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WO2025133609A1 - Polypeptide se liant au glypican-3 - Google Patents

Polypeptide se liant au glypican-3 Download PDF

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Publication number
WO2025133609A1
WO2025133609A1 PCT/GB2024/053164 GB2024053164W WO2025133609A1 WO 2025133609 A1 WO2025133609 A1 WO 2025133609A1 GB 2024053164 W GB2024053164 W GB 2024053164W WO 2025133609 A1 WO2025133609 A1 WO 2025133609A1
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seq
antigen
cell
antibody
polypeptide
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Claire RODDIE
Martin PULÉ
Harriet RODDY
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UCL Business Ltd
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UCL Business Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4261—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a polypeptide comprising an antigen-binding domain which binds to Glypican-3 (GPC3), wherein binding is not inhibited by a soluble GPC3.
  • GPC3 Glypican-3
  • the invention also relates to chimeric antigen receptors (CARs) which comprise such polypeptides.
  • CARs chimeric antigen receptors
  • Cells expressing CARs which bind GPC3 are useful in the treatment of hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • HCC BACKGROUND TO THE INVENTION
  • Hepatocellular carcinoma (HCC) accounts for 80% of all primary liver cancers and is a leading cause of cancer related mortality.
  • TKIs Protein tyrosine kinase inhibitors
  • VEGF vascular endothelial growth factor receptor 2
  • PD-1 Programmed Death Receptor-1
  • Glypican-3 is a 70 kDa, glycosylphosphatidylinositol (GPI) anchored, 580 amino acid heparan sulphate proteoglycan that is expressed in 72% of cases of HCC, where it portends to poor prognosis (Capurro et al., Gastroenterology, 2003; 125(1):89–97).
  • GPC3 is minimally expressed on other tissues including normal and cirrhotic liver. Similar to other glypicans, GPC3 can be released from the cell surface and can be found around tumours or in circulation.
  • Soluble GPC3 is found in the serum of 53% of HCC patients and is under development as a disease biomarker (Capurro et al., Gastroenterology, 2003; 125(1):89–97).
  • serum GPC3 levels are significantly elevated in HCC patients compared with healthy individuals (Capurro et al., Gastroenterology, 2003; 125(1):89–97; Xu et al., Ann Hepatol, 2019; 18(1):58–67; Liu et al., Clin Biochem, 2020; 79:54–60) and that local tumour GPC3 concentrations in HCC may also be much higher than those in normal tissues.
  • the present inventors have developed novel GPC3 binders with improved properties. Surprisingly, the present binders interact with membrane bound GPC3 but are not inhibited by soluble GPC3.
  • the binders may be useful be useful in antigen binding entities such as antibodies, chimeric antigen receptors (CARs) and bi-specific T cell engagers (BiTEs), in particular for the treatment of HCC.
  • the invention provides a polypeptide comprising an antigen-binding domain which binds to a region between amino acids S495 to H580 of Glypican-3 (GPC3), wherein binding is not inhibited by a soluble GPC3.
  • the binding between the polypeptide comprising the antigen-binding domain and the region between amino acids S495 to H580 of GPC3 is not inhibited by soluble GPC3.
  • the antigen-binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3; and a light chain variable region (VL) having CDRs: LCDR1, LCDR2 and LCDR3 selected from the following: i.
  • the antigen-binding domain comprises: i. HCDR1 - SEQ ID NO: 1, HCDR2 - SEQ ID NO: 2; HCDR3 - SEQ ID NO: 3; LCDR1 - SEQ ID NO: 4; LCDR2 - SEQ ID NO: 5; LCDR3 - SEQ ID NO: 6; or ii. HCDR1 - SEQ ID NO: 7; HCDR2 - SEQ ID NO: 8; HCDR3 - SEQ ID NO: 9; LCDR1 - SEQ ID NO: 10; LCDR2 - SEQ ID NO: 11; LCDR3 - SEQ ID NO: 12.
  • the antigen-binding domain comprises: i.
  • the antigen-binding domain comprises: i. the sequence shown as SEQ ID NO: 17, or a variant having at least 80% sequence identity thereto; or ii.
  • the invention provides a polypeptide comprising an antigen-binding domain which binds to a region between amino acids S495 to H580 of Glypican-3 (GPC3), wherein the antigen-binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3; and a light chain variable region (VL) having CDRs: LCDR1, LCDR2 and LCDR3 selected from the following: i.
