Anti-GPC3 antibodies and radioconjugates thereof

Abstract

The present disclosure relates to novel antibodies binding to GPC3 or antigen-binding fragments thereof. These anti-GPC3 antibodies or fragments thereof preferably don't comprise lysine residues within their CDRs and are therefore especially suitable for conjugation. The present disclosure further relates to targeted radionuclide conjugates such as targeted actinium-225 conjugates (TACs). Preferred are TACs of formula (I) comprising the anti-GPC3 antibodies according to the present disclosure or fragments thereof. Said antibodies or antigen binding fragments thereof, and / or said antibody conjugates and / or said TACs are useful in diagnostics and / or therapy, preferably in therapy, more preferably in treating cancer, most preferably in treating hepatocellular carcinoma (HCC). Provided are pharmaceutical compositions and kits with instructions for use comprising these antibodies, functional fragments and conjugates. The disclosure further provides methods and tools to generate these antibodies, functional fragments and conjugates.

Description

[0001] ANTI-GPC3 ANTIBODIES AND RADIOCONJUGATES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel monoclonal antibodies capable of binding to glypican-3 (GPC3) or antigen-binding fragments thereof and conjugates comprising these anti-GPC3 antibodies or fragments thereof. The present invention further relates to targeted radionuclide conjugates such as targeted actinium-225 (Ac-225) conjugates (TACs). Preferred are targeted actinium conjugates of formula (I) comprising the anti-GPC3 antibodies according to the present invention or fragments thereof. Said antibodies or antigen binding fragments thereof, and / or said antibody conjugates and / or said targeted actinium conjugates are useful in diagnostics and / or therapy, preferably in therapy, more preferably in treating hyperproliferative diseases such as cancer, most preferably in treating hepatocellular carcinoma (HCC).

[0004] Provided are pharmaceutical compositions and kits with instructions for use comprising the antibodies, functional fragments and conjugates according to the current invention.

[0005] The invention further provides methods and tools to generate the antibodies, functional fragments and conjugates according to the invention. For example, provided are polynucleotides encoding the foregoing antibodies or fragments, vectors containing the same, and cells for production.

[0006] BACKGROUND OF THE INVENTION

[0007] Glypican-3 (GPC3) is an oncofetal protein expressed in various tumors, for example hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), gastric carcinoma, ovarian carcinoma, melanomas, and pediatric embryonal tumors. GPC3 expression is particularly high in HCC with limited expression in adults and it is not expressed in viral hepatitis, cirrhosis or NASH, making HCC a highly attractive target for GPC3 based therapy. This is very important taking into account that primary liver cancer is the sixth most common malignant neoplasm and the second most common cause of cancer-related death worldwide (Torre et al, Global cancer statistics, 2012. CA Cancer J Clin. 2015 Mar;65(2):87-108).

[0008] Thus far some anti-GPC3 antibodies are already described, e.g. hYP7, hYP9.1 b (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 Acad Sci U S A. 2013 Mar 19;110(12), fully human antibodies 2-F7, 4-11 G, 7-5B, 7-8B, 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 227Th a-Therapy Reduces Tumor Burden in an Orthotopic Xenograft Murine Model of Hepatocellular Carcinoma. J Nucl Med. 2022 Ju I ;63(7) : 1033-1038). However, none of these anti-GPC3 antibodies has entered clinical phase so far.

[0009] The only clinically validated GPC3-targeting antibody is codrituzumab, a humanized version of the murine monoclonal antibody GC33 that specifically binds 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 Res Commun. 2009 Jan 9;378(2):279-84). Codrituzumab induces antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cell cytotoxicity (CDC) and inhibits tumor growth. Codrituzumab was well tolerated in phase I trials and showed antitumor effects in HCC patients. However, in a randomized phase II study, codrituzumab showed no 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 follow up clinical studies showed that codrituzumab showed prognostic merit for HCC patients with a high sensitivity related to ADCC and a high expression of GPC3 (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 clinical phase I study is recruiting testing codrituzumab in children and young adults with solid tumors (Clinical Trials.gov identifier: NCT04928677).

[0010] However, as ADCC / CDC activity of GPC3-targeting antibodies seems not to be sufficient for an effective treatment of diseases wherein expression of GPC3 plays a role, there is a need for novel therapeutics specifically targeting GPC3. GPC3 is an oncofetal protein mainly expressed in specific cancer types but not or only very minor in normal tissue which makes it a suitable target for targeted radionuclide therapy. Targeted radionuclide therapy is a promising and developing area with the potential to deliver highly cytotoxic radiation specifically to cell types associated with disease. The most common forms of radiopharmaceuticals currently authorized for use in humans employ beta-emitting and / or gamma-emitting radionuclides. However, due to their potential for more potent cell killing the use of alpha-emitting radionuclides is of increasing interest, especially for cancer therapy.

[0011] The unique mode-of-action of targeted alpha therapy (TAT) is the induction of difficult-to-repair clustered DNA double strand breaks through specific delivery of alpha-particle emitting radionuclides, like actinium-225, to tumors using tumor antigen targeting moieties such as small molecules, peptides or monoclonal antibodies. The exceptional cytotoxicity of alpha emitting radionuclides demands specific biological targeting antibodies in order to avoid unacceptable side effects. Biological targeting of the isotopes strongly depends on the stability of the chelator, the robustness of the antibody to the chelation process, and the targeting properties of the antibody to its particular target in the specific biological context.

[0012] The radiation range of typical alpha emitters in physiological surroundings is generally less than 100 micrometers, the equivalent of only a few cell diameters. This makes these sources well suited for the treatment of tumors, including micro-metastases, because they have the range to reach neighboring cells within a tumor but if they are well targeted then little of the radiated energy will pass beyond the target cells. Thus, not every cell needs to be targeted and damage to surrounding healthy tissue may be minimized at the same time (see Feinendegen et al., Radiat. Res.148:195-201 (1997)). In contrast, a beta particle has a range of 1 mm or more in water (see Wilbur D.S., Antibody Immunoconj Radiopharm. 4: 85-97 (1991 )).

[0013] The energy of alpha-particle radiation is high in comparison with that carried by beta particles, gamma rays and X-rays, typically being 5-8 MeV, or 5 to 10 times that of a beta particle and 20 or more times the energy of a gamma ray. Thus, this deposition of a large amount of energy over a very short distance gives alpha-radiation an exceptionally high linear energy transfer (LET), high relative biological efficacy (RBE) and low oxygen enhancement ratio (OER) compared to gamma and beta radiation (see Hall, "Radiobiology for the radiologist", Fifth edition, Lippincott Williams & Wilkins, Philadelphia PA, USA, 2000). This explains the exceptional cytotoxicity of alpha emitting radionuclides and also imposes stringent demands on the biological targeting of such isotopes and upon the level of control and study of alpha emitting radionuclide distribution which is necessary in order to avoid unacceptable side effects. So far, with regards to the application of alpha-emitting nuclides in radioimmunotherapy the main attention has been focused on211At,213Bi and225Ac and these three nuclides have been explored in clinical immunotherapy trials. However, the compounds currently in use, which typically employ the macrocyclic chelator DOTA (2,2',2",2'"-(1 ,4,7,10-Tetraazacyclododecane- 1 ,4,7,10-tetrayl)tetraacetic acid) are lacking in stability, leading to the dissociation of the radionuclide from the chelating macrocycle, which results in a reduced selectivity and activity to the targeted tissue and an increased toxicity to the non-targeted tissue. 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 a-Melanocyte- Stimulating Hormone Derivative for Targeted Alpha Therapy Using ISOL-Produced 225Ac. EJNMMI Radiopharm. Chem. 2019, 4, 21 ) leading to deposition of 225Ac in the liver and femur after the administration of DOTA-based conjugates. (Deal et al., Improved in Vivo Stability of Actinium-225 Macrocyclic Complexes. J. Med. Chem. 1999, 42, 2988-2992.)

[0014] Further effort was made for the identification and development of suitable chelating agents for the large Ac3+ion (Thiele et al., Actinium-225 for Targeted a Therapy: Coordination Chemistry and Current Chelation Approaches. Cancer Biother. Radiopharm. 2018, 33, 336-348). Acyclic chelators like ethylenediamine tetraacetic acid (H4EDTA) and diethylenetriamine pentaacetic acid (H5DTPA) are suffering of lability of the resulting complexes (Davis et al., Comparison of 225Actinium Chelates: Tissue Distribution and Radiotoxicity. Nucl. Med. Biol. 1999, 26, 581-589). Macrocyclic chelators as tetraazacy-clododecane-1 ,4,7,10-tetraacetic acid (H4DOTA) have shown to be more inert. However, radiolabeling of H4DOTA with 225Ac either requires high temperatures, which is incompatible with temperature-sensitive macro-molecular biomolecules such as antibodies, or long incubation times, which yields constructs with relatively low specific activities and low purity.

[0015] So far, only a few examples of GPC3-targeting radionuclide conjugates are already described. WO20221 15778 discribes targeting moieties comprising macrocyclic peptides conjugated to a metal chelator (e.g. DOTA) that can be complexed with a radioisotope, e.g. Lu177 or Ac225 for the treatment of cancer. As relatively small molecules, these peptides show a much faster tumor clearance than larger molecules, as for example antibodies, and a relatively low tumor uptake as described for RAYZ-8009, a conjugate comprising a GPC3 binding macrocyclic peptide, a linker and DOTA, by Li et al. (Li et al., Novel peptide binder to Glypican-3 for targeted radiopharmaceutical therapy of hepatocellular carcinoma. Journal of Clinical Oncology 2023, 41 :16_suppl, e16131 -e16131 ). Labadie et al. describe a227Th-labeled GPC3-targeting antibody conjugate (227Th-octapa- aGPC3) for treatment of HCC in an orthotopic murine model (Labadie et al., Glypican-3- Targeted 227Th a-Therapy Reduces Tumor Burden in an Orthotopic Xenograft Murine Model of Hepatocellular Carcinoma. J Nucl Med. 2022 Jul;63(7):1033-1038; WO2023 / 056474) for which as little as 70%227Th labeling yield was observed after 2 hours at room temperature. In addition, this radiopharmaceutical employs with227Th a radionuclide with a relatively long halflife, 18.7 days, and223Rathe first daughter of227Th also has a long half-life, leading to a slow delivery of radioactive dose compared to225Ac, which has daughters with shorter half-lives thereby delivering the radioactive dose much faster.

[0016] Bell et al. describe the codrituzumab-based actinium conjugate [225Ac]Ac-Macropa-GC33 (Bell et al., Glypican-3-Targeted Alpha Particle Therapy for Hepatocellular Carcinoma. Molecules. 2020 Dec 22;26(1 ):4), and Kadassery et al. compare [225Ac]Ac-GC33-BZmacropa to an analogous [225Ac]Ac-GC33-macropa conjugate (Kadassery et al., H2BZmacropa-NCS: A Bifunctional Chelator for Actinium-225 Targeted Alpha Therapy. Bioconjug Chem. 2022 Jun 15;33(6):1222-1231 ). Although these conjugates employ the highly potent alpha-emitting radionuclide actinium225 they are based on the GPC3-binding antibody codrituzumab (GC33) which however comprises 4 lysine residues within its CDRs. Likewise, aGPC3 as disclosed in WO2023 / 056474 (see above) comprises 4 lysine residues within its CDRs, and also the fully human antibodies 2-F74-1 1 G, 7-5B, 7-8B and 17-4D described 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) comprise at one or more lysine residues in their CDRs (2-F7: 3 lysine residues; 2- 4-11 G, 75B, 7-8B and 17-4D: 1 lysine residue).

[0017] Conjugation of chelators, like for example macropa, usually takes place via lysine residues of an antibody and may also occur within the target binding CDR regions of the antibody. Such labeling hinders the ability of the antibody to bind to its target. The presence of lysine residues in the CDRs thus means a risk of increasing the non-binding fraction of an antibody conjugate, leading to e.g. drop in internalization rate or causing toxic side effects. At a labeling degree of approximately 10 chelators per antibody, Bell et al. (2020) (see above) observed substantial non-specific liver accumulation of their codrituzumab-based actinium conjugate [225Ac]Ac- Macropa-GC33. Most likely, this finding is due to the increased non-binding fraction of the conjugate by labeling lysines in the GC33 CDRs. A further drawback for the use of the humanized anti-GPC3 antibody codrituzumab in targeted alpha conjugates is its relatively high immunogenicity potential which might cause immunogenicity related side effects.

[0018] OBJECTS OF THE INVENTION

[0019] In view of the prior art, it is an object of the present invention to provide novel therapeutic anti- GPC3 antibodies and antigen-binding fragments thereof which are especially suitable for use as conjugates for targeted radionuclide therapy that overcome the shortcomings of anti-GPC3 antibodies and GPC3-targeting radionuclide conjugates of the prior art. In particular it is an object of the present invention to provide (i) novel anti-GPC3 antibodies and antigen-binding fragments thereof having high target affinity, cross reactivity to cynomolgus antigen, low immunogenicity, high thermostability and good manufacturability by recombinant production, while the number of lysine residues in the CDRs and other sequence liabilities are as low as possible, and (ii) targeted a-particle-emitting radionuclide conjugates comprising these anti- GPC3 antibodies and antigen-binding fragments thereof, preferably targeted actinium conjugates (TACs), still having high binding affinity, which are highly stable, have a low chelator-to-antigen ratio (CAR) and can be chelated at mild conditions with high yield.

[0020] Such novel anti-GPC3 antibodies and anti-GPC3-TACs thereof would offer major advantages in the treatment of hyperproliferative diseases such as cancer, e.g. hepatocellular carcinoma (HCC).

[0021] Furthermore, such novel antibodies could also be used as imaging agents, e.g. for PET imaging when labeled via a suitable chelator with a positron emitter.

[0022] SUMMARY OF THE INVENTION

[0023] The above-mentioned object and other objects are achieved by the teaching of the present invention. The present invention is based on the discovery of novel antibodies targeting GPC3 that are especially suitable for use as conjugates for targeted radionuclide therapy and can deliver a therapeutic benefit to a subject.

[0024] Codrituzumab a humanized version of the murine monoclonal antibody GC33 is the only clinically validated GPC3-targeting antibody. However, this antibody has the drawbacks of high immunogenicity score, a high number of germline deviations, presence of lysine residues in the CDRs hindering conjugation of chelators via lysine coupling, and potential aspartate isomerization sites in the CDRs. To optimize these features codrituzumab was subjected to sequence germlining, additionally further alterations were introduced, such as removal of lysine residues in the CDR or of potential aspartate isomerization sites. Nakano et al have described the humanization and optimization of GC33 giving rise to Codrituzumab. They showed that even substitution of single amino acids led to complete loss of binding (e.g. N33D in CDR-L1 ). Thus, the expectation is that little that more of optimization could be performed without complete loss of beneficial properties. Moreover, due to the high number of deviations from human germline (32), the sequence space is far too large to be assessed systematically in its entirety to further significantly reduce its immunogenicity potential, inherent to antibodies derived from immunization of mice.

[0025] Surprisingly, despite a very high number of mutations in both VH and VL as compared to Codrituzumab, Applicants were able to identify antibody variants which a) still can be produced recombinantly, and b) show comparable or superior affinities in either binding to recombinant or cellular human GPC3, and c) still show binding to cynomolgus GPC3, and d) still show stability of the Fab part Tm above 65 °C. Yet, the number of germline deviations was dramatically reduced, which is also reflected in a much reduced in silico immunogenicity score (IPAD, Epibase) as well as in vitro immunogenicity assessment (Epibase). IPAD immunogenicity score is even more promising than scores of the fully human antibodies 2-F7, 4-11 G, 7-5B, 7-8B, 17-4D described above.

[0026] Surprisingly by combination of germlining and alterations of CDR sequence, Applicants have been able to identify novel anti-GPC3 antibodies with promising binding properties and reduced immunogenicity potential which do not contain any lysine residue in the CDRs what makes them especially suitable as targeting moieties for radiopharmaceuticals or other antibody drug conjugates which are conjugated via lysine residues.

[0027] In addition, the chelator Macropa-NCS used for the targeted actinium conjugates (TACs) of the current invention allows high values for the immunoreactive fraction (IRF) at low chelator- to-antigen (CAR) ratios, and high monomeric purity thus leading to a high fraction of radionuclide-labelled compound having affinity for GPC3. Furthermore, GPC3-TACs of the present invention show a favorable biodistribution in vivo and low accumulation in the liver, which is a sign of reduced dissociation of the radionuclide from the chelating moiety and therefore of increased stability. The antibody chelator conjugates of the present invention can be labelled with the radionuclide under very mild conditions and at reduced temperatures (i.e., room temperature), thereby reducing antibody denaturation and the amount of non-GPC3 binding fraction in the final product. Thus, the TACs of the present invention show an improved tumor to liver ratio and can therefore treat disease more effectively and selectively, reducing damage to non-targeted tissue while maintaining a high potency against cancer cells in the targeted tissue.

[0028] Besides their suitability for use as conjugates for targeted radionuclide therapy, the antibodies or fragments thereof according to the present invention can also be used for various other purposes such as imaging, antibody drug conjugates or as therapeutic agents in the absence of a payload. For example, when conjugated to chelator DFO* (as for example described in W02021051 168) and labeled with89Zr the antibodies or fragments thereof according to the present invention can be used for PET imaging purposes.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.

[0031] DEFINITIONS

[0032] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references, however, can provide one of skill in the art to which this invention pertains with a general definition of many of the terms used in this invention, and can be referenced and used so long as such definitions are consistent with the meaning commonly understood 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 ); and Lackie et al., The Dictionary of Cell & Molecular Biology (3d ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W.B. Saunders Company. Any additional technical resource available to the person of ordinary skill in the art providing definitions of terms used herein having the meaning commonly understood in the art can be consulted. For the purposes of the present invention, the following terms are further defined. Additional terms are defined elsewhere in the description. As used herein and in the appended claims, the singular forms "a," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.

[0033] In the context of the present invention, the term “comprises” or “comprising” means “including, but not limited to”. The term is intended to be open-ended, to specify the presence of any stated features, elements, integers, steps or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. The term “comprising” thus includes the more restrictive terms “consisting of” and “essentially consisting of’. In one embodiment the term “comprising” as used throughout the application and in particular within the claims may be replaced by the term “consisting of”.

[0034] In this context, the term “about” or “approximately” means within 80% to 120%, alternatively within 90% to 1 10%, including within 95% to 105% of a given value or range.

[0035] If within the present text any item is referred to as “as mentioned herein”, it means that it may be mentioned anywhere in the present text.

[0036] In the context of the present invention, the term “GPC3” refers to the glypican 3. GPC3 is an oncofetal heparan sulfate glycoprotein attached to the cell membrane by a glycophosphatidylinositol anchor (Filmus J, Capurro M. Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma. FEBS J. 2013 May;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. The single-chain GPC3 is processed by furin at the Arg358-Cys359 bond to generate the mature GPC3 consisting of a 40-kDa N-terminal subunit and a 30-kDa C-terminal subunit linked by disulfide bonds (De Cat B, Muyldermans SY, Coomans C, Degeest G, Vanderschueren B, Creemers J, Biemar F, 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 fragments.

[0037] The human GPC3 protein is encoded by the gene GPC3 (NCBI Gene ID 2719). Synonyms for GPC3 are SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1 and GTR2-2. The GPC3 protein comprises human, murine, cynomolgus and further mammalian and non-mammalian homologues.

[0038] Human GPC3 (SEQ ID NO: 321 ):

[0039] QPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNME QLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSD INVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFL QALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKY WREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPE DLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYS QKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGD DEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSM AISVVCFFFLVH

[0040] The terms “anti-GPC3 antibody” or "anti-glypican 3 antibody" and "an antibody that binds to glypican 3" or “an antibody that binds to GPC3” are used synonymously herein and refer to an antibody that is capable of binding GPC3, preferably with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting GPC3.

