WO2023036043A1 - 抗癌结合分子及其应用 - Google Patents

抗癌结合分子及其应用 Download PDF

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WO2023036043A1
WO2023036043A1 PCT/CN2022/116450 CN2022116450W WO2023036043A1 WO 2023036043 A1 WO2023036043 A1 WO 2023036043A1 CN 2022116450 W CN2022116450 W CN 2022116450W WO 2023036043 A1 WO2023036043 A1 WO 2023036043A1
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amino acid
seq
domain
acid sequence
binding molecule
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PCT/CN2022/116450
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French (fr)
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孟祥雪
史继亚
介淼星
杨敏
王雅秋
任志衡
陈立模
李文佳
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广东东阳光药业有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the disclosure belongs to the field of biotechnology, and in particular relates to a binding molecule specifically binding to 4-1BB and glypican 3 (GPC3) and an application thereof.
  • GPC3 glypican 3
  • 4-1BB (also known as CD137, TNFRSF9, etc.) is a member of the tumor necrosis factor receptor superfamily (TNFRS).
  • Antibodies against 4-1BB have the ability to activate the 4-1BB signaling pathway, and have potential medical value for tumor treatment, anti-infection, and anti-autoimmune diseases.
  • Glypican 3 (GPC3) belongs to the carcinoembryonic antigen of the glypican family and is highly expressed in a variety of cancer cells, especially hepatocellular carcinoma (HCC), melanoma, and Wilms tumor and hepatoblastoma. Therefore, there is a clinical need for novel protein drugs based on 4-1BB activating antibodies that can target tumor cells and activate the 4-1BB pathway to treat tumors.
  • the present disclosure provides a new method for targeting the tumor-associated antigen GPC3 (glypican 3) and simultaneously activating the immune pathways related to the 4-1BB signaling pathway, which can Effectively solve the side effects of 4-1BB activating antibody.
  • GPC3 tumor-associated antigen
  • a binding molecule comprising: a first domain specifically binding to 4-1BB or a fragment thereof, and a first domain specifically binding to Glypican 3 (GPC3) or the second domain of a fragment thereof.
  • GPC3 Glypican 3
  • the binding molecule further comprises a third domain comprising an Fc fragment of an immunoglobulin.
  • the binding molecule further comprises a third domain comprising an Fc fragment of an immunoglobulin.
  • the immunoglobulin is selected from IgA, IgG, IgM, IgD and IgE.
  • the immunoglobulin is IgG, such as IgG1, IgG2, IgG3 or IgG4.
  • the Fc fragment has one or more mutations among L234F, L235E, P331S, D356E and L358M, wherein the Fc fragment is numbered according to the EU index of Kabat.
  • the third domain has an amino acid sequence as shown in SEQ ID NO: 55, 62 or 69 or a variant thereof.
  • the binding molecule may include a heavy chain constant region CH 1 linked to the N-terminus of the Fc fragment.
  • the first domain comprises a first heavy chain variable region VH 1 and a first light chain variable region VL 1 .
  • the structure of the first domain is selected from Fab, Fab', F(ab') 2 , Fv or scFv.
  • the C-terminal of the VH 1 peptide chain is directly connected to the N-terminal of the VL 1 peptide chain or connected via a linker, and vice versa.
  • the VH 1 comprises: (a) HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences shown in SEQ ID NO: 1, 2 and 3, respectively; or, (b) comprising HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO:7, 8 and 9.
  • the VL1 comprises: (c) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, respectively; or, (d) comprising LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO: 10, 11 and 12.
  • the first structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 1, HCDR2 including the amino acid sequence shown in SEQ ID NO: 2, including the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR3 of the amino acid sequence shown in ID NO:3, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:4, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence of LCDR3 is shown.
  • the first structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 7, HCDR2 including the amino acid sequence shown in SEQ ID NO: 8, including the amino acid sequence shown in SEQ ID NO: 8
  • the HCDR3 of the amino acid sequence shown in ID NO:9, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:10, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:11, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:12 The amino acid sequence of LCDR3 is shown.
  • the first domain of the binding molecule of the present disclosure comprises VH 1 and VL 1 .
  • the VH 1 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO:31 or 33.
  • the VL 1 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:32 or 34.
  • the VH 1 of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 31
  • VL 1 comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO: 31
  • the amino acid sequence represented by 32 has an amino acid sequence having at least 90% sequence identity.
  • the VH 1 of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 33
  • VL 1 comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO: 33
  • the amino acid sequence shown in 34 has an amino acid sequence having at least 90% sequence identity.