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VL light chain variable region
  • the polypeptide of the invention may be an antibody or antigen-binding fragment thereof comprising an antigen-binding domain as defined in the invention.
  • the antibody or antigen-binding fragment thereof is a scFv, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, or a bi-specific T cell activator molecule, such as a bi-specific T cell engager (BiTE).
  • the antibody or antigen-binding fragment may be conjugated to a cargo or payload component.
  • the invention provides an antibody conjugate comprising the antibody or antigen-binding fragment of the invention.
  • the invention further provides a chimeric antigen receptor (CAR) comprising a polypeptide or antibody or antigen-binding fragment thereof according to the invention.
  • the CAR may comprise a transmembrane domain, preferably a CD28 transmembrane domain.
  • the polypeptide comprising an antigen-binding domain and the transmembrane domain of the CAR may be connected by a spacer, preferably the spacer comprises a CD28 hinge.
  • the CAR may comprise an intracellular T cell signalling domain, preferably a CD28 endodomain and a CD3-Zeta endodomain.
  • the CAR comprises a sequence selected from the group comprising SEQ ID NO: 19 or SEQ ID NO: 20, or a variant which has at least 80% sequence identity thereto and retains the capacity to i) bind GPC3 and ii) induce T cell signalling.
  • the invention provides a method for treating a disease which comprises the step of administering a polypeptide comprising an antigen-binding domain according to the invention, an antibody or antigen-binding fragment thereof according to the invention, an antibody conjugate according to the invention, a polynucleotide according to the invention, a vector according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention to a subject.
  • the invention provides for use of a polypeptide comprising an antigen-binding domain according to the invention, an antibody or antigen-binding fragment thereof according to the invention, an antibody conjugate according to the invention, a polynucleotide according to the invention, a vector according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention in the manufacture of a medicament for treating a disease.
  • the disease to be treated is cancer.
  • the cancer may be hepatocellular carcinoma (HCC), melanoma, ovarian clear-cell carcinomas, yolk sac tumour, neuroblastoma, hepatoblastoma, Wilms' tumor cells, rhabdoid tumors, and rhabdomyosarcomas.
  • HCC hepatocellular carcinoma
  • melanoma ovarian clear-cell carcinomas
  • yolk sac tumour neuroblastoma
  • hepatoblastoma hepatoblastoma
  • Wilms' tumor cells rhabdoi
  • the cancer is preferably HCC.
  • DESCRIPTION OF THE FIGURES Figure 1 – Shed GPC3 is clinically relevant and requires binder generation specific for this antigen characteristic.
  • constructs used to generate overexpressing cell lines include GPC3 with signal peptide and GPI anchor for both, one is a truncated version of the protein finishing at residue 495, both constructs contain eGFP as a marker of transduction and for use as target detection in coculture based assays.
  • Staining with commercial antibody clone (1G12) shows transduction of both cell lines when compared to un-transduced cells and a linear relationship between eGFP and antigen.
  • e Cell line validation of antigen density.
  • Figure 6 Reduction in binding in the presence of shed antigen.
  • Binding assay Crude supernatant comprising scFv (or supernatant from NT cells) was incubated with SupT1 GPC3+ target cells in the presence of different amounts of sGPC3 (0, 5, 10 ⁇ g/mL). Cells were washed and stained with secondary antibody. The dotted line shows median fluorescent intensity (MFI) of maximal binding.
  • MFI median fluorescent intensity
  • Heavy chain variable region refers to the fragment of the heavy chain of an antigen- binding domain or antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • Light chain variable region refers to the fragment of the light chain of an antigen-binding domain or antibody that contains three CDRs interposed between framework regions.
  • CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., 1977, J. Biol. Chem. 252:6609-6616; Kabat, 1978, Adv. Prot. Chem. 32:1-75). CDR 11 region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved ⁇ -sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, 1987, J. Mol. Biol.196:901-917). Alternatively, IMGT or EU numbering may be used. These terminologies are well recognized in the art.
  • the antigen binding domain may be defined by the presence of HCDRs and LCDRs determined according to CDR numbering schemes which are known in the art.
  • the CDRs may be defined according to the IMGT, Chothia and/or Kabat numbering schemes.