[0041] “Codrituzumab” (CAS Registry Number 1365267-33-9), is a humanized monoclonal anti-GPC3 antibody. Its preparation, isolation and purification has been described in e.g., US7, 919,086. Codrituzumab (also named “TPP-14890” thoughout the present description) comprises a heavy chain protein sequence according to SEQ ID NO. 57 and a light chain protein sequence according to SEQ ID NO.58. Further, Codrituzumab comprises at least the three CDR heavy chain sequences according to SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44 and the three CDR light chain sequences according to SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 48:

[0042] Codrituzumab CDR Heavy Chain Sequences

[0043] SEQ ID NO. 42: DYEMH

[0044] SEQ ID NO. 43: ALDPKTGDTAYSQKFKG

[0045] SEQ ID NO. 44: FYSYTY

[0046] Codrituzumab CDR Light Chain Sequences

[0047] SEQ ID NO. 46: RSSQSLVHSNRNTYLH

[0048] SEQ ID NO. 47: KVSNRFS

[0049] SEQ ID NO. 48: SQNTHVPPT

[0050] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof. Amino acids may be referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0051] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules including, but not limited to, full-length antibodies and monovalent antibodies. “Full-length antibodies” are preferably comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains which are typically inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can comprise e.g., three domains CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is typically composed of three CDRs and up to four FRs arranged from amino-terminus to carboxy-terminus e.g., in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. “Monovalent antibodies” as used herein are preferably comprised of three polypeptide chains, two heavy (H) chains and one light (L) chain which are typically inter-connected by disulfide bonds. One heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can comprise e.g., three domains CH1 , CH2 and CH3. The other heavy chain is comprised of a heavy chain constant region only. The light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is typically composed of three CDRs and up to four FRs arranged from amino-terminus to carboxy-terminus e.g., in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.

[0052] As used herein, the term "Complementarity Determining Regions” (CDRs; e.g., CDR1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1 , CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (e.g. about residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H1 ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunolological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )) and / or those residues from a "hypervariable loop" (e.g. about residues 26-32 (L1 ), 50-52 (L2) and 91 -96 (L3) in the light chain variable domain and 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 instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.

[0053] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into "subclasses" (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. A preferred class of immunoglobulins for use in the present invention is IgG. In particular embodiments, the antibody according to the present invention is an lgG1 , an lgG2, an lgG3 or an lgG4 antibody, more particularly an lgG1 antibody. Different isotypes may have different effector functions. Human light chains are classified as kappa (K) and lambda (A) light chains. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). The heavy-chain constant domains that correspond to the different classes of antibodies are called [alpha], [delta], [epsilon], [gamma], and [mu], respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. As used herein antibodies are conventionally known antibodies and functional fragments thereof.

[0054] A “functional fragment” or “antigen-binding antibody fragment” of an antibody / immunoglobulin hereby is defined as a fragment of an antibody / immunoglobulin (e.g., a variable region of an IgG) that retains the antigen-binding region. An “antigen-binding region” of an antibody typically is found in one or more hyper variable region(s) of an antibody, e.g., the CDR1 , -2, and / or -3 regions; however, the variable “framework” regions can also play an important role in antigen binding, such as by providing a scaffold for the CDRs. Preferably, the “antigen-binding region” comprises at least amino acid residues 4 to 103 of the variable light (VL) chain and 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and 4 to 11 1 of VH, and particularly preferred are the complete VL and VH chains (amino acid positions 1 to 109 of VL and 1 to 113 of VH; numbering according to WO 97 / 08320). Nonlimiting examples of “functional fragments” or “antigen-binding antibody fragments” include Fab, Fab', F(ab')2, Fv fragments, domain antibodies (dAb), complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single chain antibody fragments, diabodies, triabodies, tetrabodies, minibodies, linear antibodies (Zapata et al., Protein Eng. ,8 (10): 1057-1062 (1995)); chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), an antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or muteins or derivatives thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, such as a CDR sequence, as long as the antibody retains the desired biological activity; and multispecific antibodies such as bi- and tri-specific antibodies formed from antibody fragments (C. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001 ) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. The F(ab’)2 or Fab may be engineered to minimize or completely remove the intermolecular disulfide interactions that occur between the CHI and CL domains. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two "Fv" fragments. An "Fv" fragment is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen.

[0055] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.

[0056] Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding. For a review of Fvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteine residues from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteine residues between them.

[0057] The term "mutein" or "variant" can be used interchangeably and refers to an antibody or antigen-binding fragment that contains at least one amino acid substitution, deletion, or insertion in the variable region or the portion equivalent to the variable region, provided that the mutein or variant retains the desired binding affinity or biological activity. Variants of the antibodies or antigen-binding antibody fragments contemplated in the invention are molecules in which the binding activity of the antibody or antigen-binding antibody fragment is maintained.

[0058] A “humanized antibody” is generally defined as one that is (I) derived from a non-human source (e.g., a transgenic mouse which bears a heterologous immune system), which antibody is based on a human germline sequence; or (II) CDR-grafted, wherein the CDRs of the variable region are from a non-human origin, while one or more framework regions and / or part of the CDR sequence of the variable region are of human origin and typically the constant region (if any) is of human origin.

[0059] The term "humanized antibody" thus includes antibodies in which the variable region in either the heavy or light chain or both of a human antibody is altered by at least partial replacement of one or more CDRs from a non-human antibody of known specificity and, if necessary, by partial framework region replacement and sequence changing. In other words, an antibody in which one or more "donor" CDRs from a non-human antibody (such as mouse, rat, rabbit or non-human primate antibody) of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody." It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable domain to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site.

[0060] The framework regions (FR) within the variable region in a heavy or light chain, or both, of a humanized antibody may comprise solely residues of human origin, in which case these framework regions of the humanized antibody are referred to as "fully human framework regions." A human framework region that comprises a mixture of human and donor framework residues, and is referred to herein as a "partially human framework region." Furthermore, humanized antibodies may comprise residues that are neither found in the recipient antibody nor in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain desired affinity).

[0061] In general, the humanized antibody will thus comprise at least one, and typically two, variable regions, in which all or part of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that 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.

[0062] “Human antibodies” or “fully human antibodies” comprise human derived CDRs, i.e. CDRs of human origin. A “human” antibody is hereby defined as one that is not chimeric or “humanized” and not from (either in whole or in part) a non-human species. A human antibody or functional antibody fragment can be derived from a human or can be a synthetic human antibody. A “synthetic human antibody” is defined herein as an antibody having a sequence derived, in whole or in part, in silico from synthetic sequences that are based on the analysis of known human antibody sequences. In silico design of a human antibody sequence or fragment thereof can be achieved, for example, by analyzing a database of human antibody or antibody fragment sequences and devising an amino acid sequence utilizing the data obtained therefrom. Another example of a human antibody or functional antibody fragment is one that is encoded by a nucleic acid isolated from a library of antibody sequences of human origin {i.e., such library being based on antibodies taken from a human natural source).

[0063] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the term "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The term "monoclonal” is not to be construed as to require production of the antibody by any particular method. For example, the monoclonal antibodies to be used may be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 [1975, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be recombinant, chimeric, humanized, human, Human Engineered™, or antibody fragments, for example.

[0064] An "isolated" antibody is one that has been identified and separated from a component of the cell that expressed it. Contaminant components of the cell are materials that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0065] An "isolated" nucleic acid is one that has been identified and separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0066] As used herein, an antibody “binds specifically to”, is “specific to / for” or “specifically recognizes” an antigen of interest, e.g. GPC3, is one that binds the antigen-target with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen or one that binds an antigen-binding polypeptide target with sufficient affinity such that the antibody is useful as a reversal agent to neutralize the therapeutic activity of this antigen-binding polypeptide (e.g. an antigen-binding antibody) and does not significantly crossreact with other proteins or does not significantly cross-react with proteins other than orthologs and variants (e.g. mutant forms, splice variants, or proteolytically truncated forms) of the aforementioned target. The term "specifically recognizes" or "binds specifically to" or is "specific to / for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by an antibody, or antigen-binding fragment thereof, having a monovalent dissociation constant (KD) for the antigen of less than about 10-4M, alternatively less than about 10-5M, alternatively less than about 10-6M, alternatively less than about I O7M, alternatively less than about 10-8M, alternatively less than about 10-9M, alternatively less than about 10-10M, alternatively less than about 10-11M, alternatively less than about 10-12M, or less. An antibody “binds specifically to,” is “specific to / for” or “specifically recognizes” an antigen if such antibody is able to discriminate between such antigen and one or more reference antigen(s). In its most general form, “specific binding”, “binds specifically to”, is “specific to / for” or “specifically recognizes” is referring to the ability of the antibody to discriminate between the antigen of interest and an unrelated antigen, as determined, for example, in accordance with one of the following methods. Such methods comprise, but are not limited to, surface plasmon resonance (SPR), Western blots, ELISA-, RIA-, ECL-, IRMA- tests and peptide scans. For example, a standard ELISA assay can be carried out. The scoring may be carried out by standard color development (e.g., secondary antibody with horseradish peroxidase and tetramethyl benzidine with hydrogen peroxide). The reaction in certain wells is scored by the optical density, for example, at 450 nm. Typical background (=negative reaction) may be 0.1 OD; typical positive reaction may be 1 OD. This means the difference positive / negative is more than 5-fold, 10-fold, 50-fold, and preferably more than 100-fold. Typically, determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.

[0067] "Binding affinity" or “affinity” refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of an antibody or fragment thereof for a target can be determined using techniques well known in the art, for example by ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), flow cytometry or fluorescent polarization assays. The dissociation constant “KD” is commonly used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen) i.e., how tightly a ligand binds to a particular protein. Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules. Affinity can be measured by common methods known in the art, including those described herein. Preferably the affinity is provided as dissociation constant KD, in the alternative the affinity is provided as an EC50 value. In one embodiment, the "Kb" or "Kb value" according to this invention is measured by using surface plasmon resonance assays a Biacore T200 instrument (Cytiva). Other suitable devices are 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).

[0068] “Half maximal effective concentration” (EC50) refers to the concentration of a drug, modulator, antibody, fragment, conjugate or molecule which induces a response halfway between the baseline and maximum after a specified incubation time. In the context of affinity, the EC50 thus reflects the concentration of binder (e.g. antibody) that is needed for half-maximal binding. An EC50 can be determined if an inflection point can be determined by mathematical modeling (e.g., non-linear regression) of the dose-response curve describing the relationship between applied drug, antibody, fragment, conjugate or molecule concentration and signal. For example, if the dose-response curve follows a sigmoidal curve, an EC50 can be determined. Where the response is an inhibition, the EC50 is termed “half maximal inhibitory concentration” (IC50).

[0069] As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, or combinations thereof and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0070] The term “maturated antibodies” or “maturated antigen-binding fragments” such as maturated Fab variants or “optimized” variants includes derivatives of an antibody or antibody fragment exhibiting stronger binding - i. e. binding with increased affinity - to a given antigen such as the extracellular domain of a target protein. Maturation is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment leading to this affinity increase. The maturation process is the combination of molecular biology methods for introduction of mutations into the antibody and screening for identifying the improved binders.

[0071] The term “antibody conjugate” refers to a conjugate comprising at least one antibody moiety and one or more further molecules or moieties. The one or more further molecules or moieties may be selected without limitation from a drug, a chelator, a radioactive element, a cytotoxic agent, a further antibody or antigen-binding fragment.

[0072] The term “antibody drug conjugate” (“ADC”) refers to an antibody conjugate comprising at least one drug moiety.

[0073] The term ’’germlining” refers to replacement of residues in the variable domains of an antibody with those present in the pre-mutated germline genes to reduce the potential for immunogenicity.

[0074] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc gamma receptors (FcyRs) present on certain cytotoxic cells (e.g. NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell e.g. with cytotoxins. To assess ADCC activity of an antibody of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821 ,337 or U.S. Patent No. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMC and NK cells.

[0075] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased C1q binding are described, e.g., in US Patent No. 6,194,551 Bl and WO 1999 / 51642.

[0076] "Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues, respectively, in a candidate sequence that are identical with the nucleic acid or amino acid residues, respectively, in the reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of the sequence identity. Preferred are un-gapped alignments. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0077] Substantial sequence identity / similarity may be taken as having a sequence similarity / identity of at least 80% to the complete sequences and / or at least 90% to the specific binding regions (e.g., the CDR regions). Preferable sequence similarity or more preferably identity may be at least 92%, 95%, 97%, 98% or 99%. Sequence similarity and / or identity may be determined using the "BestFit" program of the Genetics Computer Group Version 10 software package from the University of Wisconsin. The program uses the local had algorithm of Smith and Waterman with default values: Gap creation penalty=8, Gap extension penalty=2, Average match=2.912, average mismatch 2.003. The terms “polynucleotide” or “nucleic acid”, as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.

[0078] "Sequence homology" indicates the percentage of amino acids that either is identical or that represent conservative amino acid substitutions.

[0079] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0080] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and 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 the primary transformed / transfected / transduced cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0081] As used herein, the phrase "therapeutically effective amount" is meant to refer to an amount of therapeutic or prophylactic antibody that would be appropriate to elicit the desired therapeutic or prophylactic effect or response, including alleviating some or all of such symptoms of disease or reducing the predisposition to the disease, when administered in accordance with the desired treatment regimen.

[0082] The term "pharmaceutical formulation" I “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “radionuclide” (also: “radioactive nuclide”, “radioisotope” or “radioactive isotope”) is an atom that undergoes radioactive decay. Without limitation, for example the radionuclide may be a p- emitting radionuclide (beta emitter), an a-particle-emitting radionuclide (alpha emitter), or an Auger electron emitting radionuclide (Auger electron emitter).

[0083] “ -emitting radionuclides” or “beta emitter” are radionuclides which emit beta particles. Examples of beta emitters include without limitation copper-67 (67Cu), strontium-89 (89Sr), yttrium-90 (90Y), rhodium-105 (105Rh), iodine-131 (131l), promethium-149 (149Pm), holmium-166 (166Ho), lutetium-177 (177Lu), rhenium-186 (186Re), rhenium-188 (188Re), gold-198 (198Au) and gold-199 (199Au). Jongho has reviewed various techniques for chelation of various of these radioisotypes (Jeon, Jongho. "Review of therapeutic applications of radiolabeled functional nanomaterials." International journal of molecular sciences 20.9 (2019): 2323.).

[0084] “Auger electron emitting radionuclides” or “Auger electron emitter” are radionuclides which emit Auger electrons. Examples of Auger electron emitters include without limitation bromine-77, indium-1 11 , iodine-123, and iodine-125.

[0085] “a-emitting radionuclides” or “alpha emitter” are radionuclides which emit alpha particles, i.e. 4He nuclei with a +2 charge. Non limiting examples of alpha emitters include bismuth-213 (213Bi), characterized by a half-life of 45.6 min, actinium-225 (225Ac), characterized by a halflife of 9.9 d, astatine-211 (211At), characterized by a half-life of 7.2 h, radium-223 (223Ra), characterized by a half-life of 1 1 .4 d, radium-224 (224Ra), characterized by a half-life of 3.7 d, and thorium-227 (227Th), characterized by a half-life of 18.7 d.

[0086] In the context of the present invention, the term “Ac” means an ion of at least one alphaemitting actinium isotope. Preferably, the alpha-emitting actinium isotope is 225Ac. Preferably, the ion of at the least one alpha-emitting actinium isotope is225Ac having a triple positive charge, i.e.,225Ac3+. Thus, in the context of the present invention, the term “Ac” preferably, but not exclusively, means225Ac3+.

[0087] Radionuclides can be obtained as known in the art. For example, as described in Poty, Sophie, et al,211At can be cyclotron-produced by bombarding natural bismuth with a medium energy a-particle beam using the209Bi(a,2n)211At reaction ("Poty, Sophie, et al. a-Emitters for radiotherapy: from basic radiochemistry to clinical studies — part 1 / 2." Journal of Nuclear Medicine 59.6 / 59.7 (2018): 878-884 / 1020-1027).227Th and223Ra are both available upon separation from their mutual parent,227Ac. Clinical production of223Ra uses227Ac / 227Th-based generators. Parent isotopes are loaded on actinide chromatographic resin and223Ra chloride solution is obtained after elution with 1 M HCI or HNO3, subsequent cation exchange column, evaporation, and dissolution in saline solution.

[0088] “Chelation” refers to the formation or presence of two or more separate coordinate bonds between a ligand and a single central metal atom. The ligands which are capable of forming these coordinate bonds are termed “chelators”, “chelating agents”, or “sequestering agents”.

[0089] “A chelating moiety” is a chelator which can chelate a given radionuclide or group of radionuclides, such as, without limitation, alpha emitters, beta emitters or Auger electron emitter. Various chelators are known in the art and can be used according to the current invention, e.g. as described in Price, Eric W., and Chris Orvig. "Matching chelators to radiometals for radiopharmaceuticals." Chemical Society Reviews 43.1 (2014): 260-290, incorporated herein in its entirety.

[0090] In the context of the present invention the chelating moiety comprises for example an 18- membered chelating moiety as for example the macrocyclic chelator “macropa” (N,N'-bis[(6- carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6). The macrocyclic chelator macropa has been described, for example, in Thiele et al., Angew. Chem. Int. Ed. 2017, 56(46), 14712 and references therein. Macropa chelators and derivatives thereof, as for example “Macropa-NCS” (6-[[16-[(6-carboxy-2-pyridyl)methyl]-1 ,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl]methyl]- 4-[2-(4-isothiocyanatophenyl)ethoxy]pyridine-2-carboxylic acid), have also been described in WO2018 / 183906 and W02020 / 106886 and references therein.

[0091] The chelating moiety for complexation of actinium (or “actinium-complexating chelating moiety”) may terminate in an isothiocyanate or carboxylic acid moiety in order to facilitate its coupling with the targeting moiety (e.g an anti-GPC3 antibody or an antigen-binding fragment thereof according to the invention). When the macropa chelator is used, one of the pyridine rings of the macropa chelator is typically substituted so that it can be coupled to the targeting. This can be a simple functional group (e.g., a carboxylic acid -COOH) or a more complex chemical structure, as long as it is capable of being coupled to a targeting moiety (e.g. an anti- GPC3 antibody or an antigen-binding fragment thereof according to the invention). For example, the substituent on the pyridine ring of the macropa chelator can terminate in an isothiocyanate or carboxylic acid moiety.

[0092] A “targeting moiety” as used herein is any chemical structure binding to a biological target, such as GPC3 or a cell expressing GPC3. The targeting moiety localizes itself, e.g. as part of a bigger structure at a specific site where the presence is required to exert the intended effect, e.g. delivery of radioactive decay in case of a TAC. Thus, a tissue targeting group or moiety serves to provide greater localization of a molecule or conjugate to at least one desired site in the body of a subject in comparison with the concentration of an equivalent complex not comprising the targeting moiety. The targeting moiety can be for example selected without limitation from 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 current invention, the targeting moiety is preferably an antibody or antigen-binding fragment thereof (i.e. the anti-GPC3-antibodies or fragments thereof of the present invention).

[0093] A “targeted actinium conjugate” (also “TAG”) is a conjugate comprising a targeting moiety (i.e., the anti-GPC3 antibodies or fragments thereof of the present invention) and at least one chelator or chelating group for complexation of actinium (e.g. macropa, preferably Macropa- NCS), and optionally comprise actinium.

[0094] The term “DOTA” refers to 2,2’,2”,2”’-(1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10- tetrayl)tetraaceticacid or 1 ,4,7,10-tetra-azacycloododecane-N,N',N",N'"-tetraacetic acid and derivatives thereof, which can chelate a radionuclide. DOTA is a chelator arranged for complexation of an a-particle-emitting radionuclide, such as212Bi,213Bi,225Ac,227Th. Chelators such as DOTA form stable complexes with a chelated As-225, which exists in the 3+ oxidation state. In order to achieve sufficient labeling of DOTA-coupled antibodies, the complexation step should preferably be performed as a two-step process or directly at elevated temperatures.

