-89-DFO*-TPP-29537 at different time points.

[0397] After in vivo imaging, to assess the in vitro biodistribution of Zr-89-DFO*-TPP-29537, imaging mice were sacrificed (3 mice at each time point), and blood, tumors, liver, kidneys, spleen, femurs, and tibias were collected. Zr-89 activity was then determined using a Perkin Elmer Wizard II gamma counter.

[0398] Simultaneously, mice carrying HEP-3B tumors were intravenously (IV) injected with 250 kBq / kg Ac-225-Macropa-TPP-29537, with a protein dose of 0.75 mg / kg. Animals were sacrificed at 0.5, 2, 4, 24, 72, and 168 hours post-treatment. Accumulated Ac-225 levels in blood, tumors, liver, kidneys, spleen, and femur were measured using a germanium detector HPGe (GEM-F8250-LB-C, Ortec).

[0399] like Figure 15As shown, Ac-225-Macropa-TPP-29537 and Zr-89-DFO*-TPP-29537 exhibited comparable in vitro biodistribution and similar hematogenous clearance up to the last time point of the study (168 hours). Tumor accumulation of Ac-225-Macropa-TPP-29537 and Zr-89-DFO*-TPP-29537 was similar from pi up to 72 hours post-injection (pi), after which Ac-225-Macropa-TPP-29537 showed a higher %ID / g, most likely due to tumor shrinkage resulting from the therapeutic effect of Ac-225-Macropa-TPP-29537.

[0400] In vitro biodistribution and in vivo PET imaging quantification of Zr-89-DFO*-TPP-29537 indicated comparable uptake in the heart / blood, kidneys, liver, and tumors. Figure 15 ).

[0401] In summary, these results demonstrate the suitability of Zr-89-DFO*-TPP-29537 as a PET imaging pairing with Ac-225-Macropa-TPP-29537 for early development.

[0402] sequence

Claims

1. An isolated anti-GPC3 antibody or an antigen-binding fragment capable of binding to GPC3, comprising: i) Containing a heavy chain antigen-binding region comprising H-CDR1 of SEQ ID NO: 102, H-CDR2 of SEQ ID NO: 103, and H-CDR3 of SEQ ID NO: 104, and a light chain antigen-binding region comprising L-CDR1 of SEQ ID NO: 106, L-CDR2 of SEQ ID NO: 107, and L-CDR3 of SEQ ID NO: 108; or ii) Contains a heavy chain antigen-binding region comprising H-CDR1 of SEQ ID NO: 122, H-CDR2 of SEQ ID NO: 123, and H-CDR3 of SEQ ID NO: 124, and a light chain antigen-binding region comprising L-CDR1 of SEQ ID NO: 126, L-CDR2 of SEQ ID NO: 127, and L-CDR3 of SEQ ID NO: 128; or iii) Contains a heavy chain antigen-binding region comprising H-CDR1 (SEQ ID NO: 142), H-CDR2 (SEQ ID NO: 143), and H-CDR3 (SEQ ID NO: 144), and a light chain antigen-binding region comprising L-CDR1 (SEQ ID NO: 146), L-CDR2 (SEQ ID NO: 147), and L-CDR3 (SEQ ID NO: 148); or iv) Contains a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 162, H-CDR2 of SEQ ID NO: 163, and H-CDR3 of SEQ ID NO: 164, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 166, L-CDR2 of SEQ ID NO: 167, and L-CDR3 of SEQ ID NO:

168.

2. The isolated antibody or antigen-binding fragment according to claim 1, comprising: i) Containing a variable heavy chain domain of SEQ ID NO: 101 and a variable light chain domain of SEQ ID NO: 105; or ii) Containing a variable heavy chain domain of SEQ ID NO: 121 and a variable light chain domain of SEQ ID NO: 125; or iii) Containing a variable heavy chain domain of SEQ ID NO: 141 and a variable light chain domain of SEQ ID NO: 145; or iv) Contains a variable heavy chain domain of SEQ ID NO: 161 and a variable light chain domain of SEQ ID NO:

165.

3. The isolated antibody or antigen-binding fragment according to any one of the preceding claims, wherein the monoclonal antibody is an IgG antibody.

4. The isolated antibody or antigen-binding fragment according to any one of the preceding claims, wherein the monoclonal antibody is an IgG1 antibody.