  • the CDRs of the antigen-binding domain are defined according to the IMGT numbering scheme. It may be possible to introduce one or more mutations (substitutions, additions or deletions) into each CDR without negatively affecting binding activity.
  • Each CDR may, for example, have one, two or three amino acid mutations.
  • Identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % identity between two or more sequences.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleotide sequences Research 12:387).
  • Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid – Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching.
  • the percentage identity between two polypeptide sequences may be readily determined by BLAST which is freely available at http://blast.ncbi.nlm.nih.gov. Once the software has produced an optimal alignment, it is possible to calculate % identity. The software typically does this as part of the sequence comparison and generates a numerical result. 12
  • the sequence may have one or more deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent molecule. These sequences are encompassed by the present invention. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the activity is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the invention further provides an antigen-binding domain which binds to a region between amino acids S495 to H580 of GPC3, wherein binding is not inhibited by a soluble GPC3.
  • entity comprising an antigen- binding domain which binds to a region between amino acids S495 to H580 of GPC3, wherein binding is not inhibited by a soluble GPC3.
  • the GPC3 gene encodes a 580 amino acid, 70 kDa precursor protein, which is cleaved by furin between residues Arg358 and Ser359 to generate an N-terminal subunit ( ⁇ 40 kDa) and a C-terminal subunit ( ⁇ 30 kDa).
  • the two subunits can be connected by one or more disulphide bonds.
  • the C-terminal subunit comprises heparan sulfate modification at two sites (Ser495 and Ser509).
  • GPC3 binds to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor; Ser560 of GPC3 inserts into the lipid bilayer and anchors the protein to the bilayer by phosphatidylinositol.
  • GPI glycosylphosphatidylinositol
  • the amino acid sequence for human GPC3 is (SEQ ID NO: 22): 13 MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPK GPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPS LTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRD LKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVK PCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGK LTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRR
  • the signal peptide (residues 1 to 24), which may be cleaved in the mature protein, is shown in italics. Reference to amino acid positions in SEQ ID NO: 22 throughout refers to the full length polypeptide sequence, including signal peptide.
  • the antigen-binding domain according to the present invention binds to a region between amino acids S495 to H580 of GPC3. In some embodiments, the antigen-binding domain according to the present invention binds to an epitope solely within (i.e. that consists within) amino acids S495 to H580 of GPC3, presented below as SEQ ID NO: 23.
  • the antigen-binding domain according to the present invention binds to a region within SEQ ID NO: 23.
  • N-terminal region of GPC3 SEQ ID NO: 23: SGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPL KLLTSMAISVVCFFFLVH
  • the antigen-binding domain according to the present invention binds to a region within positions 495 to 580 of SEQ ID NO: 22, or a variant having at least 80% identity thereto.
  • the antigen-binding domain according to the present invention binds to an epitope solely within positions 495 to 580 of SEQ ID NO: 22, or a variant having at least 80% identity thereto. In some embodiments, the antigen-binding domain according to the present invention binds to a region within positions 495 to 580, 500 to 580, 505 to 580, 510 to 580, 515 to 580, 520 to 580, 525 to 580, 530 to 580, 535 to 580, 540 to 580, 545 to 580, 550 to 580, 555 to 580, 560 to 580, 565 to 580, 570 to 580, or 575 to 580 of SEQ ID NO: 22, or a variant having at least 80% identity thereto.
  • the antigen-binding domain according to the present invention binds to a region within positions 551 to 580, 552 to 580, 553 to 580, 554 to 580, 555 to 580, 556 to 580, 557 to 580, 558 to 580, 559 to 580, 560 to 580, 561 to 580, 562 to 580, 563 to 580, 564 to 580, 565 to 580 of SEQ ID NO: 22, or a variant having at least 80% identity thereto. In some embodiments, the antigen-binding domain according to the present invention binds to a region within positions 554 to 580 of SEQ ID NO: 22, or a variant having at least 80% identity thereto.
  • the antigen-binding domain according to the present invention binds to an epitope solely within positions 554 to 580 of SEQ ID NO: 22, or a variant having at least 80% identity thereto. In some embodiments, the antigen-binding domain according to the present invention binds to a region within positions 564 to 580 of SEQ ID NO: 22, or a variant having at least 80% identity thereto. In some embodiments, the antigen-binding domain according to the present invention binds to an epitope solely within positions 564 to 580 of SEQ ID NO: 22, or a variant having at least 80% identity thereto.