[0095] In the context of the present invention, the term CAR refers to the Chelator-to-Antigen Ratio, which is a measure of the specific activity of the radiolabeled compound (e.g., the targeted actinium conjugates of the present invention).

[0096] In the context of the present invention, the term IRF refers to the Immunoreactive Fraction i.e., the fraction of the labelled product (i.e., the targeted actinium conjugates of the present invention) which is 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 assessing the immunoreactive fraction.

[0097] ANTI-GPC3 ANTIBODIES ACCORDING TO THE PRESENT INVENTION According to a first aspect, the present invention relates to isolated antibodies or antigenbinding fragments thereof capable of binding to GPC3.

[0098] The antibodies can be, for example, IgG antibodies, such as a human lgG1 , lgG2, lgG3, or lgG4, or a mouse lgG1 , lgG2a, lgG2b or lgG2c. In some highly preferred embodiments, the isolated antibodies or antigen-binding fragment thereof according to the current aspect are humanized IgG 1 antibodies. In some further preferred embodiments, the antibodies or antigenbinding fragments thereof according to the current aspect are a Fab', F(ab')2, Fab, Fv, rlgG, or scFv fragments.

[0099] In some preferred embodiments, the antibodies or antigen-binding fragments thereof according to the current aspect is binding to human GPC3. In some of these preferred embodiments the antibodies or antigen-binding fragments thereof according to the current aspect cross-react with GPC3 from other species e.g. monkey, macaca fascicularis (cynomolgus monkey), macaca mulatta (rhesus macaque), rodent, mouse, rat, horse, bovine, pig, dog, cat and / or camel GPC3, preferable with cynomolgus GPC3.

[0100] According to some preferred embodiments, the antibodies or antigen-binding fragments have a binding affinity KD for human GPC3 of SEQ ID 321 which is below 2E-07 M, preferably below 1 E-08 M, 1 E-09 M, 10E-10 M or 1 E-10 M. According to some embodiments, which may be the same or different, the antibodies or antigen-binding fragments have a binding affinity KDto cynomolgus GPC3 of SEQ ID 323 which is below 2E-07 M, preferably below 1 E-08 M, 1 E-09 M, 10E-10 M or 1 E-10 M.

[0101] Preferably, the antibodies or antigen-binding fragments bind human and cynomolgus GPC3 with a KD in the same order of magnitude, e.g. with a KD below 2E-07 M, preferably below 1 E- 08 M, 1 E-09 M, 10E-10 M or 1 E-10 M.

[0102] In further preferred embodiments, the antibodies or antigen-binding fragments thereof bind to human GPC3 of SEQ ID 321 with an EC50 which is below 1 E-07 M preferably below 10 E-08 M, 1 E-08 M, 10E-9 M, or 1 E-09 M. According to some embodiments, which may be the same or different, the antibodies or antigen-binding fragments have a EC50 to cynomolgus GPC3 of SEQ ID 323 which is below 2E-07 M, preferably below 10 E-08 M, 1 E-08 M, 10E-9 M, or I E- 09 M.

[0103] Preferably, the antibodies or antigen-binding fragments bind human and cynomolgus GPC3 with a EC50 in the same order of magnitude, e.g. with a EC50 below 2E-07 M, preferably below 10 E-08 M, 1 E-08 M, 10E-9 M, or 1 E-09 M. In some preferred embodiments the antibodies or antigen-binding fragments thereof according to the current aspect have a low number of germline deviations from the closest germline light chain IGKV2-28-IGKJ2 (SEQ ID NO:325) and the closest germline heavy chain identified outside of CDR-H3 IGHV1 -46-IGHJ4 (SEQ-ID NO: 324) which is below 32 preferably below 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 5, 4, 3, 2, or 1.

[0104] In further preferred embodiments the antibodies or antigen-binding fragments thereof according to the current aspect have a low risk for immunogenicity as reflected by an IPAD score (as predicted using the v4.03 algorithm as described in Example 6) below 1000, preferably below 900, 800, 700, 600, or 550.

[0105] In further preferred embodiments the antibodies or antigen-binding fragments thereof according to the current aspect comprise a low number of lysine residues within their CDRs. Preferably the number of lysine residues within the six CDRs of the antibodies or antigenbinding fragments thereof is below 4, more preferably below 3, even more preferably below 2, and most preferably below 1 . According to a highly preferred embodiment the antibodies or antigen-binding fragments thereof according to the current aspect comprise no lysine residue within their six CDRs.

[0106] The isolated antibodies or antigen-binding fragments according to the present invention may exhibit any combination of the above-described characteristics.

[0107] In some preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise: i) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 82, an H-CDR2 comprising SEQ ID NO: 83, and an H-CDR3 comprising SEQ ID NO: 84 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 86, an L-CDR2 comprising SEQ ID NO: 87, and an L-CDR3 comprising SEQ ID NO: 88 (TPP-29312), or ii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L- CDR3 comprising SEQ ID NQ:108 (TPP-29537), or iii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H-CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L-CDR2 comprising SEQ ID NO: 127, and an L- CDR3 comprising SEQ ID NO:128 (TPP-29538), or iv) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H-CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L-CDR2 comprising SEQ ID NO: 147, and an L- CDR3 comprising SEQ ID NO:148 (TPP-29636), or v) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H-CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L-CDR2 comprising SEQ ID NO: 167, and an L- CDR3 comprising SEQ ID NO:168 (TPP-29638), or vi) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 182, an H-CDR2 comprising SEQ ID NO: 183, and an H-CDR3 comprising SEQ ID NO: 184 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 186, an L-CDR2 comprising SEQ ID NO: 187, and an L- CDR3 comprising SEQ ID NO:188 (TPP-29642).

[0108] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise: i) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L- CDR3 comprising SEQ ID NO:108, or ii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H-CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L-CDR2 comprising SEQ ID NO: 127, and an L- CDR3 comprising SEQ ID NO:128, or iii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H-CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L-CDR2 comprising SEQ ID NO: 147, and an L- CDR3 comprising SEQ ID NO:148, or iv) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H-CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L-CDR2 comprising SEQ ID NO: 167, and an L- CDR3 comprising SEQ ID NO:168.

[0109] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L-CDR3 comprising SEQ ID NQ:108 (TPP-29537).

[0110] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H-CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L-CDR2 comprising SEQ ID NO: 127, and an L-CDR3 comprising SEQ ID NO:128 (TPP-29538).

[0111] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H-CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L-CDR2 comprising SEQ ID NO: 147, and an L-CDR3 comprising SEQ ID NO:148 (TPP-29636).

[0112] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H-CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L-CDR2 comprising SEQ ID NO: 167, and an L-CDR3 comprising SEQ ID NO:168 (TPP-29638).

[0113] In further preferred embodiments the isolated antibodies or antigen-binding fragments according to the present invention comprise: 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.

[0114] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise: 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.

[0115] In further preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise: i) a variable heavy chain domain comprising SEQ ID NO: 81 and a variable light chain domain comprising SEQ ID NO: 85; or ii) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or iii) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or iv) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or v) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165; or vi) a variable heavy chain domain comprising SEQ ID NO: 181 and a variable light chain domain comprising SEQ ID NO: 185.

[0116] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise: i) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or ii) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or iii) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or iv) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165.

[0117] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105.

[0118] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125.

[0119] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145.

[0120] In some highly preferred embodiments, the isolated antibodies or antigen-binding fragments according to the present invention comprise a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165.

[0121] In particular embodiments, the isolated antibodies according to the present invention are IgG 1 , lgG2, lgG3 or an lgG4 antibodies. Most particularly, the isolated antibodies according to the present invention are lgG1 antibodies.

[0122] In further preferred embodiments the isolated antibodies or antigen-binding fragments according to the present invention comprise: i) a heavy chain having at least 90 %, 95 %, 98 %, 99 % or 100 % sequence identity with SEQ ID NO: 97 and / o 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) a heavy chain having at least 90 %, 95 %, 98 %, 99 % or 100 % sequence identity with SEQ ID NO: 197 and / or a light chain having at least 90 %, 95 %, 98 %, 99 % or 100 % sequence identity with SEQ ID NO: 198.

[0123] In some highly preferred embodiments, the isolated antibodies according to the present invention comprise: 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) 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.

[0124] In some preferred embodiments, the isolated antibodies according to the present invention comprise: i) a heavy chain comprising SEQ ID NO: 97 and a light chain comprising SEQ ID NO: 98; or ii) a heavy chain comprising SEQ ID NO: 1 17 and a light chain comprising SEQ ID NO: 1 18; or iii) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or iv) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or v) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178; or vi) a heavy chain comprising SEQ ID NO: 197 and a light chain comprising SEQ ID NO: 198.

[0125] In some highly preferred embodiments, the isolated antibodies according to the present invention comprise: i) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 1 18; or ii) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or iii) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or iv) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178. In some highly preferred embodiments, the isolated antibodies according to the present invention comprise a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118 (TPP-29537).

[0126] In some highly preferred embodiments, the isolated antibodies according to the present invention comprise a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138 (TPP-29538).

[0127] In some highly preferred embodiments, the isolated antibodies according to the present invention comprise a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158 (TPP-29636).

[0128] In some highly preferred embodiments, the isolated antibodies according to the present invention comprise a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178 (TPP-29638).

[0129] In particular embodiments, the antigen-binding fragments according to the present invention are scFv, Fab, Fab’ fragment or a F(ab’)2 fragments.

[0130] In particular embodiments, the isolated antibodies or antigen-binding fragments according to the present invention are monoclonal antibodies or antigen-binding fragments.

[0131] In particular embodiments, the isolated antibodies or antigen-binding fragments according to the present invention are human, humanized or chimeric antibodies or antigen-binding fragments, more particularly humanized antibodies or antigen-binding fragments.

[0132] In particular embodiments, the isolated antibodies or antigen-binding fragments according to the present invention are monospecific antibodies. In particular other embodiments, the isolated antibodies or antigen-binding fragments according to the present invention are multispecific antibodies that bind to GPC3 and at least one further antigen, such bispecific, trispecific or tetraspecific antibodies.

[0133] Amino acid sequences of preferred antibodies according to the present invention are listed in Table 1 .

[0134] Table 1 : Amino acid sequences of preferred antibodies according to the present invention

[0135] Nucleic acid sequences of preferred antibodies according to the present invention are listed in Table 2.

[0136] Table 2: Nucleic acid sequences of preferred antibodies according to the present invention

[0137] PEPTIDE VARIANTS

[0138] Antibodies or antigen-binding fragments of the invention are not limited to the specific peptide sequences provided herein. Rather, the invention also embodies variants of these polypeptides. With reference to the instant disclosure and conventionally available technologies and references, the skilled worker will be able to prepare, test and utilize functional variants of the antibodies disclosed herein, while appreciating these variants having the ability to bind to GPC3 fall within the scope of the present invention.

[0139] A variant can include, for example, an antibody that has at least one altered complementary determining region (CDR) (hyper-variable) and / or framework (FR) (variable) domain / position, vis-a-vis a peptide sequence disclosed herein.

[0140] By altering one or more amino acid residues in a CDR or FR region, the skilled worker routinely can generate mutated or diversified antibody sequences, which can be screened against the antigen, for new or improved properties, for example.

[0141] A further preferred embodiment of the invention is an antibody or antigen-binding fragment in which the VH and VL sequences are selected as shown in Table 1 . The skilled worker can use the data in T able 1 to design peptide variants that are within the scope of the present invention. It is preferred that variants are constructed by changing amino acids within one or more CDR regions; a variant might also have one or more altered framework regions. Alterations also may be made in the framework regions. For example, a peptide FR domain might be altered where there is a deviation in a residue compared to a germline sequence.

[0142] Alternatively, the skilled worker could make the same analysis by comparing the amino acid sequences disclosed herein to known sequences of the same class of such antibodies, using, for example, the procedure described by Knappik A., et al., JMB 2000, 296:57-86. Furthermore, variants may be obtained by using one antibody as starting point for further optimization by diversifying one or more amino acid residues in the antibody, preferably amino acid residues in one or more CDRs, and by screening the resulting collection of antibody variants for variants with improved properties. Particularly preferred is diversification of one or more amino acid residues in CDR3 of VL and / or VH. Diversification can be done e.g. by synthesizing a collection of DNA molecules using trinucleotide mutagenesis (TRIM) technology (Virnekas B. et al., Nucl. Acids Res. 1994, 22: 5600.). Antibodies or antigen-binding fragments thereof include molecules with modifications / variations including but not limited to e.g. modifications leading to altered half-life (e.g. modification of the Fc part or attachment of further molecules such as PEG), altered binding affinity or altered ADCC or CDC activity.

[0143] CONSERVATIVE AMINO ACID VARIANTS

[0144] Polypeptide variants may be made that conserve the overall molecular structure of an antibody peptide sequence described herein. Given the properties of the individual amino acids, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0145] For example, (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophane, 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. Substitutions typically may be made within groups (a)-(d). In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in a-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in p-pleated sheets. Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns. Some preferred substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist readily can construct DNAs encoding the conservative amino acid variants.

[0146] GLYCOSYLATION VARIANTS Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 using Kabat EU numbering of the CH2 domain of the Fc region; see, e.g., Wright et al. Trends Biotechnol. 15: 26-32 (1997).

[0147] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the expression system (e.g. host cell) and / or by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0148] In one embodiment of this invention, aglycosyl antibodies having decreased effector function or antibody derivatives are prepared by expression in a prokaryotic host. Suitable prokaryotic hosts for include but are not limited to E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0149] In one embodiment, antibody variants are provided having decreased effector function, which are characterized by a modification at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody. In one embodiment of present invention, the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at the site. Thus, in one preferred embodiment of this invention, the aglycosyl antibodies or antibody derivatives are prepared by mutation of the heavy chain glycosylation site, - i.e., mutation of N297 using Kabat EU numbering and expressed in an appropriate host cell.

[0150] In another embodiment of the present invention, aglycosyl antibodies or antibody derivatives have decreased effector function, wherein the modification at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody or antibody derivative comprises the removal of the CH2 domain glycans, - i.e., deglycosylation. These aglycosyl antibodies may be generated by conventional methods and then deglycosylated enzymatically. Methods for enzymatic deglycosylation of antibodies are well known in the art (e.g. Winkelhake & Nicolson (1976), J Biol Chem. 251 (4) :1074-80).

[0151] In another embodiment of this invention, deglycosylation may be achieved using the glycosylation inhibitor tunicamycin (Nose & Wigzell (1983), Proc Natl Acad Sci USA, 80(21 ):6632-6). That is, the modification is the prevention of glycosylation at the conserved N- linked site in the CH2 domains of the Fc portion of said antibody.

[0152] In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function.

[0153] Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include Okazaki et al. J Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

[0154] Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); and WO 2004 / 056312), and knockout cell lines, such as alpha-1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)).

[0155] Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GIcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 01 1878; US Patent No. 6,602,684; and US 2005 / 0123546.

[0156] Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO1997 / 30087; WO1998 / 58964; and W0 1999 / 22764.

[0157] FC REGION VARIANTS

[0158] In certain embodiments, one or more amino acid modifications (e.g. a substitution) may be introduced into the Fc region of an antibody (e.g., a human IgG 1 , lgG2, lgG3 or lgG4 Fc region) provided herein, thereby generating an Fc region variant.

[0159] In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity) but retains FcRn binding ability. In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1 q binding and / or Complement Dependent Cytotoxicity (CDC).

[0160] In certain embodiments, the invention contemplates an antibody variant that possesses an increased or decreased half-live. Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs 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 with one or more substitutions therein which improve binding of the Fc region to FcRn.

[0161] DNA MOLECULES ACCORDING TO THE PRESENT INVENTION

[0162] The present invention also relates to an isolated nucleic acid sequence that encodes the antibody or antigen-binding fragment according to the present invention. The isolated nucleic acid sequence encoding the antibody or antigen-binding fragment according to the present invention can for instance be produced by techniques described in Sambrook et al., 1989, and Ausubel et al., 1989, or alternatively, by chemically synthesis, (e.g. techniques described in Oligonucleotide Synthesis (1984, Gait, ed., IRL Press, Oxford)). The DNA sequences used for the antibodies expressed are given in Table 2. These sequences are optimized in certain cases for mammalian expression. DNA molecules of the invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the invention may be described by reference to their physical properties in hybridization. The skilled worker will recognize that DNA can be used to identify its complement and, since DNA is double stranded, its equivalent or homolog, using nucleic acid hybridization techniques. It also will be recognized that hybridization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe. For guidance regarding such conditions see, Sambrook et al., 1989 supra and Ausubel et al., 1995 (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

[0163] Structural similarity between two polynucleotide sequences can be expressed as a function of "stringency" of the conditions under which the two sequences will hybridize with one another. As used herein, the term "stringency" refers to the extent that the conditions disfavor hybridization. Stringent conditions strongly disfavor hybridization, and only the most structurally related molecules will hybridize to one another under such conditions. Conversely, non-stringent conditions favor hybridization of molecules displaying a lesser degree of structural relatedness. Hybridization stringency, therefore, directly correlates with the structural relationships of two nucleic acid sequences.

[0164] Hybridization stringency is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size and the presence of agents which disrupt hydrogen bonding. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen bonding. Hybridization typically is performed in two phases: the “binding” phase and the “washing” phase.

[0165] FUNCTIONALLY EQUIVALENT DNA VARIANTS

[0166] Yet another class of DNA variants within the scope of the invention may be described with reference to the product they encode. These functionally equivalent polynucleotides are characterized by the fact that they encode the same peptide sequences due to the degeneracy of the genetic code.

[0167] It is recognized that variants of DNA molecules provided herein can be constructed in several different ways. For example, they may be constructed as completely synthetic DNAs. Methods of efficiently synthesizing oligonucleotides are widely available. See Ausubel et al., section 2.1 1 , Supplement 21 (1993). Overlapping oligonucleotides may be synthesized and assembled in a fashion first reported by Khorana et al., J. Mol. Biol. 72:209-217 (1971 ); see also Ausubel et al., supra, Section 8.2. Synthetic DNAs preferably are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector. As indicated, a method of generating variants is to start with one of the DNAs disclosed herein and then to conduct site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typical method, a target DNA is cloned into a single-stranded DNA bacteriophage vehicle. Single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide alteration(s). The 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. In addition, various methods are available that increase the probability that the progeny phage will be the desired mutant. These methods are well known to those in the field and kits are commercially available for generating such mutants.

[0168] RECOMBINANT DNA CONSTRUCTS AND EXPRESSION

[0169] The present invention further provides recombinant DNA constructs comprising one or more of the nucleotide sequences according to the present invention. The recombinant constructs of the present invention can be used in connection with a vector, such as a plasmid, phagemid, phage or viral vector, into which a DNA molecule encoding an antibody of the invention or antigen-binding fragment thereof or variant thereof is inserted.

[0170] Thus, in one aspect, the present invention relates to a vector comprising a nucleic acid sequence according to the present invention.

[0171] An antibody, antigen binding portion, or variant thereof provided herein can be prepared by recombinant expression of nucleic acid sequences encoding light and heavy chains or portions thereof in a host cell. To express an antibody, antigen binding portion, or variant thereof recombinantly a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and / or heavy chains or portions thereof such that the light and heavy chains are expressed in the host cell. Standard recombinant DNA methodologies are used to prepare and / or obtain nucleic acids encoding the heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al..

[0172] In addition, the nucleic acid sequences encoding variable regions of the heavy and / or light chains can be converted, for example, to nucleic acid sequences encoding full-length antibody chains, Fab fragments, or to scFv. The VL- or VH-encoding DNA fragment can be operatively linked, (such that the amino acid sequences encoded by the two DNA fragments are in-frame) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker. The sequences of human heavy chain and light chain constant regions are known in the art (see e.g., Kabat, E. A., el al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.