5. The isolated antibody or antigen-binding fragment according to any one of the preceding claims, comprising: i) The heavy chain containing SEQ ID NO: 117 and the light chain containing SEQ ID NO: 118; or ii) a heavy chain containing SEQ ID NO: 137 and a light chain containing SEQ ID NO: 138; or iii) A heavy chain containing SEQ ID NO: 157 and a light chain containing SEQ ID NO: 158; or iv) The heavy chain containing SEQ ID NO: 177 and the light chain containing SEQ ID NO:

178.

6. An antibody conjugate comprising a monoclonal antibody or antigen-binding fragment according to any one of claims 1-5.

7. The antibody conjugate according to claim 6, comprising a radionuclide, a cytotoxic agent, an organic compound, a protein toxin, an immunomodulator, other antibodies or antigen-binding fragments, a fluorescent portion, or a chimeric antigen receptor.

8. The antibody conjugate according to any one of claims 6 or 7, wherein the antibody or its antigen-binding fragment binds to the remainder of the molecule via the terminal amino group of a lysine residue of the monoclonal antibody or its antigen-binding fragment.

9. A targeted actinium conjugate (TAC) of formula (I) Wherein [Ab] is a monoclonal antibody or antigen-binding fragment thereof capable of binding GPC3 according to any one of claims 1-5.

10. The targeted actinide conjugate of claim 9, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of a lysine residue of the monoclonal antibody or its antigen-binding fragment.

11. A targeted actinium conjugate (TAC) of formula (I) [Ab] is a monoclonal antibody or its antigen-binding fragment capable of binding GPC3, comprising: a heavy chain antigen-binding region containing H-CDR1 of SEQ ID NO: 102, H-CDR2 of SEQ ID NO: 103, and H-CDR3 of SEQ ID NO: 104, and a light chain antigen-binding region containing L-CDR1 of SEQ ID NO: 106, L-CDR2 of SEQ ID NO: 107, and L-CDR3 of SEQ ID NO: 108, wherein [Ab] binds to the remainder of the molecule via the terminal amino group of a lysine residue.

12. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment according to any one of claims 1-5, or an antibody conjugate according to claims 6-8, or a targeted actinide conjugate according to any one of claims 9-11, and optionally one or more pharmaceutically acceptable excipients.

13. An antibody or antigen-binding fragment thereof according to any one of claims 1-5, or an antibody conjugate according to any one of claims 6-8, or a targeted actinide conjugate according to any one of claims 9-11, or a pharmaceutical composition according to claim 12, for the treatment of a disease.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the antibody conjugate according to any one of claims 6-8, or the targeted actinide conjugate according to any one of claims 9-11, or the pharmaceutical composition according to claim 12, wherein the disease is characterized by ectopic expression of GPC3.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the antibody conjugate according to any one of claims 6-8, or the targeted conjugate according to any one of claims 9-11, or the pharmaceutical composition according to claim 12, wherein the disease is cancer, preferably hepatocellular carcinoma.

16. The isolated antibody or antigen-binding fragment according to any one of claims 1-5, or the conjugate according to any one of claims 6-8, or the targeted actinide conjugate according to any one of claims 9-11, or the pharmaceutical composition according to claim 12, for use simultaneously, separately, or sequentially in combination with one or more other therapeutically active compounds.

17. The isolated antibody or its antigen-binding fragment according to any one of claims 1-5, or the antibody conjugate according to any one of claims 6-8, or the targeted actinide conjugate according to any one of claims 9-11, used as a diagnostic agent or imaging agent.

18. An isolated nucleic acid sequence encoding an antibody or antigen-binding fragment according to any one of claims 1-5.

19. A vector comprising the nucleic acid sequence according to claim 18.

20. An isolated cell for preparing an antibody or antigen-binding fragment according to any one of claims 1-5.

21. A method for preparing an antibody or antigen-binding fragment according to any one of claims 1-5, an antibody conjugate according to any one of claims 6-8, or a targeted actinide conjugate according to any one of claims 9-11, comprising culturing the cells according to claim 20 and optionally purifying the antibody or antigen-binding fragment.

22. A kit comprising an isolated antibody or antigen-binding fragment according to any one of claims 1-5, or an antibody conjugate according to any one of claims 6-8, or a targeted actinide conjugate according to any one of claims 9-11, or a pharmaceutical composition according to claim 12, and instructions for use.

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