  • the antigen-binding domain according to the present invention binds to a region within positions 495 to 510 of SEQ ID NO: 22, or a variant having at least 80% identity thereto.
  • the variant sequence may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 22.
  • the antigen-binding domain may bind to a linear epitope.
  • the antigen-binding domain may bind to a conformational epitope.
  • the antigen-binding domain may bind to post-translational modifications, such as heparin sulfate, N-linked glycans and/or the GPI anchor.
  • GPC3 can be ‘shed’ from the cell at the furin cleavage site.
  • GPC3 can be released from the cell surface to the extracellular environment after cleavage by lipase activities that cleave the GPI anchor and/or protease activities that cleave the membrane proximal region.
  • soluble GPC3 refers to both the free N-terminal subunit of GPC3 (i.e. amino acids 1-358) and other soluble truncated versions of GPC3, such 15 as lacking the GPI anchor, that is not associated with cell membrane and/or that is not capable of associating with cell membrane.
  • soluble GPC3 refers to amino acids 1 to 494 of SEQ ID NO: 22. In some embodiments, soluble GPC3 refers to amino acids 1 to 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, or 563 of SEQ ID NO: 22.
  • the soluble GPC3 protein may not include the signal peptide (amino acids 1-24 of SEQ ID NO: 22), which may be cleaved during translation and processing to generate the mature form.
  • soluble GPC3 refers to amino acids 25-494 of SEQ ID NO: 22.
  • soluble GPC3 refers to amino acids 25-550 of SEQ ID NO: 22.
  • the antigen-binding domain according to the present invention binds to a region between amino acids S495 to H580 of GPC3, wherein binding is not inhibited by a soluble GPC3.
  • not inhibited is meant that binding is not reduced by the presence of soluble GPC3 as compared to the absence of soluble GPC3.
  • the polypeptide comprising an antigen-binding domain of the invention is able to bind a region between amino acids S495 to H580 of GPC3 in the presence of about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, or about 100 ⁇ g/mL soluble GPC3.
  • binding in the presence of soluble GPC3 may be reduced by about 0-15%, 0-10%, 0-5% or less, relative to conditions in which soluble GPC3 is not present.
  • a binder that is inhibited by soluble GPC3 may demonstrate > 15% reduction in binding to GPC3 in the presence of soluble GPC3 as compared to the absence of soluble GPC3.
  • binding in the presence of soluble GPC3 may be reduced by about 15-25%, 15- 16 50%, 15-75% or more.
  • Binding in the presence of soluble GPC3 may be reduced by about at least 15%, at least 25%, at least 50% or at least 75%.
  • a quantitative assessment or measurement of binding affinity may be determined or measured using methods know in the art, such as by surface plasmon resonance, for example by using the Biacore® system.
  • KD equilibrium dissociation constant
  • Ka (1/Ms) association rate constant
  • Kd dissociation rate constant
  • SPR Surface Plasmon Resonance
  • Binding affinity can also be determined using methods such as fluorescence quenching, isothermal titration calorimetry.
  • the cytotoxicity of a cell for example a cytolytic immune cell, which comprises a CAR according to the present invention may not be reduced in the presence of soluble GPC3 as compared to the absence of soluble GPC3.
  • a cytolytic immune cell expressing a CAR according to the invention may have cytotoxic activity that is reduced by ⁇ 20% in the presence of soluble GPC3, as compared to in the absence of soluble GPC3.
  • cytotoxicity in the presence of soluble GPC3 may be reduced by about 0-20%, 0-15%, 0-10%, 0-5% or less, relative to conditions in which soluble GPC3 is not present.
  • a cell expressing a CAR comprising a binder that is inhibited by soluble GPC3 may demonstrate > 20% reduction in cytotoxicity in the presence of soluble GPC3 as compared to the absence of GPC3.
  • antibody means a protein or polypeptide having an antigen binding site or antigen-binding domain which comprises at least one complementarity determining region CDR.
  • the antibody may comprise 3 CDRs and have an antigen binding site which is 17 equivalent to that of a domain antibody (dAb).
  • the antibody may comprise 6 CDRs and have an antigen binding site which is equivalent to that of a classical antibody molecule.
  • the remainder of the polypeptide may be any sequence which provides a suitable scaffold for the antigen binding site and displays it in an appropriate manner for it to bind the antigen.