[0173] To create a polynucleotide sequence that encodes a scFv, the VH- and VL-encoding nucleic acids can be operatively linked to another fragment encoding a flexible linker such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., 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).

[0174] To express the antibodies, antigen binding fragments thereof 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 example, DNA encoding the desired polypeptide can be inserted into an expression vector which is then transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples for prokaryotic host cells are e.g. bacteria, examples for eukaryotic hosts cells are yeasts, insects and insect cells, plants and plant cells, transgenic animals, or mammalian cells. Introduction of the recombinant construct into the host cell can be carried out using standard techniques such as calcium phosphate transfection, DEAE dextran mediated transfection, electroporation, transduction or phage infection.

[0175] In some embodiments, the DNAs encoding the heavy and light chains are inserted into separate vectors. In other embodiments, the DNA encoding the heavy and light chains is inserted into the same vector. It is understood that the design of the expression vector, including the selection of regulatory sequences is affected by factors such as the choice of the host cell, the level of expression of protein desired and whether expression is constitutive or inducible.

[0176] Thus, in a further aspect, the present invention relates to an isolated cell expressing the antibody or antigen-binding fragment according to the present invention and / or comprising the nucleic acid according to the present invention or the vector according to the present invention. The isolated cell can be virtually any cell for which expression vectors are available. The isolated cell can for example a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.

[0177] In a further aspect, the present invention relates to a method of producing the isolated antibody or antigen-binding fragment according to the present invention comprising culturing of the cell according to the present invention. In particular embodiments, the cell according to the present invention is cultivated under suitable conditions for antibody expression and the antibody or antigen-binding fragment is recovered. In particular embodiments, the antibody or antigenbinding fragment is purified, particularly to at least 95% homogeneity by weight.

[0178] Bacterial Expression

[0179] Useful expression vectors for bacterial use are constructed by inserting a DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include but are not limited to E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0180] Bacterial vectors may be, for example, bacteriophage-, plasmid- or phagemid-based. These vectors can contain a selectable marker and a bacterial origin of replication derived from commercially available plasmids typically containing elements of the well-known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed / induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0181] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Therefore, an embodiment of the present invention is an expression vector comprising a nucleic acid sequence encoding for the novel antibodies of the present invention.

[0182] Antibodies of the present invention or antigen-binding fragments thereof or variants thereof include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic host, including, for example, E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably, from E. co / / cells.

[0183] Mammalian Expression

[0184] Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Expression of the antibodies may be constitutive or regulated (e.g. inducible by addition or removal of small molecule inductors such as Tetracyclin in conjunction with Tet system). For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. 5,168,062 by Stinski, U.S. 4,510,245 by Bell et al. and U.S. 4,968,615 by Schaffner et al.. The recombinant expression vectors can also include origins of replication and selectable markers (see e.g., U.S. 4,399,216, 4,634,665 and U.S. 5,179,017). Suitable selectable markers include genes that confer resistance to drugs such as G418, puromycin, hygromycin, blasticidin, zeocin / bleomycin or methotrexate or selectable marker that exploit auxotrophies such as Glutamine Synthetase (Bebbington et al., Biotechnology (N Y). 1992 Feb;10(2):169-75), on a host cell into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, neo gene confers resistance to G418, the bsd gene from Aspergillus terreus confers resistance to blasticidin, puromycin N-acetyl-transferase confers resistance to puromycin, the Sh ble gene product confers resitance to zeocin, and resistance to hygromycin is conferred by the E. coli hygromycin resistance gene (hyg or hph). Selectable markers like DHFR or Glutamine Synthetase are also useful for amplification techniques in conjunction with MTX and MSX.

[0185] Transfection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, nucleofection, calcium-phosphate precipitation, lipofection, polycation-based transfection such as polyethlylenimine (PEI)-based transfection and DEAE-dextran transfection.

[0186] Suitable mammalian host cells for expressing the antibodies, antigen binding fragments thereof or variants thereof provided herein include Chinese Hamster Ovary (CHO cells) such as CHO-K1 , CHO-S, CHO-K1 SV [including dhfr- CHO cells, described in llrlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun;33(2):405- 12, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601 -621 ; and other knockout cells exemplified in Fan et al., Biotechnol Bioeng. 2012 Apr;109(4):1007-15], NS0 myeloma cells, COS cells, HEK293 cells, HKB1 1 cells, BHK21 cells, CAP cells, EB66 cells, and SP2 cells.

[0187] Expression might also be transient or semi-stable in expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB11 , Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1 , CHO-K1 SV, CHOEBNALT85, CHOS-XE, CHO-3E7 or CAP-T cells (for instance Durocher et al., Nucleic Acids Res. 2002 Jan 15;30(2):E9).

[0188] In some embodiments, the expression vector is designed such that the expressed protein is secreted into the culture medium in which the host cells are grown. The antibodies, antigen binding fragments thereof or variants thereof can be recovered from the culture medium using standard protein purification methods.

[0189] PURIFICATION

[0190] Antibodies of the invention or antigen-binding fragments thereof or variants thereof 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, phospho-cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001 ), e.g., Chapters 1 , 4, 6, 8, 9, 10, each entirely incorporated herein by reference.

[0191] Antibodies of the present invention or antigen-binding fragments thereof or variants thereof include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present invention can be glycosylated or can be nonglycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.

[0192] In preferred embodiments, the antibody is purified (1 ) to greater than 95% by weight of antibody as determined e.g. by the Lowry method, UV-Vis spectroscopy or by by SDS-Capillary Gel electrophoresis (for example on a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device), and in further preferred embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0193] ANTIBODY CONJUGATES

[0194] In addition to naked antibodies or antigen-binding fragment thereof, various antibody- or antibody fragment-based conjugates can be designed using the antibodies or antigen binding fragments disclosed herein.

[0195] In one aspect, the present invention relates to antibody conjugates targeting GPC3, comprising the isolated antibodies or antigen binding fragments according to the present invention. These conjugates may be conjugates for diagnosis, therapy, research applications, and various other purposes. The antibodies or antigen binding fragments thereof may for example be conjugated to radionuclides, cytotoxic agents, organic compounds, protein toxins, immunomodulators such as cytokines, fluorescent moieties, cells, further antibodies or antigen binding fragments thereof.

[0196] In some other embodiments, the conjugate comprises a cytotoxic agent, e.g. to form an antibody drug conjugate (ADC). For example, the cytotoxic agent may be selected from an auristatin, a maytansinoid, a kinesin-spindle protein (KSP) inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor or a pyrrolobenzodiazepine derivative.

[0197] The cytotoxic and / or cytostatic agent of the ADC may be any agent known to inhibit the growth and / or replication of, and / or kill cells. Numerous agents having cytotoxic and / or cytostatic properties are known in the literature. Non-limiting examples of classes of cytotoxic and / or cytostatic agents include, by way of example and not limitation, cell cycle modulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, alkylating agents, DNA cross-linking agents, intercalating agents, mitochondria inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA / DNA antimetabolites and antimitotic agents.

[0198] Linkers linking the cytotoxic and / or cytostatic agent(s) to the targeting moiety of an ADC may be long, short, flexible, rigid, hydrophilic or hydrophobic in nature, or may comprise segments that have different characteristics, such as segments of flexibility, segments of rigidity, etc. The linker may be chemically stable to extracellular environments, for example, chemically stable in the blood stream, or may include linkages that are not stable and release the cytotoxic and / or cytostatic agents in the extracellular milieu. A wide variety of linkers useful for linking drugs to antigen binding moieties such as antibodies in the context of ADCs are known in the art.

[0199] The number of cytotoxic and / or cytostatic agents linked to the targeting moiety of an anti-GPC3 ADC (drug-to-antibody ratio: DAR) can vary and will be limited only by the number of available attachments sites on the targeting moiety and the number of agents linked to a single linker. Typically, a linker will link a single cytotoxic and / or cytostatic agent to the targeting moiety of the ADC. In embodiments of ADCs which include more than a single cytotoxic and / or cytostatic agent, each agent may be the same or different.

[0200] In some other embodiments, the conjugate is a chimeric antigen receptor conjugate engineered for GPC3 targeting. Recently, CAR T cells have gained attention from their clinical successes and expedited FDA approvals, cf. W02020 / 102240, incorporated herein in its entirety. In the CAR T cell approach, T cells are engineered to express CARs that are specific for an antigen present on tumor cells. These engineered T cells are then re-administered to the same patient. Upon injection, CAR T cells recognize the targeted antigen on target cells to induce target cell death. T cells expressing chimeric antigen receptors (CAR T cells) thus constitute a novel modality for medical uses such as tumor treatment. The chimeric antigen receptor (CAR) is a genetically engineered receptor that is designed to target a specific antigen, for example, a tumor antigen. This targeting can result in cytotoxicity against the tumor, for example, such that CAR T cells expressing CARs can target and kill tumors via the specific tumor antigens.

[0201] According to the present invention, the antibodies or antigen binding fragments provided for GPC3 can be used to engineer CAR T cells for specific recognition of GPC3 expressing cells. Preferably, the CARs according to the current invention comprise a co-stimulation domain (e.g., CD137, CD28 or CD134) to achieve prolonged activation of T cells in vivo. Addition of a co-stimulation domain enhances the in vivo proliferation and survival of T cells containing CARs, and initial clinical data have shown that such constructs are promising therapeutic agents in the treatment of diseases, such as cancer. According to the current invention, the CAR T cells can be used to treat any disease with local or systemic aberrant presence of cells expressing GPC3.

[0202] 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.

[0203] In some preferred embodiments, the first binding moiety of the bispecific antibody is an antibody or antigen binding fragment according to the present invention and the second binding moiety of the bispecific antibody is the same or a different antibody or antigen binding fragment according to the present invention.

[0204] In some further embodiments, the first binding moiety of the bispecific antibody is an antibody or antigen binding fragment according to the present invention and the second binding moiety of the bispecific antibody is an antibody or antigen binding fragment binding to a cell-surface protein such as a cell type-specific antigen. In some of these embodiments, the second binding moiety of the bispecific antibody is an antibody or antigen binding fragment targeting 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, Cemiplimab, Dostarlimab, or Ipilimumab. In some other of these embodiments, the second binding moiety of the bispecific antibody is HER2 targeting antibody, such as Trastuzumab, Pertuzumab and / or Margetuximab.

[0205] Techniques for making bi- or multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having 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 ). Multispecific antibodies may also be made by cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)), using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol, 148(5): 1547- 1553 (1992)), using diabody technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), using single- chain Fv (sFv) dimers (see,e.g. Gruber et al., J. Immunol, 152:5368 (1994)), by preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991 ) and by controlled Fab arm exchange (cFAE) according to Labrijn AF et al. Proc Natl Acad Sci USA 2013; 110:5145- 50.

[0206] In some other embodiments, the conjugate comprises a detectable moiety. Examples of detectable moieties include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, nonradioactive paramagnetic metal ions and reactive moieties.

[0207] The detectable substance can be coupled or conjugated either directly to the antibody or fragment thereof or indirectly, e.g. through a linker known in the art or another moiety, using techniques known in the art. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; US4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, p-galactosidase, acetylcholinesterase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[0208] Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, 1311, 1 11 1n or 99mTc.

[0209] In some embodiments the conjugate may comprise a radionuclide, such as a beta particle, an alpha particle, a gamma particle or an Auger electron emitter. For example, the radionuclide may be selected from the group consisting of43Sc,44Sc,47Sc,89Zr,90Y,111In,149Tb,152Tb,155Tb,161Tb,166Ho,177Lu,186Re,188Re,212Bi,213Bi,225Ac,227Th, and232Th. Suitable beta emitters according to the current invention are for example67Cu,89Sr,89Zr,90Y,105Rh,1311,149Pm,166Ho,177Lu,186Re,188Re,198Au. In some preferred embodiments of the current invention, the radionuclide is89Zr. Suitable Auger electron emitting radionuclides are for example67Ga,71Ge,77Br,99mTc,103Pd,111In,123l,125l,140Nd,178Ta,193Pt,195mPt,197Hg. Suitable alpha emitters, according to the current invention are for example211At,212Pb,213Bi,223Ra,224Ra,225Ac, or227Th.

[0210] In some preferred embodiments, the radionuclide is an a-particle-emitting radionuclide such as225Ac.

[0211] In some particularly preferred embodiments, the radionuclide is225Ac.

[0212] In some preferred embodiments, the conjugate furthermore comprises a chelator for immobilization of the radionuclide, e.g. as described elsewhere herein.

[0213] TARGETED RADIONUCLIDE CONJUGATES ACCORDING TO THE PRESENT INVENTION

[0214] According to a further aspect, the present invention relates to targeted radionuclide conjugates comprising the anti-GPC3 antibodies or antigen-binding fragments thereof according to the present invention and a chelating moiety for chelating a radionuclide.

[0215] In some embodiments of this aspect of the present invention the chelating moiety is capable of complexing an alpha-emitting radionuclide, a beta-emitting radionuclide, a gamma-emitting radionuclide or an Auger electron emitter.

[0216] In some preferred embodiments of this aspect of the present invention the chelating moiety is capable of complexing an alpha-emitting radionuclide. I some embodiments the alpha-emitting radionuclide may be for example, but is not limited to, actinium (225Ac3+), radium (233Ra2+), bismuth (213Bi3+,212Bi3+, or211Bi3+), lead (212Pb2+or212Pb4+), thorium (227Th4+or226Th4+), dysprosium (152Dy3+), or astatine (211At+or217At+), In some highly preferred embodiments of present aspect of the invention the alpha-emitting radionuclide complexed to a chelating moiety is actinium (225Ac), preferably225Ac3+.

[0217] In some preferred embodiments of this aspect of the present invention the chelating moiety capable of complexing actinium comprises an 18-membered chelating moiety. In some further preferred embodiments of the present invention, the chelating moiety comprises the macrocyclic macropa chelator N,N'-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6 . In some highly preferred embodiments of the present invention, the chelating moiety comprises the chelator Macropa-NCS (6-[[16-[(6-carboxy-2-pyridyl)methyl]-1 ,4,10,13-tetraoxa-7,16- diazacyclooctadec-7-yl]methyl]-4-[2-(4-isothiocyanatophenyl)ethoxy]pyridine-2-carboxylic acid) (in the Examples abbreviated as “Macropa”) as for example described in W02020 / 106886.

[0218] In some preferred embodiments of the present invention the chelating moiety capable of complexing actinium terminates in an isothiocyanate or carboxylic acid moiety in order to facilitate its coupling with an antibody or an antigen-binding fragment thereof according to the present invention. When the macropa chelator is used, one of the pyridine rings of the macropa chelator is typically substituted so that it can be coupled to the anti-GPC3 antibody or an antigen-binding fragment thereof according to the invention. This can be a simple functional group (e.g., a carboxylic acid -COOH) or a more complex chemical structure, as long as it is capable of being coupled to an anti-GPC3 antibody or an antigen-binding fragment thereof. In particular embodiments of the present invention, the substituent on the pyridine ring of the macropa chelator preferably terminates in an isothiocyanate or carboxylic acid moiety.

[0219] In accordance with this aspect the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3.

[0220] In further embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof. In further embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3, selected from an isolated antibody or antigen-binding fragment according to the first aspect of the present invention comprising: i) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 82, an H-CDR2 comprising SEQ ID NO: 83, and an H-CDR3 comprising SEQ ID NO: 84 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 86, an L-CDR2 comprising SEQ ID NO: 87, and an L-CDR3 comprising SEQ ID NO: 88, or ii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L- CDR3 comprising SEQ ID NQ:108, or iii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H-CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L-CDR2 comprising SEQ ID NO: 127, and an L- CDR3 comprising SEQ ID NO:128, or iv) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H-CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L-CDR2 comprising SEQ ID NO: 147, and an L- CDR3 comprising SEQ ID NO:148, or v) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H-CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L-CDR2 comprising SEQ ID NO: 167, and an L- CDR3 comprising SEQ ID NO:168, or vi) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 182, an H-CDR2 comprising SEQ ID NO: 183, and an H-CDR3 comprising SEQ ID NO: 184 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 186, an L-CDR2 comprising SEQ ID NO: 187, and an L- CDR3 comprising SEQ ID NO:188.

[0221] In further embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3, selected from an isolated antibody or antigen-binding fragment according to the first aspect of the present invention comprising: i) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 82, an H-CDR2 comprising SEQ ID NO: 83, and an H-CDR3 comprising SEQ ID NO: 84 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 86, an L-CDR2 comprising SEQ ID NO: 87, and an L-CDR3 comprising SEQ ID NO: 88, or ii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L- CDR3 comprising SEQ ID NQ:108, or iii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H-CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L-CDR2 comprising SEQ ID NO: 127, and an L- CDR3 comprising SEQ ID NO:128, or iv) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H-CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L-CDR2 comprising SEQ ID NO: 147, and an L- CDR3 comprising SEQ ID NO:148, or v) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H-CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L-CDR2 comprising SEQ ID NO: 167, and an L- CDR3 comprising SEQ ID NO:168, or vi) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 182, an H-CDR2 comprising SEQ ID NO: 183, and an H-CDR3 comprising SEQ ID NO: 184 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 186, an L-CDR2 comprising SEQ ID NO: 187, and an L- CDR3 comprising SEQ ID NO:188, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0222] In some preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3, selected from an isolated antibody or antigen-binding fragment comprising: i) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L- CDR3 comprising SEQ ID NQ:108, or ii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H-CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L-CDR2 comprising SEQ ID NO: 127, and an L- CDR3 comprising SEQ ID NO:128, or iii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H-CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L-CDR2 comprising SEQ ID NO: 147, and an L- CDR3 comprising SEQ ID NO:148, or iv) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H-CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L-CDR2 comprising SEQ ID NO: 167, and an L- CDR3 comprising SEQ ID NO:168.

[0223] In some preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3, selected from an isolated antibody or antigen-binding fragment comprising: i) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L- CDR3 comprising SEQ ID NO:108, or ii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H-CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L-CDR2 comprising SEQ ID NO: 127, and an L- CDR3 comprising SEQ ID NO:128, or iii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H-CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L-CDR2 comprising SEQ ID NO: 147, and an L- CDR3 comprising SEQ ID NO:148, or iv) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H-CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L-CDR2 comprising SEQ ID NO: 167, and an L- CDR3 comprising SEQ ID NO:168, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0224] In some highly preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L- CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L-CDR3 comprising SEQ ID NO:108.

[0225] In some highly preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H- CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L- CDR2 comprising SEQ ID NO: 127, and an L-CDR3 comprising SEQ ID NO:128.

[0226] In some highly preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H- CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L- CDR2 comprising SEQ ID NO: 147, and an L-CDR3 comprising SEQ ID NO:148.

[0227] In some highly preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H- CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L- CDR2 comprising SEQ ID NO: 167, and an L-CDR3 comprising SEQ ID NO:168.

[0228] In some highly preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L- CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L-CDR3 comprising SEQ ID NQ:108, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0229] In some highly preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H- CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L- CDR2 comprising SEQ ID NO: 127, and an L-CDR3 comprising SEQ ID NO:128, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0230] In some highly preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H- CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L- CDR2 comprising SEQ ID NO: 147, and an L-CDR3 comprising SEQ ID NO:148, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0231] In some highly preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H- CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L- CDR2 comprising SEQ ID NO: 167, and an L-CDR3 comprising SEQ ID NO:168, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0232] In further preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 selected from an isolated antibody or antigen-binding fragment comprising i) a variable heavy chain domain comprising SEQ ID NO: 81 and a variable light chain domain comprising SEQ ID NO: 85; or ii) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or iii) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or iv) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or v) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165; or vi) a variable heavy chain domain comprising SEQ ID NO: 181 and a variable light chain domain comprising SEQ ID NO: 185.

[0233] In further preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 selected from an isolated antibody or antigen-binding fragment comprising i) a variable heavy chain domain comprising SEQ ID NO: 81 and a variable light chain domain comprising SEQ ID NO: 85; or ii) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or iii) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or iv) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or v) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165; or vi) a variable heavy chain domain comprising SEQ ID NO: 181 and a variable light chain domain comprising SEQ ID NO: 185, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof. In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 selected from an isolated antibody or antigen-binding fragment comprising i) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or ii) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or iii) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or iv) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165.

[0234] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 selected from an isolated antibody or antigen-binding fragment comprising i) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or ii) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or iii) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or iv) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165. wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0235] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105.

[0236] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125. In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145.

[0237] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165.

[0238] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0239] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0240] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0241] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165 wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0242] In particular embodiments, the isolated monoclonal antibodies comprised in the targeted actinium conjugates according to the present invention are lgG1 , lgG2, lgG3 or an lgG4 antibodies. Most particularly, the isolated monoclonal antibodies according to the present invention are lgG1 antibodies.

[0243] In further preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 selected from an isolated antibody or antigen-binding fragment comprising i) a heavy chain comprising SEQ ID NO: 97 and a light chain comprising SEQ ID NO: 98; or ii) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118; or iii) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or iv) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or v) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178; or vi) a heavy chain comprising SEQ ID NO: 197 and a light chain comprising SEQ ID NO: 198.

[0244] In further preferred embodiments the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 selected from an isolated antibody or antigen-binding fragment comprising i) a heavy chain comprising SEQ ID NO: 97 and a light chain comprising SEQ ID NO: 98; or ii) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118; or iii) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or iv) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or v) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178; or vi) a heavy chain comprising SEQ ID NO: 197 and a light chain comprising SEQ ID NO: 198, wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0245] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 selected from an isolated antibody or antigen-binding fragment comprising i) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118; or ii) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or iii) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or iv) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178.

[0246] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 selected from an isolated antibody or antigen-binding fragment comprising i) a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118; or ii) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or iii) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or iv) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178 wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0247] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118 (TPP-29537).

[0248] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138 (TPP-29538). In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158 (TPP-29636).

[0249] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178 (TPP-29638).

[0250] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain comprising SEQ ID NO: 117 and a light chain comprising SEQ ID NO: 118 (TPP-29537), wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigenbinding fragment thereof.

[0251] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138 (TPP-29538), wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigenbinding fragment thereof.

[0252] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158 (TPP-29636), wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigenbinding fragment thereof.

[0253] In some highly preferred embodiments, the present invention relates to targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178 (TPP-29638), wherein [Ab] is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigenbinding fragment thereof.

[0254] The targeted actinium conjugates (TACs) of formula (I) of the present invention comprise a chelating moiety comprising a complexed actinium atom (Ac).

[0255] In the targeted actinium conjugates (TACs) of formula (I) of the present invention, the actinium (Ac) atom is shown as being complexed to four oxygen and two nitrogen atoms of the macrocyclic ring as well as to two carboxylate groups. While this is the assumed complexation pattern for the actinium atom, this depiction includes all possible and conceivable cases in which one or more of the bonds is not present, e.g., where the actinium atom is not bound to all heteroatoms of the macrocyclic ring, or to the carboxylate groups, etc.

[0256] The anti-GPC3 antibody or an antigen-binding fragment thereof is preferably coupled to the rest of the molecule forming the targeted actinium conjugates of the present invention through a Lysine residue of the antibody. Thus, the actinium-complexating chelating moiety and the anti-GPC3 antibody or the antigen-binding fragment thereof can be coupled via an amide or thiourea moiety. When using the preferred macrocyclic chelator macropa, a terminal isothiocyanate or carboxylic acid moiety is coupled with a terminal amino group of a Lysine residue in the anti-GPC3 antibody or an antigen-binding fragment thereof, leading to the formation of a thiourea or an amide group, respectively. Thus, the preferred actinium- complexating chelating moiety comprising a macropa chelator is attached to an anti-GPC3 antibody or an antigen-binding fragment thereof via a thiourea or an amide bond derived from a terminal amino group of a Lysine residue of said anti-GPC3 antibody or an antigen-binding fragment thereof.

[0257] SYNTHESIS OF TARGETED ACTINIUM CONJUGATES ACCORDING TO THE INVENTION In a further aspect, the present invention relates to a process for the preparation of a targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof binding to GPC3, said method comprising

[0258] (i) coupling a chelating moiety of formula (II) with a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3, and (ii) contacting the resulting product with an aqueous solution comprising an ion of at least one alpha-emitting actinium isotope.

[0259] In a further embodiment, the present invention relates to a process for the preparation of a targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3, said method comprising

[0260] (i) coupling a chelating moiety of formula (II) with a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 via a Lysine residue of the monoclonal antibody or antigenbinding fragment thereof, and (ii) contacting the resulting product with an aqueous solution comprising an ion of at least one alpha-emitting actinium isotope.

[0261] In a further embodiment, the present invention relates to a process for the preparation of a targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is an anti-GPC3 antibody selected from TPP-29312, TPP-29537, TPP-29538,

[0262] TPP-29636, TPP-29638, and TPP-29642 or an antigen-binding fragment thereof, said method comprising

[0263] (i) coupling a chelating moiety of formula (II) with an anti-GPC3 antibody selected from TPP-29312, TPP-29537, TPP- 29538, TPP-29636, TPP-29638, and TPP-29642 or an antigen-binding fragment thereof via a Lysine residue of the anti-GPC3 antibody or antigenbinding fragment thereof, and (ii) contacting the resulting product with an aqueous solution comprising an ion of at least one alpha-emitting actinium isotope.

[0264] In a further embodiment, the present invention relates to a process for the preparation of a targeted actinium conjugate (TAC) of formula (I) wherein [Ab] is the anti-GPC3 antibody is selected from TPP-29537, TPP-29538, TPP-29636, orTPP-29638 or an antigen-binding fragment thereof, said method comprising

[0265] (i) coupling a chelating moiety of formula (II) with the anti-GPC3 antibody is selected from TPP-29537, TPP-29538, TPP- 29636, orTPP-29638or an antigen-binding fragment thereof via a Lysine residue of the monoclonal antibody or antigen-binding fragment thereof, and

[0266] (ii) contacting the resulting product with an aqueous solution comprising an ion of at least one alpha-emitting actinium isotope.

[0267] In step (i), a chelating moiety of formula (II) is coupled with a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3. Preferably, the anti-GPC3 antibody is selected from TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 or an antigen-binding fragment thereof. More preferably, the anti-GPC3 antibody is selected from TPP-29537, TPP-29538, TPP-29636 and TPP-29638 or an antigen-binding fragment thereof. Most preferably, the anti-GPC3 antibody is TPP-29537.

[0268] Preferably, the chelating moiety of formula (II) is coupled to the anti-GPC3 antibody or an antigen-binding fragment thereof via a Lysine residue of the monoclonal antibody, thus forming a thiourea moiety that links the chelating moiety with the anti-GPC3 antibody or antigen-binding fragment thereof.

[0269] Preferably, the coupling in step (i) is carried out under basic pH, i.e., at a pH higher than 7. More preferably, the coupling in step (i) is carried out at a pH of from 8 to 10, most preferably at a pH of 9.

[0270] After step (i), a antibody drug conjugate (ADC) of formula (III) is generated. Thus, in further embodiments, the present invention relates to a antibody drug conjugate of formula (III) wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3.

[0271] Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof is selected from TPP- 29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 or an antigenbinding fragment thereof. More preferably, the anti-GPC3 antibody is selected from TPP- 29537, TPP-29538, TPP-29636 and TPP-29638 or an antigen-binding fragment thereof. Most preferably, the anti-GPC3 antibody is TPP-29537.

[0272] In step (ii), the product resulting from the coupling in step (i) is contacted with an aqueous solution comprising an ion of at least one alpha-emitting actinium isotope. Preferably, the alpha-emitting actinium isotope is225Ac. Preferably, the ion of at least one alpha-emitting actinium isotope is225Ac having a triple positive charge, i.e.,225Ac3+. The present invention allows for the execution of step (ii) at room temperature, i.e., at a temperature that is lower than the one required for the preparation of the corresponding DOTA conjugates, which require higher temperatures (60°C).

[0273] THERAPEUTIC METHODS

[0274] Therapeutic methods involve administering to a subject in need of treatment a therapeutically effective amount of an antibody or an antigen-binding fragment thereof or a variant thereof and / or a targeted actinium conjugate (TAC) contemplated by the invention.

[0275] Uses

[0276] In a further aspect, the antibodies and / or targeted actinium conjugates (TACs) of the present invention may be used for the treatment of disease in a human or in a non-human animal subject. In particular, the antibodies and / or targeted actinium conjugates (TACs) of the present invention may be used for the treatment of hyperplastic and / or neoplastic diseases, such as cancer. In one embodiment, the antibodies and / or targeted actinium conjugates (TACs) of the present invention have specificity and / or binding affinity for GPC3.

[0277] The various aspects of the invention as described herein relate to treatment of disease, particularly for the selective targeting of diseased tissue, as well as relating to complexes, conjugates, medicaments, formulation, kits etc. useful in such methods. In all aspects, the diseased tissue may reside at a single site in the body (for example in the case of a localized solid tumor) or may reside at a plurality of sites (for example where several joints are affected in arthritis or in the case of a distributed or metastasized cancerous disease).

[0278] The antibodies and / or targeted actinium conjugates (TACs) of the present invention preferably have binding affinity for glypican-3 (GPC3). GPC3 expression in tumor cells has been observed in different types of diseases, in particular 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 neoplasms suitable for treatment using the preferred antibodies and / or targeted actinium conjugates (TACs) of the present invention include hepatocellular carcinoma (HCC), lung cancer including squamous non-small cell lung cancer, neuroendocrine prostate cancer, Merkel cell carcinoma, pediatric indications (as for example pediatric solid embryonal tumors (including the majority of hepatoblastomas), Wilms tumors, rhabdoid tumors, certain germ cell tumor subtypes (as Yolk sac + Chorio carcinoma) and some rhabdomyosarcomas), melanoma, ovarian cancer, colorectal carcinoma, esophageal carcinoma, cervical carcinoma, head and neck carcinoma, renal cell carcinoma, breast cancer (including triple negative breast carcinoma), stomach cancer, sarcomas, bladder carcinomas.

[0279] In some embodiments, the present invention includes a method of using an antibody and / or targeted actinium conjugate (TAC) of the present invention for the treatment of diseases, more preferably hyperproliferative diseases, more particularly wherein the hyperproliferative disease is cancer, and yet even more particularly wherein the cancer disease is hepatocellular carcinoma (HCC). In some embodiments, the present invention includes a method of using an antibody and / or targeted actinium conjugate (TAC) of the present invention for the treatment of diseases, preferably hyperproliferative diseases, more preferably cancer, characterized by an ectopic expression of GPC3 in the tumor cells.

[0280] In some embodiments, the present invention includes a method of treating a hyperproliferative disease, more particularly cancer, more particularly hepatocellular carcinoma, said method comprising administering an effective amount of at least one antibody and / or targeted actinium conjugate (TAC) of the present invention to a subject in need thereof. In some embodiments, the present invention includes a method of treating a hyperproliferative disease characterized by an ectopic expression of GPC3 in the tumor cells, more particularly wherein the hyperproliferative disease is cancer, more particularly hepatocellular carcinoma, said method comprising administering an effective amount of at least one antibody and / or targeted actinium conjugate (TAC) and a further pharmaceutical agent of the present invention to a subject in need thereof.

[0281] In some embodiments, the present invention provides an antibody and / or targeted actinium conjugate (TAC), as described supra, for use in the treatment and / or prophylaxis of diseases, in particular hyperproliferative disorders, more particularly cancer, even more particularly wherein the cancer disease is hepatocellular carcinoma. In some embodiments, the present invention includes an antibody and / or targeted actinium conjugate (TAC), as described supra, for use in the treatment and / or prophylaxis of hyperproliferative diseases characterized by an ectopic expression of GPC3 in the tumor cells.

[0282] In some embodiments, the present invention includes an antibody and / or targeted actinium conjugate (TAC) of the present invention for use in a method of inhibiting proliferation of a cell and / or the induction of apoptosis in a cell, comprising contacting the cell with an antibody and / or targeted actinium conjugate (TAC). In some embodiments, the present invention includes an antibody and / or targeted actinium conjugate (TAC) of the present invention for use in a method of inhibiting proliferation of tumor cells and / or the induction of apoptosis in tumor cells, comprising contacting the tumor cell with an antibody and / or targeted actinium conjugate (TAC), wherein GPC3 is overexpressed in the tumor cells.

[0283] In some embodiments, the present invention includes an antibody and / or targeted actinium conjugate (TAC) for use in a method of treating a hyperproliferative disease, more particularly wherein the hyperproliferative disease is cancer, and yet even more particularly wherein the cancer disease is hepatocellular carcinoma. In some embodiments, the present invention includes an antibody and / or targeted actinium conjugate (TAC for use in a method of treating a hyperproliferative disease characterized by an ectopic expression of GPC3 in the tumor cells.

[0284] In some embodiments, the present invention includes the use of an antibody and / or targeted actinium conjugate (TAC) for the treatment and / or prophylaxis of a hyperproliferative disease. In some embodiments, the present invention includes the use of an antibody and / or targeted actinium conjugate (TAC) for the manufacture of a medicament for the treatment and / or prophylaxis of a hyperproliferative disease characterized by an ectopic expression of GPC3 in the tumor cells.

[0285] In some embodiments, the present invention includes the use of an antibody and / or targeted actinium conjugate (TAC) for the manufacture of a medicament for the treatment of a hyperproliferative disease, particularly cancer and more particularly hepatocellular carcinoma.

[0286] The disorders mentioned above have been well characterized in humans, but also exist with a similar etiology in other animals, including mammals, and can be treated by administering pharmaceutical compositions according to the present invention.

[0287] The antibodies or antigen-binding fragments thereof according to the present invention or the targeted actinium conjugates (TACs) according to the present invention or a variant thereof might be co-administered with known medicaments, and in some instances the antibody or antigen-binding fragment thereof might itself be modified. For example, an antibody or an antigen-binding fragment thereof or a variant thereof or a TAC could be conjugated to a drug or to another peptide or protein to potentially further increase efficacy.

[0288] The antibodies or antigen-binding fragments thereof according to the present invention or the targeted actinium conjugates (TACs) according to the present invention may be administered as the sole pharmaceutical agent or in combination with one or more additional therapeutic agents where the combination causes no unacceptable adverse effects.

[0289] Thus, in a further aspect, the present invention relates to the antibodies or antigen-binding fragments thereof according to the present invention or the targeted actinium conjugates (TACs) according to the present invention or the pharmaceutical composition according to the present invention for use in simultaneous, separate, or sequential combination with one or more further therapeutically active compounds having biological activity, preferably against cancer, especially hepatocellular cancer..

[0290] Non-limiting examples of therapeutically active compounds to be used in combination with the antibodies or antigen-binding fragments thereof according to the present invention or the targeted actinium conjugates (TACs) according to the present invention are listed below. These compounds are defined by their classes, thus allowing a skilled person to identify such compounds belonging to said class.

[0291] In the context of the present invention, a TK inhibitor refers to a compound which is a pharmacological inhibitor of a tyrosine kinase. Tyrosine kinases are receptors responsible for the activation of many proteins by signal transduction cascades. Examples thereof include, but are not limited to, sorafenib, lenvatinib, regorafenib or cabozantinib.

[0292] In the context of the present invention, an VEGFR inhibitor refers to a compound which is a pharmacological inhibitor of vascular endothelial growth factor receptor (VEGFR) signaling that plays a role in the regulation of tumor-induced angiogenesis. Examples thereof include, but are not limited to, bevacizumab and ramucirumab.

[0293] In the context of the present invention, a chemotherapeutic agent refers to a compound suitable for chemotherapy of GPC3 positive cancers, especially HCC. Examples thereof include, but are not limited to, doxorubicin, epirubicin, or cisplatin.

[0294] Examples for early-stage pipeline drugs which may be used in the context of the present invention in combination with the antibodies or antigen-binding fragments thereof according to the present invention or the targeted actinium conjugates (TACs) according to the present invention include, but are not limited to tivozanib (AVEO Oncology), namodenoson (Can-Fite BioPharma), GT90001 (Kintor Pharmaceutical), ARTEMIS T-cell therapy (Eureka Therapeutics), MTL-CEBPA (MiNA Therapeutics), fostroxacitabine bralpamide / fostrox (Medivir), or various TIG IT inhibitors.

[0295] In the context of the present invention, a transarterial chemoembolization agent refers to a compound suitable for intraarterial application into an artery that specifically supply blood to an intrahepatic HCC lesion. Examples thereof include, but are not limited to, doxorubicin, epirubicin, or cisplatin, doxorubicin releasing drug elution beads, lipiodol, Yttrium-90 (beta- emitter)-labeled glass or resin microspheres or embolization agents likegelatin sponge [Gelfoam] or degradable starch microspheres. In the context of the present invention, external beam radiotherapy refers to radiation therapy in which an external source of ionizing radiation is pointed at and / or applied to a particular part of the body of a subject.

[0296] In the context of the present invention, the term “combination” means not only a dosage form which contains all the components (i.e., the anti-GPC3 antibodies or fragments thereof and / or the anti-GPC3-TACs according to the present invention) and a further pharmaceutical agent, also called fixed-dose combinations), and combination packs containing the components separate from one another, but also components which are administered simultaneously or sequentially, as long as they are employed for the prophylaxis or treatment of the same disease.

[0297] The amount of the administered active ingredient can vary widely according to such considerations as the particular compound and dosage unit employed, the mode and time of administration, the period of treatment, the age, sex, and general condition of the patient treated, the nature and extent of the condition treated, the rate of drug metabolism and excretion, the potential drug combinations and drug-drug interactions, and the like.

[0298] PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

[0299] In a further aspect, the present invention relates to pharmaceutical compositions comprising the isolated antibodies or antigen-binding fragments according to the present invention or the targeted actinium conjugates according to the present invention. To treat any of the foregoing disorders, pharmaceutical compositions for use in accordance with the present invention may be formulated in any conventional manner using one or more physiologically acceptable carriers, excipients, or auxiliaries. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).

[0300] The isolated antibodies or antigen-binding fragments according to the present invention or the targeted actinium conjugates according to the present invention can be administered by any suitable means, which can vary, depending on the type of disorder being treated. Possible administration routes include oral, parenteral, and topical administration. Methods of parenteral delivery include intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In addition, the isolated antibodies or antigen-binding fragments according to the present invention or the targeted actinium conjugates according to the present invention may be administered by pulse infusion, with, e.g., declining doses . Preferably, administration is by injections, most preferably by intravenous injection. The amount to be administered will depend on a variety of factors such as the clinical symptoms, weight of the individual, whether other drugs are administered, and the like. The skilled artisan will recognize that the route of administration will vary depending on the disorder or condition to be treated.

[0301] The pharmaceutical composition according to the present invention comprises the antibody or antigen-binding fragment according to the present invention or the targeted actinium conjugate according to the present invention alone or in combination with at least one other agent, such as a stabilizing compound. The antibody or antigen-binding fragment according to the present invention or the targeted actinium conjugate according to the present invention may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. In particular embodiments, the pharmaceutical composition according to the present invention may comprise one or more further pharmaceutically active compounds, in particular one or more further pharmaceutically active compounds that are suitable to treat GPC3 associated disorders and / or disorders associated with ischemic events due to partial or complete vessel occlusion. Any of these agents can be administered to a patient alone, or in combination with other agents or drugs, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers. In particular embodiments, the pharmaceutically acceptable carrier is pharmaceutically inert.

[0302] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.

[0303] Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores can be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e. dosage.