  • the antibody may be a whole immunoglobulin molecule or a part thereof such as a Fab, F(ab)’ 2 , Fv, single chain Fv (ScFv) fragment, Nanobody or single chain variable domain (which may be a VH or VL chain, having 3 CDRs).
  • the antibody may be a bifunctional antibody, for example a bispecific antibody.
  • the antibody may be non-human, chimeric, humanised or fully human. Descriptions of an antibody of the present invention provided herein are generally applicable to an antigen binding fragment thereof.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the antibody may be a synthetic antibody.
  • the antibody is a monoclonal antibody.
  • an antigen-binding fragment examples include, but are not limited to, a single chain antibody (scFv), a single-domain antibody (sdAb), an antigen-binding fragment (Fab), a camelid antibody (VHH), a variable region (Fv), a heavy chain variable region (VH), a light chain variable region (VL), and a complementarity determining region (CDR).
  • the antibody may be a full-length, classical antibody.
  • the antibody may be an IgG, IgM or IgA molecule.
  • the antibody is a full monoclonal antibody.
  • Antibodies may be obtained by techniques comprising immunizing an animal with a target antigen and isolating the antibody from serum.
  • Monoclonal antibodies 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. Pat. No.4,816,567).
  • the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.
  • the antibody may also be a chimeric or humanized antibody or fragment thereof.
  • an antibody comprising an antigen-binding domain according to the invention may be obtained by immunising an animal with truncated GPC3, such as residues S359-H580 of GPC3.
  • the invention provides a method for obtaining an antibody comprising an antigen-binding domain as defined in the invention.
  • the method may comprise the steps of: 18 i. Immunising an animal with residues S359 to H580 of GPC3 (for example, amino acids 359 to 580 of SEQ ID NO: 22); ii. Isolating an antibody and/or nucleic acid sequence encoding an antibody from said animal.
  • the method may further comprises screening to identify an antibody for binding to residues S495 to H580 of GPC3 (for example, amino acids 495 to 580 of SEQ ID NO: 22).
  • the present invention further provides an antibody generated by the method of the invention.
  • the antibody of fragment according to the invention may prove useful in any method which relies on a high affinity binding interaction between an antigen-binding domain and a cognate target.
  • the antibody of fragment according to the invention may be used as a detection antibody and/or a capture antibody.
  • the antibody of fragment according to the invention may be used a therapeutic antibody, for example, as a therapeutic antibody that targets GPC3 protein or a cell expressing GPC3.
  • a non-limiting example therefore for the application of the antibody of fragment according to the invention is the use in the treatment of cancers characterized by expression and/or overexpression of GPC3.
  • the present invention also encompasses fragments of any antibody or protein or polypeptide as defined herein. It will be appreciated that a fragment comprises an amino acid sequence that is shorter than the full-length sequence of an antibody or protein or polypeptide, but retains full biological activity and/or antigenic nature of the full-length sequence of the antibody or protein or polypeptide. It will also be appreciated that said fragment retains the same binding affinity of the full-length sequence of the antibody or protein or polypeptide.
  • IMMUNE CELL ENGAGERS In one embodiment, the present invention provides an immune cell activator molecule.
  • a T cell activator molecule or an NK cell activator molecule for example, a T cell activator molecule or an NK cell activator molecule.
  • the present invention provides a T cell activator molecule which is a bispecific molecule (i.e. a bi-specific T cell engager (BiTE)) which comprises an antigen- binding domain as described herein as a first domain, and a T cell activating domain as a second domain.
  • Bi-specific T cell engaging molecules are a class of bi-specific antibody-type molecules that have been developed, primarily for the use as anti-cancer drugs. They direct a host's immune system, more specifically the T cells' cytotoxic activity, against a target cell, such as a cancer 19 cell.
  • the cell-containing sample may be isolated from a subject or from other sources, for example as described above.
  • the cells may be isolated from a subject’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).

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Abstract

La présente invention concerne un polypeptide comprenant un domaine de liaison à l'antigène qui se lie au glypican-3 (GPC3), la liaison n'étant pas inhibée par un GPC3 soluble. L'invention concerne également des récepteurs antigéniques chimériques (CAR) qui comprennent lesdits polypeptides.
PCT/GB2024/053164 2023-12-20 2024-12-19 Polypeptide se liant au glypican-3 Pending WO2025133609A1 (fr)

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