[0304] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

[0305] Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles 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 agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0306] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0307] The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, drageemaking, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0308] The pharmaceutical composition may be provided as a salt and can be formed with acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be liquid formulation or a lyophilized powder thereof in histidine or phosphate or Tris buffer, sucrose and / or mannitol at a pH range of 4.5 to 7.5 optionally comprising additional substances like polysorbate that is combined with buffer prior to use.

[0309] After pharmaceutical compositions comprising a compound of the invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of anti-GPC3 antibodies or antigen-binding fragment thereof or anti-GPC3-TACs, such labeling would include amount, frequency and method of administration.

[0310] THERAPEUTICALLY EFFECTIVE DOSE

[0311] Pharmaceutical compositions suitable for use according to the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose, e.g., treatment of a particular disease state characterized by an ectopic expression of GPC3.

[0312] The determination of an effective dose is well within the capability of those skilled in the art. Determining a therapeutically effective amount of the novel antibody of this invention or an antigen-binding fragment thereof or a variant thereof, largely will depend on particular patient characteristics, route of administration, and the nature of the disorder being treated. General guidance can be found, for example, in the publications of the International Conference on Harmonization and in 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 a therapeutically effective amount will depend on such factors as toxicity and efficacy of the medicament. Toxicity may be determined using methods well known in the art and found in the foregoing references.

[0313] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, or in animal models, usually mice, rabbits, dogs, pigs or monkeys. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0314] A therapeutically effective dose refers to that amount of antibody or antigen-binding fragment thereof or that amount of antibody conjugate, that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED5o (the dose therapeutically effective in 50% of the population) and LD5o (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED50 / LD50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0315] The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance / response to therapy. Long-acting pharmaceutical compositions might be administered for example every 3 to 4 days, every week, once every two weeks, or once every three weeks, depending on halflife and clearance rate of the particular formulation.

[0316] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 10 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature. See U.S. Pat. No. 4,657,760; 5,206,344; or 5,225,212.

[0317] KITS

[0318] In a further aspect, the present invention relates to kits comprising the isolated antibodies or antigen-binding fragments according to the present invention or the targeted actinium conjugates according to the present invention and instructions for use. In particular embodiments, the kits comprise one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.

[0319] DIAGNOSTIC METHODS According to a further aspect the antibodies or antigen-binding fragments according to the present invention or the antibody conjugates according to the present invention may be utilized, as such or in compositions, in research and diagnostics, or as analytical reference standards, and the like.

[0320] Anti-GPC3 antibodies or antigen-binding fragments thereof or the antibody conjugates according to the present invention can be used for detecting the presence of GPC3. Thus, in a further aspect, the present invention relates to the isolated antibodies or antigen-binding fragments according to the present invention or the antibody conjugates according to the present invention for use as a diagnostic agent.

[0321] Detection of expression of GPC3 generally involves contacting a biological sample (tumor, cells, tissue, or body fluid of an individual) with one or more antibodies or fragments according to the current invention (optionally conjugated to a detectable moiety) and detecting whether the sample is positive for GPC3, or whether the sample has altered (e.g., reduced or increased) expression as compared to a control sample.

[0322] In certain embodiments, provided conjugate comprises one or more detectable moieties, i.e., is “labeled” with one or more such moieties. In some such embodiments, a conjugate of the present disclosure is useful in diagnostic or imaging applications, e.g., diagnosing or imaging cancer. Any of a wide variety of detectable moieties may be used in labeled antibody conjugates described herein. Suitable detectable moieties comprise, without limitation: various ligands, radionuclides; fluorescent dyes (e.g. fluorescent resonance energy dyes (FRET) as i.e.Cy5, Cy7); chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g, gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (such as, for example, dyes, colloidal gold, and the like); biotin; dioxigenin; haptens; and proteins for which antisera or monoclonal antibodies are available.

[0323] According to certain preferred embodiments of the present invention, the detectable moiety can be fluorescent dyes as e.g. Alexa Flour dyes (Invitrogen), or fluorescent resonance energy dyes (FRET) as e.g. the pH sensitive dye CypHer5E-NHS-Ester (Cytiva) which can be used for direct labeling of antibodies or antigen-binding fragments via lysine residues.

[0324] PET IMAGING According to a further aspect the antibodies or antigen-binding fragments according to the present invention and / or conjugates thereof may be utilized as imaging agents, preferably as PET imaging agents.

[0325] 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 emitter. When combined with computed tomography (CT), PET / CT allows for the precise localization of the positron emitter within the anatomical framework of the body. Multiple positron emitters are routinely used in the clinic, such as C-1 1 , F-18, Ga-68 and Zr- 89. Preferably, Zr-89 is used as positron emitter in combination with antibodies or antigenbinding fragments according to the present invention or the antibody conjugates according to the present invention, due to its long half-life (78.4 hours) and efficient labeling reaction in gentle conditions.

[0326] Suitable chelators for Zr-89 in PET imaging applications include, but are not limited to, for example DFO (Desferrioxamine), N-succinyl-desferrioxamine-tetrafluorophenol ester (N-suc- DFO-TFP ester) p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS, also known as DFO- Ph-NCS), desferrioxamine-maleimide (DFO-maleimide), desferrioxamine-squaramide (DFO- sq), or DFO*. All these chelators can be conjugated with 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 present invention, the chelators are conjugated the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

[0327] DFO* is an extended version of DFO and89Zr-DFO* complexes are shown to exhibit increased in vitro stability and improved in vivo properties compared to DFO complexes (W02021051 168) and therefore is a preferred chelator for Zr-89 when conjugated with antibodies or antibody fragments according to the present invention for use in PET imaging.

[0328] DFO* refers to 5,11 ,16,22-Tetraazahexacosanediamide, N1-[5-(acetylhydroxyamino)pentyl]- N26,5,16-trihydroxy-N26-[5-[[[(4-isothiocyanatophenyl)amino]thioxomethyl]amino]pentyl]- 4,12,15,23-tetraoxo (CAS no.1810009-29-0):

[0329] and can according to some embodiments of the present invention be conjugated to an antibody or an antigen binding fragment thereof [Ab], preferably to a monoclonal antibody or an antigenbinding fragment thereof binding to GPC3. Preferably, the anti-GPC3 antibody is selected from TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 or an antigenbinding fragment thereof. More preferably, the anti-GPC3 antibody is selected from TPP- 29537, TPP-29538, TPP-29636 and TPP-29638 or an antigen-binding fragment thereof.

[0330] According to certain highly preferred embodiments of the present invention, DFO* is coupled to the anti-GPC3 antibody or an antigen-binding fragment thereof according to the present invention via a lysine residue of the monoclonal antibody, thus forming a thiourea moiety that links the chelating moiety with the anti-GPC3 antibody or antigen-binding fragment thereof.

[0331] One skilled in the art, utilizing the preceding information, can utilize the present invention to its fullest extent.

[0332] SHORT DESCRIPTION OF THE FIGURES

[0333] Figure 1 : FACS binding of anti-GPC3 antibodies TPP-14890 and TPP-29537 to human GPC3 transiently transfected into CHO cells. TPP-5657 is an isotype control antibody.

[0334] Figure 2: FACS binding of anti GPC3 antibodies TPP-14890 and TPP-29537 to cynomolgus GPC3 transiently transfected into CHO cells. TPP-5657 is an isotype control antibody.

[0335] Figure 3: FACS binding of anti GPC3 antibodies TPP-14890 and TPP-29537 to mouse GPC3 transiently transfected into CHO cells. TPP-5657 is an isotype control antibody.

[0336] Figure 4: FACS binding of anti GPC3 antibodies TPP-14890 and TPP-29537 transiently mock transfected CHO cells. TPP-5657 is an isotype control antibody. Figure 5: Calculated vesicle area per cell at 5 hours after addition of directly labelled antibodies to HepG2 cells. TPP-5657 is an isotype control antibody.

[0337] Figure 6: Calculated vesicle area per cell at 5 hours after addition of Fabfluor labelled antibodies to HepG2 cells. TPP-5657 is an isotype control antibody.

[0338] Figure 7: Antibody binding capacities on cell surface determined by Flow Cytometry with Alexa 488 labeled TPP-14890 on cell lines HFF-1 , SU-DHL-10, PLC / PRF / 5, HepG2, Hep3B2 and primary fibroblasts HCF and HPF.

[0339] Figure 8: Antibody binding capacities on cell surface determined by Flow Cytometry with Alexa 488 labeled TPP-29537 on cell lines HFF-1 , SU-DHL-10, PLC / PRF / 5, HepG2, Hep3B2 and primary fibroblasts HCF and HPF.

[0340] Figure 9: 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)] x 100.

[0341] Figure 10: In vitro cytotoxicity data of Ac-225-Macropa-TPP-29537 (CAR 0.5 and CAR 1 1 ), Ac- 225-Macropa-TPP- 14890 (CAR 0.7 and CAR 10) and Ac-225-Macropa-isotype-control (CAR 0.7) inHepG2 cells (A) and in HEP-3B cells (B).

[0342] Figure 11 : Biodistribution of A) Ac-225-Macropa-TPP-29537 (CAR 0.7) and B) 225Ac- Macropa-isotype-control (CAR 0.7) in different tissues in the human hepatoma xenograft model Huh-7. Animals were dosed with total antibody dose of 0.75 mg / kg, 500kBq / kg single dose i.v. Accumulated Ac-225 is given in % of injected dose per gram (%ID / g).

[0343] Figure 12: A) In vivo efficacy of Ac-225-Macropa-TPP-29537 (CAR 0.7) and Ac-225-Macropa- isotype-control (CAR 0.7) in the human hepatoma xenograft model Huh-7. Tumors were measured using a digital caliper, and the results are presented as tumor area (mm2), calculated as longest x shortest diameter of the tumors. B) % Survival of in vivo efficacy study in Huh-7 tumor model.

[0344] Figure 13: 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)] x 100.

[0345] Figure 14. Coronal PET / CT MIP of imaged HEP-3B tumor bearing mice with Zr-89-DFO*-TPP- 29537. 2 h n=1 , 4 h n=1 , 24 h n=3, 72 h n=3 and 168 h n=2.

[0346] Figure 15: Ex vivo and in vivo PET / CT quantification of Zr-89-DFO*-TPP-29537 and ex vivo biodistribution of Ac-225-Macropa-TPP-29537 in the HEP-3B tumor model. Ex vivo biodistribution data: n=3 per time point. Imaging based analysis: 2 h n=1 , 4 h n=1 , 24 h n=3, 72 h n=3 and 168 h n=2. Data expressed as mean+SD

[0347] EXPERIMENTAL SECTION

[0348] Chemical names were generated using the BIOVIA Draw 2019 from Dassault Systemes. In some cases, generally accepted names of commercially available reagents were used in place of BIOVIA Draw generated names.

[0349] The following Table lists the abbreviations used in this paragraph and in the Examples section as far as they are not explained within the text body. Other abbreviations have their meanings customary per se to the skilled person.

[0350] Table 3. Abbreviations

[0351] The following table lists the abbreviations used herein.

[0352] 225Ac actin ium-225

[0353] Ac-225 actinium-225

[0354] ACC antibody-chelator conjugate

[0355] CAR chelator-to-antibody ratio

[0356] CT computed tomography

[0357] DCTA 1 ,2-diaminocyclohexanetetraacetic acid

[0358] DMA N,N-dimethylacetamide

[0359] DMSO dimethyl sulfoxide

[0360] Edll 5-Ethynyl-2’-deoxyuridine

[0361] ESI electrospray ionization

[0362] EtOH ethanol

[0363] FPLC fast protein liquid chromatography

[0364] FSC forward scatter

[0365] GPC3 glypican-3

[0366] HPGe high purity germanium

[0367] HPLC high performance liquid chromatography iTLC instant thin layer chromatography

[0368] IRF immunoreactive fraction mAb monoclonal antibody min minutes

[0369] MS mass spectrometry

[0370] NaCI sodium chloride nm nanometer nmol nanomole

[0371] PBS phosphate buffered saline

[0372] PET positron emission tomography

[0373] RAC radioactive concentration

[0374] RCP radiochemical purity

[0375] RPMI Roswell Park Memorial Institute 1640 medium

[0376] SEC size exclusion chromatography

[0377] SCC side scatter

[0378] TAC targeted actinium conjugate (Ac-225 labelled ACC)

[0379] TFA trifluoroacetic acid

[0380] TOF time of flight

[0381] UPLC ultra performance liquid chromatography

[0382] UV ultraviolet

[0383] All reagents, for which the synthesis is not described in the experimental part, are either commercially available, or known compounds or may be formed from known compounds by known methods by a person skilled in the art.

[0384] The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way. The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.

[0385] Anti-GPC3 antibodies and targeted radionuclide conjugates thereof

[0386] Example 1 : Generation of anti-GPC3 antibodies and reference compounds

[0387] All antibodies were expressed in HEK293 cells using standard transient transfection procedures and purified from the cell culture supernatant via Protein-A and size exclusion chromatography.

[0388] In some examples, prior art antibody sequences such as those of TPP-14890 (Codrituzumab) were randomly altered. Only few of the resulting antibodies showed improved or mostly unchanged behavior. Only examples with improved or mostly unchanged characteristics are included herein.

[0389] Prior art antibody sequences such as those of TPP-14890 were furthermore subjected to sequence germlining and further alterations were introduced, such as removal of lysine residues in CDRs or potential aspartate isomerization sites. The resulting antibodies (TPP- 29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, TPP-29642) were characterized with regard to monovalent binding affinity (KD) as assessed by surface plasmon resonance (SPR) on human and cynomolgus GPC3 as described in Example 2, and cellular binding (EC50) to CHO cells transfected with either human or cynomolgus GPC3 by flow cytometry as described in Example 3. Furthermore, the melting temperature of the Fab (Tm Fab) was determined by Differential Scanning Calorimetry (DSC) as described in Example 4. All these data are summarized in Table 4.

[0390] Table 4: Summary table comparing properties of antibodies according to the invention to TPP- 14890.

[0391] Surprisingly, despite a very high number of mutations in both VH and VL as compared to TPP- 14890, variants were identified which a) still could be produced recombinantly, b) showed comparable or superior affinities in either binding to recombinant or cellular human GPC3, c) still showed binding to cynomolgus GPC3, d) still showed stability of the Fab part Tmabove 65 °C, and e) showed expression rates above 145 mg / L in a standard HEK293E expression system (as listed in Table 4) hinting for a good manufacturability. Yet, the number of germline deviations was dramatically reduced, which is also reflected in a much reduced in silico immunogenicity score (IPAD, Epibase) as well as in vitro immunogenicity assessment (Epibase). Furthermore, the number of lysine residues within the CDRs was reduced from four lysine residues in the CDRs of TPP-14890 to one or even no lysine residue in the CDRs of the antibodies according to the invention (Table 4). Reducing the number of lysine residues within the CDRs lowers the risk, that chelators are coupled to the antibody within the CDR regions. Binding of chelators within the CDRs would affect binding of the antibody conjugate to its target GPC3. For variants TPP-29537, TPP-29538, TPP-29636 and TPP-29638 which do not contain any lysine residue in the CDRs, respectively this risk is completely avoided.

[0392] Example 2: SPR Comparison of TPP-14890 and antibodies according to the present invention To assess the binding kinetics and affinity of the anti-GPC3-antibodies as well as their crossreactivity to cynomolgus GPC3, binding assays were conducted using surface plasmon resonance (SPR). SPR-based experiments were run on a Biacore T200 instrument (formerly GE Healthcare Inc., now Cytiva) at 25°C using anti-human Fc capture antibodies covalently coupled to CM5 sensor chip (Cytiva).

[0393] Anti-human Fc capture antibodies were covalently coupled using the amine coupling kit (Cytiva) following the instructions of the manufacturer to approximately 5.000 RU. Capture Level, RMax as well as resulting % Activity showed comparable behavior for all variants in SPR (data not shown). Analytes (human and cynomolgus GPC3, R&D Systems, catalog No. 2119- GP and 10509-GP, respectively) were injected in multicycle kinetics mode in a titration series from 0.039 nM - 200 nM. The experiments were run in HBS-EP+ (Cytiva). Association time was set to 300 s, the dissociation time to 1.200 s and the flow rate was set to 30 pl / min. Obtained data were double referenced (subtraction of the reference cell and the 0 nM concentration signal) and fitted to a 1 :1 Langmuir binding isotherm (Rl set to constant) using the Biacore T200 evaluation software. Results are shown in Table 5.

[0394] Table 5: Kinetic data of TPP-14890 and antibodies according to present invention using SPR.

[0395] As can be seen from the kinetic data, TPP-29312, TPP-29537, TPP-29538, and TPP-29636 show affinity (KD) in a comparable order of magnitude as TPP-14890. TPP-29638 and TPP- 29642 show a slightly higher affinity compared to TPP-14890 and the other variants.

[0396] Example 3: GPC3 antibody binding to CHO cells transiently transfected with human, mouse or cynomolgus GPC3

[0397] To assess binding of the anti-GPC3 antibodies to cell bound human, cynomolgus or murine GPC3, the binding of TPP-14890 and antibodies according to the present invention to CHO cells transiently transfected with human, cynomolgus or murine GPC3 was assessed by FACS.

[0398] Transient transfection of CHO cells using GPC3 expression constructs

[0399] CHO-3E7 cells were grown in Freestyle F17 (fisherscientific, #10388033), 4mM Glutamax, 0.1 % Pluronic, 5% CO2, 37°C, 75 rpm in 35ml spin tubes (TPP, #87050). Human GPC3 (SEQ ID NO:321 ), mouse GPC3 (SEQ ID NO:322) or macaca fascicularis (cynomolgus monkey) GPC3 (SEQ ID NO:323) expression plasmid, previously generated by recombinant DNA technology, were transfected into CHO-3E7 cells at a cell density of 3x105cells per ml, and then incubated for three days at 37°C, 5% CO2.

[0400] Analysis of antibody binding to GPC3 transfected CHO cells by FACS

[0401] Five pl of a 500 pg / ml solution of the test antibody in PBS pH 7.4 were added into a well of a microtiter plate (Eppendorf, #0030601300). Then, a 1 :5 dilution series was prepared down to 0.0064 pg / ml. Next, 45 pl of a solution containing 2.5x104GPC3 transfected CHO cells, preincubated for 30 min on ice, were added to each well. After the microtiter plate were incubated for 45 min on ice, the plate was centrifuged for 5 min at 400xg and 4°C. Supernatants were discarded and the cell resuspended in 110 pl ice cold FACS buffer (PBS, 3% FCS), centrifuged again and resuspended for another wash in 1 10 pl ice cold FACS buffer. After a third round of centrifugation, the cells were resuspended in 50 pl FACS buffer, containing anti human Fab-Dimer specific and Alexa Fluor 488 labeled secondary reagent (Dianova, #109- 456-097) diluted 1 :200. After an incubation for 40 min on ice, the plate was centrifuged for 5 min at 400xg at 4°C. The supernatant was removed, and the cells resuspended in 1 10 pl FACS buffer for a final centrifugation step. Finally, the cells were resuspended in FACS buffer containing propidiumiodid (1 pig / ml), shaken at 2500 RPM for 5 sec and subjected for FACS measurement on an iQue3 instrument, using 4 sec sip as instrument setting. Results for TPP- 14980 and TPP-29537 as well as the none-binding isotype control TPP-5657 are shown in Figure 1 , Figure 2, Figure 3, and Figure 4.

[0402] EC50 values were calculated from the FACS binding curves using non-linear regression analysis integrated in the software GraphPad Prism. Results have been determined for all antibody variants as described here and are summarized in Table 6.

[0403] Table 6: EC50 values of TPP-14890 and antibodies according to present invention on CHO cells transiently transfected with human, cynomolgus or murine GPC3

[0404] In summary, TPP-14890, TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 show comparable FACS binding properties: All antibodies show strong binding to human and cynomolgus GPC3, whereas no binding to murine GPC3 was detectable. The isotype control antibody TPP-5657 does not show any binding to human, murine or cynomolgus GPC3.

[0405] Example 4: Thermostability of anti-GPC3 antibodies

[0406] Thermostability of various anti-GPC3 antibodies was determined using Differential Scanning Calorimetry (DSC) using a VP-Capillary DSC system (Malvern Instruments GmbH, former MicroCai Inc.) with a cell volume of 0.137 ml. All samples were diluted in PBS buffer to a final concentration of 0.5 mg / mL, and a buffer control without protein was used as a reference. The samples were scanned from 20 °C to 95 °C at a scan rate of 120 °C / h. The data were analyzed using Origin 7.0 Data analysis (OriginLab Corp.). Results are shown in Table 6.

[0407] Table 6: Fab melting temperatures of various GPC3 antibodies.

[0408] All antibodies according to the present invention show Tm values of Fab well above 65 °C and thus are well suited for a clinical development for human therapy.

[0409] Example 5: Germlining of antibody sequences

[0410] Prior art antibody TPP-14890 carries multiple deviations from the human germline as described in the IMGT database (cf. http: / / www.imgt.org / ).

[0411] TPP-14890 carries 15 deviations from the closest germline light chain identified (IGKV2-28- IGKJ2, TPP-35038; consecutive numbering; SEQ ID NO:325): I2V, L30V, G34R, Y35N, N36T, D39H, L55K, G56V, A60F, M94S, A96N, L97T, Q98H, T99V, Y101 P.

[0412] TPP-14890 carries 17 deviations from the closest germline heavy chain identified outside of CDR-H3 (IGHV1 -46-IGHJ4, TPP-35037; consecutive numbering; SEQ-ID NO: 324): S31 D, Y33E, I50A, 151 L, N52D, S54K, G55T, S57D, S59A, A61 S, Q65K, M70L, R72A, T74K, V79A, R87T, A97T.

[0413] A lower number of germline deviations of an antibody in comparison to its closest human germline sequence, reduces the murine content of the antibody or a phage-display derived human antibody without affecting antigen binding, thereby increasing the overall “humanness” and leading to a reduced risk of immunogenicity caused by those molecules (Hwang et al.; Methods, Volume 36, Issue 1 , 2005, Pages 35-42, ISSN 1046-2023). Details are described in Table 7. Table 7: Number of germline deviations in Light and Heavy Chain (excluding CDR-H3) and respective mutations as compared to human reference sequence for TPP-14890 family.

[0414] Compared to the TPP-14890 antibody, the antibodies according to the present invention carry a reduced number of amino acid deviations from germline. In fact, TPP-14890 has 32 germline deviations in total from human germline. In contrast, the antibodies according to the invention have only 14-16 germline deviations in total from human germline leading to a reduced risk of immunogenicity in human therapy.

[0415] In summary, TPP-29312, TPP-29537, TPP-29538, TPP-29636, TPP-29638, and TPP-29642 have significant lower numbers of germline deviations than the already humanized prior art antibody TPP-14890, thereby further decreasing the risk of immunogenic reaction upon use in human therapy. Example 6: in silico immunogenicity assessment

[0416] The potential for provoking an immunogenicity reaction in human therapy was assessed using two independent in silico prediction methods: IPAD score

[0417] The IPAD v4.03 algorithm method is relying on NetMHClIpan algorithm (Nielsen et al., Quantitative predictions of peptide binding to any HLA-DR molecule of known sequence: NetMHClIpan. PLoS Comput Biol. 2008 Jul 4;4(7):e1000107), which predicts peptide binding to HLA class II molecules. Briefly, VH and VL sequences are run through this algorithm and filtered for non-germline peptides. An HLA score is calculated by multiplying the number of predicted HLA binding peptides with the HLA frequency in % and a scaling factor (0.19352). The IPAD score is then generated by the sum of all individual HLA scores. Using this methodology, IPAD scores between 600 and 1000 are comparable to marketed fully human and humanized antibodies, scores lower than 600 compare favorably, whereas scores above 1000 indicate a higher risk for immunogenicity.

[0418] Table 8: Comparison of IPAD scores of prior art antibodies and of antibodies according to the present invention As depicted in Table 8, TPP-14890 has an IPAD score of 1129, which indicates a higher risk for immunogenicity as opposed to the antibodies according to the present invention 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, TPP-29636 have IPAD scores even below 600. The fully human antibodies 2-F7, 4-1 1 G, 7-5B, 7-8B and 17-4D show comparable or worse IPAD scores compared to antibodies according to present invention. YP7 is a murine antibody and has the highest IPAD score. The humanized variant hYP7 is significantly improved but still shows a worse IPAD score compared to antibodies according to present invention. Likewise, hYP9.1 b (also a humanized antibody) shows worse IPAD scores compared to antibodies according to present invention and is in a similar range as hYP7.

[0419] Epibase in silico Immunogenicity platform (Lonza)

[0420] Epibase in silico (IS) is a T-cell epitope screening platform from Lonza that analyzes and predicts the potential immunogenicity of proteins.

[0421] The sequences of TPP-14890 and TPP-29537 were subjected to immunoprofiling using the Epibase platform’s “HLA class II - Global v4.0” settings, and the putative HLA class II restricted epitope contents of the antibodies were compared. Profiling was performed at the allotype level for 43 DRB1 , 8 DRB3 / 4 / 5, 22 DQ and 12 DP, i.e. 86 HLA class II receptors in total. The DRB1 scores (sum of the products of critical epitope counts multiplied by allele frequency of affected allotype) are shown in Table 9.

[0422] Table 9: DRB1 risk scores of the antibodies tested in global population.

[0423] TPP-29537 has a lower number of critical DRB1 epitopes (25 epitopes), while TPP-14890 has a higher number of critical DRB1 epitopes (30 epitopes). Similarly, for the DRB3 / 4 / 5 family, TPP-29537 has the lowest epitope count (10 epitopes) and TPP-14890 has the higher with 12 epitopes. Both antibodies, TPP-14890 and TPP-29537, do not have any critical DQ epitopes. For the DP family, both TPP-29537 and TPP-14890 have 3 DP epitopes. It is evident from Table that TPP-29537 has the lower DRB1 risk score with only 785.9 as compared to TPP-14890, which has an DRBI risk score of 924.7.

[0424] In summary, the DRB1 risk score for TPP-29537 is comparable to that of fully human therapeutic antibodies, and the epitope counts are at the upper end of human antibodies, while DRB1 risk score for TPP-14890 is comparable to that of humanized therapeutic antibodies and epitope counts are at the lower end of humanized antibodies range. Thus, considering the overall critical epitope count and the population frequencies of the affected HLA allotypes, TPP-14890 carries a greater immunogenic risk than TPP-29537 in human therapeutic applications.

[0425] In summary, both independently used in silico prediction methods for provoking an immunogenicity reaction in human therapy, IPAD and Epibase indicated a reduced immunogenicity potential for TPP-29537 as compared to TPP-14890. Thus, from a patient safety perspective, TPP-29537 is more favorable than TPP-14890.

[0426] Example 7: in vitro immunogenicity assessment

[0427] To further confirm the in-silico immunogenicity predictions, the potential for provoking an immunogenicity reaction in human therapy was also assessed using an in vitro immunogenicity assessment method. To this end, antibody chelator conjugates of TPP-14890 and TPP-29537 resulting in the conjugates TPP-29537-Macropa and TPP-14890-Macropa were prepared using the chelator Macropa-NCS (as described in Example 10) and subjected to the “Epibase in vitro PBMC Proliferation Assay” (Lonza).

[0428] The assay implements Lonza’s in vitro T cell assay platform for the assessment of T cell proliferation using human peripheral blood mononuclear cell (PBMCs) from healthy donors. The T cell response, induced by test molecules, was assessed in PBMC from 31 healthy human donors with HLA-DRB1 frequencies resembling global population frequencies. The PBMC assay utilizes flow cytometry to detect the number of proliferating cells after stimulation with test molecules.

[0429] PBMC were seeded in 96 well plates. Test proteins, diluted in assay media, were added to the cells. Assay media alone was used as blank and KLH (Keyhole Limpet Hemocyanin) was used as naiive positive control. PBMCs were incubated for 7 days in 5% CO2 and at 37 °C. On day 7, PBMCs were labelled for CD3 and CD4 markers and DNA-incorporated 5-Ethynyl-2’- deoxyuridine (EdU) was used as proliferation marker. KLH was used as a positive control and was a potent driver of CD4+ T cell proliferation, indicating that the assay performed as expected: 31 / 31 donors (100%) showed a significant T cell response (SI >2.00 and p< 0.05) to KLH compared to the blank condition (Mean SI 28.94 and p< 0.0001 ). Results are shown in Table 10. Table 10: Donor response of each test condition compared to blank.

[0430] SI = Stimulation Index, p-value relates to the single sample t-test hypothesis that the frequency distribution of SI for a given antigen represents a distribution with a mean SI value of 1 .

[0431] In line with in-silico predictions, more donors responded to TPP-14890 antibody chelator conjugate (5 donors equivalent to 16%) than to TPP-29537 antibody chelator conjugate (2 donors equivalent to 6%), even when testing two different batches.

[0432] In conclusion, TPP-14890 antibody chelator conjugate raised significant CD4+ T cell responses in 16% of the donors, suggesting a moderate risk for immunogenicity. In contrast, TPP-29537 antibody chelator conjugate had responses in only 6% of the donors. Thus, TPP- 29537 antibody chelator conjugate can be ranked bearing only a low risk (< 10% of donors responding) for immunogenicity in human therapy. Thus, from a patient safety perspective, TPP-29537 is more favorable than TPP-14890.

[0433] Example 8: anti-GPC3 antibody internalization into HepG2 cells The following section describes characterization of different anti-GPC3 antibodies regarding their internalization profile.

[0434] Internalization is a process in which an antibody, after binding to its specific tumor target, is taken up into the tumor cell. Internalization is an essential prerequisite for the effectiveness of antibody drug conjugates (ADCs), since the toxin bound to the antibody is only released inside the cell and develops its cytotoxic effect. Internalization is not a prerequisite for the effect of TACs, since the radioactive radiation can kill the target cell after binding of the TAC to the receptor even without internalization. However, internalization can also be beneficial here as it may contribute to improved tumor retention.

[0435] Internalization of directly CypHer5E labelled anti-GPC3 antibodies.

[0436] In a first step, the antibodies TPP-29537 and TPP-14890 were directly labelled at a concentration of 1 mg / ml with the pH sensitive dye CypHer5E-NHS-Ester (Cytiva, PA 15401 ), which was coupled to lysine residues of the antibodies according to the instruction of the manufacturer.

[0437] After labelling the dye to protein ratio was estimated to be 21.5 for TPP-29537 and 16.4 for TPP-14890.

[0438] HepG2 cells (20,000 cells / well) were plated onto Greiner pCLEAR 96-well microtiter plates (Greiner, #655090) in 100 pl EMEM medium (ATCC, 30-2003) containing 10% FCS. After an overnight incubation at 37°C and 5% CO2, 10 pg Hoechst 33342 (Invitrogen, #H3570) staining solution in 10 pl medium was added to each well and incubated for 10 min in the dark. Next, the medium was removed and replaced by 90 pl fresh medium as well as the respective antibody of interest (10 pL per well, final concentration of 2 pg / mL). Cells were then incubated at 37°C and 5 % CO2 in the incubator. At 0.15-, 1 -, 3-, 5-, and 24-hours pictures were taken by the high content analysis system ImageXpress (Molecular Devices). Images were analyzed using the software MetaXpress (Molecular Devices) applying the Transfluor module (settings “Vesicles 5_10_500” and “Nuclear stain 6_32_100”). The vesicle area per cell was calculated for the individual antibodies and values obtained at 5 hours are shown in Figure 5.

[0439] Internalization of Fabfluor labelled antibodies.

[0440] HepG2 cells (20,000 cells / well) were plated onto Corning BioCoat pCIear 96-well MTP (356640) in 100 pl EMEM medium (ATCC, 30-2003) containing 10% FCS and incubated over night at 37°C and 5% CO2. The next day, 5 pl of a 10 pg / ml solution of Incucyte® human Fabfluor-pH antibody labeling reagent (Satorius, #4722), a dye labeled Fab fragment binding to Fc containing proteins, was mixed with 5 pl of 20 pg / ml solutions of individual test antibodies for 20 min at room temperature. Then, 90 pl EMEM medium was added and the mixture incubated for 5 additional minutes on a shaker. In addition, 10 pg Hoechst 33342 (Invitrogen, #H3570) staining solution in 10 pl medium was added to each well of the plate containing the cells and incubated for 10 min in the dark. Then, the medium from the tissue culture plate was removed and replaced by 90 pl of the antibody - Fabfluor mixtures. Cells were then incubated at 37°C and 5 % CO2 in the incubator. At 0.15-, 1 -, 3-, 5, and 24-hours pictures were taken by the high content analysis system ImageXpress (Molecular Devices). Images were analyzed using the software MetaXpress (Molecular Devices) applying the Transfluor module (settings “Vesicles 5_10_500” and “Nuclear stain 6_32_100”). The vesicle area per cell was calculated for the individual antibodies and values obtained at 5 hours are shown in Figure 6.

[0441] It becomes obvious that TPP-14890 shows lower internalization compared to TPP-29537 using direct labeling of the antibodies with CypHer5E via covalent labeling of lysine residues (see Figure 5). In contrast using Fabfluor non-covalent labeling, both anti-GPC3 antibodies show comparable internalization rates (see Figure 6). These differences in the internalization rates can be explained by the fact that direct labeling using lysine coupling chemistry also leads to labeling of one or more of overall four lysine residues within the CDRs of TPP-14890 which results in an affected binding and therefore in a lowered internalization rate. As antibody TPP- 29537 according to the present invention contains no lysine residue within its CDRs binding and internalization rates are not affected. In the case of Fabfluor labeling both anti-GPC3 antibodies show comparable internalization rates, because no lysine labeling steps are used in this setup. This example shows that the antibodies according to the invention are more suitable for applications based on lysine chemistry conjugation, e.g., ADCs or targeted actinium conjugates, than prior art antibodies comprising lysine residues within their CDRs, as e.g., antibody TPP-14890.

[0442] Example 9: Cell surface binding of GPC3 antibodies to different cell lines

[0443] Cell surface binding of TPP-14890, TPP-29537 and the isotype control TPP-754 was investigated by flow cytometry using different cultured cells. The cell lines SU-DHL-1 , HFF-1 , HepG2, Hep3B2, 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 pulmonary fibroblasts (HPF) were purchased from Promocell and cultured according to the manufacturer’s instructions. For surface staining cells were detached using non enzymatic dissociation solution (Gibco #13151 -014). Cells were washed twice in ice cold FACS buffer (PBS -I- with 3% FCS from Sigma #F2442). All subsequent steps were conducted on ice to prevent antibody internalization. 200,000 cells in 10Opil were transferred per well to 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 using A10235, Alexa Fluor 488 from Invitrogen which couples to primary amino groups present on lysine residues. Degree of labeling was determined following the manufacturer’s instructions. TPP-14890 had a degree of labeling (DOL) of 6.07; TPP-29537 DOL=7.22 and TPP-754 DOL=3.4. All antibodies were diluted in FACS buffer to reach a final concentration of 10 pg / mL. Staining was done for 30 min at 4°C shaking on a Titramaxl 00 in the dark. Cells were washed with PBS- / - and centrifuged at 800g for 2 min at 4°C. Subsequently the supernatant was carefully removed. Cells were resuspended in FACS buffer containing 1 :1000 Sytot Blue (Invitrogen S34857). Cells were measured on a BD Biosciences FACSCanto™ II Flow Cytometry System and analyzed using the FlowJo_v10.6.1 software (BD Biosciences). Living, single cells were gated using forward scatter (FSC), side scatter (SCC) and Pacific Blue gating. Along with the cells MESF beads (Quantum 488C, Lot.: 14506) were measured. The measured fluorescent signals were converted into an ABC-value (Antibody Binding Capacity) with help of the MESF beads following the manufacturer’s instructions. After calculation of ABC-values the isotype control ABC values were subtracted from the values of the target antibodies. Results were plotted as ABC (antibody binding capacity on the cell surface), see Figure 7, Figure 8, and Table 1 1.

[0444] Table 11 : ABC values for various cell lines determined using TPP-14890 or TPP-29537

[0445] It becomes obvious that in case of HepG2 and Hep3B2 the Alexa Fluor 488 labeled TPP- 29537 detects around 20-fold higher ABC value as compared to the Alexa Fluor 488 labeled TPP-14890. For PLC / PRF / 5 the Alexa Fluo r488 labeled TPP-29537 detects an ABC value of 14495,1 whereas the Alexa Fluor 488 labeled TPP-14890 is not able to detect any GPC3 on this cell line having lower amounts of GPC3 on its surface. For the cell lines with ABC = 0, the calculated ABC value for the GPC3 antibody was lower than for the isotype control antibody in that respective 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 with Alexa Fluor 488 using lysine coupling chemistry also leads to labeling of one or more of overall four lysine residues within the CDRs of the prior art antibody TPP-14890 which results in an affected binding to GPC3. As anti-GPC3 antibody TPP-29537 according to the present invention contains no lysine residue within its CDRs binding and resulting ABC values are not affected.

[0446] Example 10: Preparation of Targeted Actinium Conjugates (TACs)

[0447] Conjugation of Macropa to GPC3 antibodies (ACCs)

[0448] 6-[[16-[(6-carboxy-2-pyridyl)methyl]- 1 ,4,10,13-tetraoxa-7, 16-diazacyclooctadec-7-yl]methyl]- 4-[2-(4-isothiocyanatophenyl)ethoxy]pyridine-2-carboxylic acid (Macropa-NCS; in the following abbreviated as “Macropa”) was dissolved in DMA to 10 mg / ml. Anti-GPC3 antibodies TPP- 29537 and TPP-14890 were dissolved in PBS to 10 mg / mL and the pH was adjusted to 9 with 1 M carbonate buffer, respectively. Chelator and antibody were mixed and incubated on a thermomixer shaking at 350 rpm at RT for two hours. Molar ratio of chelator and antibody was selected depending on the desired Chelator to antibody ratio (CAR). Products were purified by FPLC (column: HiLoad 16 / 600 Superdex 200 pg column; flow: 1 mL / min; in 0.1 M Acetate buffer (pH 5); detection: LIV 280 / 254 nm). Concentration and monomeric purity were determined by SEC-LIV (as described below and shown in Table 12). CAR was determined by SEC-MS using positive electrospray ionization (as described below) and the following equation: CAR=[(A*An) / A]0, A=lntensity of all signals of the maximum entropy envelope, An=signals of the maximum entropy envelope containing chelators. CARs of the different ACCs were determined as shown in Table 12.

[0449] Table 12: Chelator to antibody ratios (CARs), concentration and monomeric purity of different Antibody Chelator Conjugates

[0450] ACC characterization by size exclusion chromatography and mass spectrometry

[0451] CAR of the ACCs was determined by SEC-MS (Water Acquity HPLC connected to Waters XEVO TOF; running buffer: 50 / 50 / 0.1 (v / v / v) ACN / Water / TFA; flow rate: 0.06 mL / min, column for on-line desalting: Waters Acquity BEH SEC, 1.7 pm, 2.1 x 150 mm; ESI+ mode) by using MS peak heights in percentage of major peak height for the components mAb, mAb + 1 chelator, mAb + 2 chelators, mAb + 3 chelators etc. and using the formula CAR = Sum(n*An) / Sum An, where n equals the number of chelators and An equals the intensity of the antibody conjugate with n chelators. Purity and concentration of the ACC were determined by SEC-LIV (Agilent 1260 Infinity HPLC system, running buffer: 10% DMSO / PBS; flow rate: 0.3 mL / min, column: Waters Acquity BEH SEC, 1.7 pm, 4.6 x 300 mm, detection: UV at 280 nm). The method separates higher aggregates, dimers and fragments from the ACC monomer.

[0452] Radiolabelling of Targeted Actinium-225 Conjugates (TACs)

[0453] Actinium-225 (Ac-225) was added to TPP-14890-Macropa and TPP-29537-Macropa in 0.1 M Acetate buffer (pH 5) at a specific activity of 2 MBq / mg and a radioactive concentration of 1 - 2.5 kBq / pL. The mixtures were incubated for one hour at room temperature followed by determination of radiochemical purity (RCP) by iTLC.

[0454] Instant thin-layer chromatography (ITLC):

[0455] An iTLC was preformed to measure radiochemical purity (RCP) of the Ac-225-Macropa-GPC3 conjugate solutions. In brief, an iTLC.SG. plate was cut to obtain 11 cm x 1 cm strips. Application point (1 cm), cut point (4 cm) and end of run point (10 cm) was marked and 2 pl of TAC was applied oniTLC strips. The strips were developed in 3 mL mobile phase in LSC vials. Free Ac-225 migrated on the strip with the solvent while complexed Ac-225 stayed at the application point. When the liquid front had reached the marked end point, the strips were removed from the vial and placed on an aluminum foil to dry. The strips were cut in two at 4 cm: the shorter “application” half (A) and the longer “front” half (F). Each part was carefully folded and placed at the bottom of scintillation vials. After 6 hours secular equilibrium was obtained of actinium-225 and daughters. The application part was counted for 60 sec and front part counted for 120 sec in a high purity Germanium detector (Ortec). The radiochemical purity (% RCP) of the samples (defined as [(radioactivity of application section) / (total radioactivity on strip)] x 100) was 99.9% for all samples described in Table 12.

[0456] Example 11 : Comparison of GPC3-TACs in immunoreactive fraction assay (I RF)

[0457] Binding of each radiolabeled sample to GPC3-coated M-270-carboxy Dynabeads (Thermo Fisher Scientific) was determined by immunoreactive fraction (IRF) assay. IRF is assessed by measuring the fraction of radiolabeled antibodies bound to magnetic antigen-coated beads, compared to the unbound fraction still in the supernatant. Binding was assessed directly after radiolabeling.

[0458] In brief, a decreasing amount of magnetic beads from 400 to 0.1 pg was placed in 2 mL Eppendorf tubes and resuspended in 50 pL buffer (PBS / 3% BSA). The radiolabeled compounds were diluted to 5 Bq / pL in buffer and added to each bead-containing tube (50Bq total). Incubation lasted for 60-90 min while shaking the tubes on a Thermomixer (Eppendorf) at 750 rpm at room temperature. After completion of the binding step, 40 pL of buffer was added to each tube and a magnet (DynaMag-2) was used to separate the beads from the supernatant. The radioactivity in the supernatant and on the beads of each sample was determined using a high purity Germanium detector (Ortec) after at least 6 hours, to allow daughter nuclides to be in secular equilibrium. Total binding was defined as [(activity in beads - activity in supernant) / (total activity)]x 100. The unspecific binding was obtained from the same calculation from blocked samples, and specific binding was defined as [total binding - unspecific binding]. Results are shown in Figure 9, and Table 13.

[0459] Table 13: CAR, EC50 (pg beads) and MAX % IRF of Ac-225-Macropa-TPP-29537 and Ac- 225-Macropa-TPP-14890.

[0460] For Ac-225-Macropa-TPP-29537 comparable EC50 and max % IRF were determined for both low and high CAR conjugates, whereas for the high CAR Ac-225-Macropa-TPP- 14890 conjugate comprising the prior art anti-GPC3 antibody TPP-14890 there was a clear decrease in EC50 value compared to the low CAR version, indicating an impaired binding potency to human GPC3 peptide.

[0461] Example 12: In vitro cytotoxicity

[0462] 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 1 1 was determined in Human Hepatocellular Carcinoma (HCC) cell lines expressing different levels of GPC3. HepG2 and Hep3B2.1 -7 cells were seeded at an appropriate cell density in the cell media MEM + 10% FCS. HepG2 cells have higher GPC3 expression on the cell surface than Hep3B2.1 -7 cells. One day after seeding the compounds at the specific activity of 2 MBq / mg and radioactive concentration of 5 kBq / ml were titrated in parallel with a radiolabeled isotype control. Cells were incubated for 6 days and subsequentially cell viability was determined using Hoechst and PI staining and analyzed on Operetta CLS High Content Analysis System (Perkin Elmer). Resulting IC50 values are shown in Table 14, Figure 10 A, and Figure 10B. Table 15 shows the Isotype / Targeting ratio of the different TACs (a higher Isotype / Targeting ratio indicates higher specific cytotoxicity).

[0463] Table 14: IC50 values of TPP-14890-TAC and TPP-29537-TAC with low and high CARs

[0464] Table 15 1 : IC50 Ratio (Isotype / Targeting) of TPP-14890-TAC and TPP-29537-TAC with low and high CARs

[0465] Strong and specific cytotoxicity was observed in HepG2 and Hep3B2.1 -7 cell lines. Highest specific cytotoxicity was determined for TPP-29537-TAC CAR 0.5 shown with highest IC50 ratio. The high CAR TPP-29537 conjugate demonstrated similar IC50 as the low CAR TPP- 14890-TAC. TPP-14890-TAC CAR 10 showed significantly lower specific cytotoxicity especially in the Hep3B2.1 -7 cell line, indicating less efficient cytotoxicity compared to the other tested compounds, indicating an impaired binding of high CAR TPP-14890-TAC due to labeling of one or more of the overall four lysine residues within the CDRs of prior art antibody TPP-14890.

[0466] Example 13: Biodistribution study of GPC3-TACs

[0467] Biodistribution of Ac-225-Macropa-TPP-29537 (CAR 0.7) and Ac-225-Macropa-isotype-control (CAR 0.7) was investigated in the GPC3-low expressing human hepatocellular xenograft model Huh-7 (ABC: 22.000). Female RJ:NMRI-Fox1 nu / nu mice were inoculated with 2 x 106Huh-7 cells on the right flank. Animals were pre-treated with 200ug / mouse unspecific lgG2a antibody 24 hours prior to hot dosing. Animals were treated with 500kBq / kg Ac-225-macropa- TPP-29537 and Ac-225-Macropa-isotype-control at a total protein dose of 0.75 mg / kg, respectively. The animals were sacrificed 72, 168, 336, and 504 hours post treatment and tumors and organs were isolated at the respective timepoints for Ac-225-Macropa-TPP-29537 For Ac-225-Macropa-isotype-control the last time point, 504 hours, was excluded due animals reaching humane endpoint. Accumulated Ac-225 in blood, tumor, liver, kidney, spleen, and femur was determined using Germanium detector HPGe (GEM-F8250-LB-C, Ortec). The results are presented in Figure 11 A and Figure 11 B.

[0468] For Ac-225-Macropa-TPP-29537 treated animals, the data indicate specific, strong and persistent tumor accumulation whereas accumulation in normal organs was generally low and transient with clearance over time.

[0469] Example 14: In vivo efficacy study comparing TPP-29537-TAC and isotype-TAC

[0470] The in vivo efficacy of Ac-225-Macropa-TPP-29537 was evaluated in the human hepatoma model Huh-7. Mice xenografted with the GPC3-low expressing human hepatoma model Huh- 7 were treated with a single dose IV of 500kBq / kg with a total antibody dose of 0.75mg / kg. Ac- 225-Macropa-TPP-29537 (CAR 0.7), and Ac-225-Macropa-isotype-control (CAR 0.7). Animals were pre-treated with 200ug / mouse unspecific lgG2a antibody 24 h prior to hot dosing. The mice remaining at day 25 post initial dosing, received a repeat dose of 250kBq / kg and 0.75 mg / kg total protein dose.

[0471] As presented in Fehler! Verweisquelle konnte nicht gefunden werden., Ac-225-Macropa- TPP-29537 had antitumor activity, and significantly inhibited Huh-7 tumor growth compared to Ac-225-Macropa-isotype-control (Figure 12A). The Ac-225-Macropa-TPP-29537 mice also had significantly better survival than the vehicle and Ac-225-Macropa-isotype-control mice (Figure 12B). No body weight loss was observed in the treatment groups.

[0472] Example 15: Preparation and Characterization of Zr-89-DFO*-TPP-29537 for imaging

[0473] To conduct in vivo positron emission tomography (PET) imaging anti-GPC3 antibody TPP- 29537 was conjugated to DFO* and radiolabeled with Zirconium-89.

[0474] Conjugation and purification of DFO*-TPP-29537:

[0475] 5,1 1 ,16,22-Tetraazahexacosanediamide,N1 -[5-(acetylhydroxyamino)pentyl]-N26,5,16- trihydroxy-N26-[5-[[[(4-isothiocyanatophenyl)amino]thioxomethyl]amino]pentyl]-4,12,15,23- tetraoxo- (p-Phe-NCS-DFO* (DFO*), ABX advanced chemical compounds, 7272) was dissolved in DMSO to 10 mg / ml. Anti-GPC3 antibody TPP-29537 was dissolved in WFI to 10 mg / mL. The pH of the antibody was adjusted to 9 with 1 M carbonate buffer. p-Phe-NCS-DFO* was added to the antibody and the mixture was incubated on a thermomixer shaking at 1 hour at 37°C. The conjugate was purified by PD-10 column according to manufacturer’s instructions (Cytiva, 17085101). The monomeric 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 equation: CAR=[(A*An) / A]0, A=lntensity of all signals of the maximum entropy envelope, An=signals of the maximum entropy envelope containing chelators. Monomeric purity and CAR were determined to be 99.2 % and 1 .2, respectively.

[0476] Radiolabelling of DFO*-TPP-29537 with Zirconium-89

[0477] Zirconium-89 in 1 M oxalic acid was added to DFO*-TPP-29537 in 1 M HEPES, pH 7.3, and labeled with a specific activity of 34 MBq / mg and a RAC of 103 MBq / mL. After incubation for 30 minutes at 37°C, the sample was purified by PD-10 column according to manufacturer’s instructions (Cytiva, 17085101 ) and buffer exchanged to formulation buffer (10 mM L-Histidine, 130 mM Glycine, 5% (m / v) Sucrose in water for injection) and diluted to RAC of 24 MBq / mL.

[0478] Immunoreactive fraction assay Immunoreactive fraction (IRF) determination was used to evaluate the binding properties of the radiolabeled immunoreactive conjugate monoclonal antibody (mAb) product Zr-89-DFO*- TPP-29537. Shortly after completion of the radiolabeling, the binding of Zr-89-DFO*-TPP- 29537 to GPC3 peptide-coated M-270-carboxy Dynabeads was determined. In short, 0.36- 800 pg beads were placed in 2 mL Eppendorf tubes in 90 pL buffer (PBS / 3% BSA). The radiolabeled compounds were diluted to 5 Bq / pL in buffer and 10 pL were added to each beadcontaining tube. The samples were run in triplicate. The binding lasted for 60 min while shaking the tubes on a Thermomixer (Eppendorf) at 750 rpm at room temperature. After completion of the binding step, a magnet (DynaMag-2) was used to separate the beads from half of the supernatant. The radio activities in the supernatant and beads of each sample were determined by using a high purity Germanium detector (Ortec). Total binding was defined as [(activity in beads - activity in supernatant) / (total activity)] x 100. Figure 13 shows the radiolabeled Zr-89-DFO*-TPP-29537 binding potency to GPC3 peptide conjugated to beads.

[0479] The number of antigen coated beads selected to give an excess of epitope for binding is achieved with 50-100 pg GPC3-peptide coated beads. In summary, an IRF% ~ 90% was determined in a single point assay.

[0480] Example 16: Biodistribution of Ac-225-Macropa-TPP-29537 and its imaging pair Zr-89- DFO*-TPP-29537

[0481] 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. In addition, Zr- 89-DFO*-TPP-29537 in vivo PET imaging quantification was performed to investigate its potential as an imaging pair for Ac-225-Macropa-TPP-29537 to be used in early development. Female RJ:NMRI-Fox1 nu / nu mice were inoculated with 4.1 x 106HEP-3B cells on the right flank. Animals were pre-treated with 200ug / mouse unspecific lgG2a antibody 24 hours prior to hot dosing.

[0482] Zr-89-DFO*-TPP-29537 was injected as a slow bolus into the tail vein of the animals as described in Table 16. PET / CT imaging was performed 2 h (n=1 ), 4 h (n=3), 24 h (n=3), 72 h (n=3) and 168 h (n=2) post injection. The imaging acquisitions were performed by MILabs Vector6 PET / SPECT / CT scanner using HE-UHR-RM collimator, 4-mouse bed, total acquisition time of 60 minutes, spiral acquisition mode and fixed field of view, determined during the first acquisition. Table 16: Overview of groups in in vivo and ex vivo biodistribution study of Zr-89-DFO*-TPP- 29537 and ex vivo biodistribution of Ac-225-Macropa-TPP-29537

[0483] The dosing was split up, to avoid having more than one mouse in the scanner at the time.

[0484] To assess the in vivo biodistribution, image analysis was performed using PMOD software. CT volumes of interest (sphere VOI) were applied to heart, liver, and kidneys. Tumor was contoured on the CT and a restricting VOI (10% threshold) was applied on the PET images. Data was decay corrected to the injection time point. Figure 14 shows the coronal PET / CT MIP of imaged HEP-3B tumor bearing mice with Zr-89-DFO*-TPP-29537 at different time points.

[0485] After the in vivo imaging, to assess the ex vivo biodistribution of Zr-89-DFO*-TPP-29537, the imaged mice, 3 mice per time point, were sacrificed and blood, tumor, liver, kidneys, spleen and femur and tibia bones were collected, and Zr-89 activity was determined using Perkin Elmer Wizard II gammacounter.

[0486] In parallel, HEP-3B tumor bearing mice were injected IV with 250kBq / kg Ac-225-Macropa- TPP-29537 at a protein dose of 0.75 mg / kg. The animals were sacrificed 0.5, 2, 4, 24, 72 and 168 hours post treatment. Accumulated Ac-225 in blood, tumor, liver, kidney, spleen, and femur was determined using Germanium detector HPGe (GEM-F8250-LB-C, Ortec).

[0487] As depicted in Figure 15, Ac-225-Macropa-TPP-29537and Zr-89-DFO*-TPP-29537 showed comparable ex vivo biodistribution, with similar blood clearance up to the last time point in the study (168 h). Tumor accumulation of Ac-225-Macropa-TPP-29537 and Zr-89-DFO*-TPP- 29537 was similar up to 72 h p.i., whereafter Ac-225-Macropa-TPP-29537 %ID / g was higher, most likely caused by tumor shrinkage resulting from Ac-225-Macropa-TPP-29537 efficacy.

[0488] Ex vivo biodistribution and in vivo PET imaging quantification of Zr-89-DFO*-TPP-29537 demonstrated comparable uptake in heart / blood, kidney, liver, and tumor (Figure 15).

[0489] In summary, these results demonstrate the suitability of Zr-89-DFO*-TPP-29537 as a PET imaging pair for Ac-225-Macropa-TPP-29537 to be used in early development. SEQUENCES

Claims

CLAIMS1. An isolated anti-GPC3 antibody or antigen-binding fragment capable of binding to GPC3 comprising: i) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H-CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L-CDR3 comprising SEQ ID NQ:108, or ii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 122, an H-CDR2 comprising SEQ ID NO: 123, and an H-CDR3 comprising SEQ ID NO: 124 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 126, an L-CDR2 comprising SEQ ID NO: 127, and an L-CDR3 comprising SEQ ID NO:128, or iii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 142, an H-CDR2 comprising SEQ ID NO: 143, and an H-CDR3 comprising SEQ ID NO: 144 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 146, an L-CDR2 comprising SEQ ID NO: 147, and an L-CDR3 comprising SEQ ID NO:148, or iv) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 162, an H-CDR2 comprising SEQ ID NO: 163, and an H-CDR3 comprising SEQ ID NO: 164 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 166, an L-CDR2 comprising SEQ ID NO: 167, and an L-CDR3 comprising SEQ ID NO:168.

2. An isolated antibody or antigen-binding fragment according to claim 1 comprising: i) a variable heavy chain domain comprising SEQ ID NO: 101 and a variable light chain domain comprising SEQ ID NO: 105; or ii) a variable heavy chain domain comprising SEQ ID NO: 121 and a variable light chain domain comprising SEQ ID NO: 125; or iii) a variable heavy chain domain comprising SEQ ID NO: 141 and a variable light chain domain comprising SEQ ID NO: 145; or iv) a variable heavy chain domain comprising SEQ ID NO: 161 and a variable light chain domain comprising SEQ ID NO: 165.

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

4. An isolated antibody or antigen-binding fragment according to any one of the preceding claims wherein the monoclonal antibody is an IgG 1 antibody.

5. An isolated antibody or antigen-binding fragment according to any one of the preceding claims comprising i) a heavy chain comprising SEQ ID NO: 1 17 and a light chain comprising SEQ ID NO: 1 18; or ii) a heavy chain comprising SEQ ID NO: 137 and a light chain comprising SEQ ID NO: 138; or iii) a heavy chain comprising SEQ ID NO: 157 and a light chain comprising SEQ ID NO: 158; or iv) a heavy chain comprising SEQ ID NO: 177 and a light chain comprising SEQ ID NO: 178.

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

7. An antibody conjugate according to claim 6 comprising a radionuclide, a cytotoxic agent, an organic compound, a protein toxin, an immunomodulator, a further antibody or antigen-binding fragment, a fluorescent moiety or a chimeric antigen receptor.

8. An antibody conjugate according to any one of claims 6 or 7, wherein the antibody or antigen-binding fragment thereof is bound to the to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.

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

10. A targeted actinium conjugate according to claim 9 wherein [Ab] is bound to the rest of the molecule via a terminal amino group of a lysine residue of the monoclonal antibody or antigen-binding fragment thereof.1 1 . A targeted actinium conjugate (TAC) of formula (I)wherein [Ab] is a monoclonal antibody or an antigen-binding fragment thereof capable of binding to GPC3 comprising a heavy chain antigen-binding region that comprises a H- CDR1 comprising SEQ ID NO: 102, an H-CDR2 comprising SEQ ID NO: 103, and an H- CDR3 comprising SEQ ID NO: 104 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 106, an L-CDR2 comprising SEQ ID NO: 107, and an L-CDR3 comprising SEQ ID NO:108, wherein [Ab] is bound to the to the rest of the molecule via a 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 actinium 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 actiniumconjugate according to any one of claims 9 - 1 1 or a pharmaceutical composition according to claim 12 for use in the treatment of a disease.

14. 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 actinium conjugate according to any one of claims 9 - 1 1 or a pharmaceutical composition according to claim 12 for use according to claim 13, wherein the disease is characterized by an ectopic expression of GPC3.

15. 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 actinium conjugate according to any one of claims 9 - 1 1 or a pharmaceutical composition according to claim 12 for use according to any one of claims 13 or 14 wherein the disease is cancer, preferably hepatocellular cancer.

16. An 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 a targeted actinium conjugate according to any one of claims 9 - 11 or the pharmaceutical composition according to claim 12 for use in simultaneous, separate, or sequential combination with one or more further therapeutically active compounds.

17. 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 actinium conjugate according to any one of claims 9 - 1 1 for use as a diagnostic or imaging agent.

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

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

20. An isolated cell arranged for production of an antibody or antigen-binding fragment according to any one of claims 1 - 5.21 . A method for producing an 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 targetedactinium conjugate according to any one of claims 9 - 11 , comprising culturing a cell according to claim 20 and optionally purification of the antibody or antigen-binding fragment.

22. A kit comprising 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 a targeted actinium conjugate according to any one of claims 9 - 1 1 or the pharmaceutical composition according to claim 12 and instructions for use.

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