  • the second domain of the binding molecule of the present disclosure comprises a second heavy chain variable region VH 2 and a second light chain variable region VL 2 .
  • the structure of the second domain is selected from Fab, Fab', F(ab') 2 , Fv or scFv.
  • the C-terminal of the VH 2 peptide chain is directly connected to the N-terminal of the VL 2 peptide chain or connected via a linker, and vice versa.
  • the VH 2 includes: (e) HCDR1, HCDR2 and HCDR3 including the amino acid sequences shown in SEQ ID NO:13, 14 and 15 respectively; (f) including the amino acid sequences shown in SEQ ID NO:13, 14 and 15 respectively; HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in ID NOs: 19, 20 and 21; or, (g) HCDR1, HCDR2 and HCDR3 including the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27, respectively.
  • the VL 2 includes: (h) LCDR1, LCDR2 and LCDR3 including the amino acid sequences shown in SEQ ID NO: 16, 17 and 18; (i) including SEQ ID NO: 16, 17 and 18 respectively; LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in ID NOs: 22, 23 and 24; or, (j) LCDR1, LCDR2 and LCDR3 including the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, respectively.
  • the second structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 13, HCDR2 including the amino acid sequence shown in SEQ ID NO: 14, including the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 of the amino acid sequence shown in ID NO:15, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:16, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:17, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:18 The amino acid sequence of LCDR3 is shown.
  • the second structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 19, HCDR2 including the amino acid sequence shown in SEQ ID NO: 20, including the amino acid sequence shown in SEQ ID NO: 20
  • the HCDR3 of the amino acid sequence shown in ID NO:21, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:22, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:23, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:24 The amino acid sequence of LCDR3 is shown.
  • the second structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 25, HCDR2 including the amino acid sequence shown in SEQ ID NO: 26, including the amino acid sequence shown in SEQ ID NO: 26
  • the HCDR3 of the amino acid sequence shown in ID NO:27, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:28, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:29, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:30 The amino acid sequence of LCDR3 is shown.
  • the second domain of the binding molecule of the present disclosure comprises VH 2 and VL 2 .
  • the VH 2 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO:35, 37 or 39.
  • the VL 2 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:36, 38 or 40.
  • the VH 2 of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 35
  • VL 2 comprises an amino acid sequence identical to that shown in SEQ ID NO: 35
  • the amino acid sequence shown in 36 has an amino acid sequence having at least 90% sequence identity.
  • the VH 2 of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 37
  • VL 2 comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO: 37
  • the amino acid sequence represented by 38 has an amino acid sequence having at least 90% sequence identity.
  • the VH 2 of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 39
  • VL 2 comprises an amino acid sequence identical to the amino acid sequence shown in SEQ ID NO: 39
  • the amino acid sequence shown at 40 has an amino acid sequence having at least 90% sequence identity.
  • the first domain, the second domain and/or the third domain are directly connected or connected through a linker.
  • the first heavy chain variable region and the first light chain variable region of the first domain may be connected directly or through a linker.
  • the second heavy chain variable region and the second light chain variable region of the second domain are connected directly or through a linker.
  • the linker used in the binding molecule of the present disclosure has a sequence shown as (G n S) z , wherein n and z are each independently an integer of 1-4.
  • the linker sequence used in the present disclosure can be GS, GSGS (SEQ ID NO: 57), GGGGS (SEQ ID NO: 58), GGGGSGS (SEQ ID NO: 59), GGGGSGGGGSGGGGS (SEQ ID NO: 60) or Amino acid sequence shown by GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 61).
  • a binding molecule of the present disclosure comprises a first domain, a second domain and a third domain connected in the following order: first domain-third domain-second domain; Second domain-third domain-first domain; first domain-second domain-third domain; or, second domain-first domain-third domain.
  • a binding molecule of the present disclosure may comprise two identical peptide chains that form a tetravalent homodimer.
  • one peptide chain of a binding molecule of the disclosure has an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41, 42, or 43.
  • a binding molecule of the present disclosure may comprise a heterotetramer formed of a long peptide chain and a short peptide chain, wherein the long peptide chain has at least 90% of the amino acid sequence shown in SEQ ID NO:44. Sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:45.
  • the binding molecules of the present disclosure include heterotetramers that may include long peptide chains and short peptide chains, wherein the long peptide chains have at least 90 % sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:47.
  • a binding molecule of the present disclosure may comprise a heterotetramer formed of a long peptide chain and a short peptide chain, wherein the long peptide chain has at least 90% of the amino acid sequence shown in SEQ ID NO:48. Sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:49.
  • a binding molecule of the present disclosure may comprise a heterotetramer formed of a long peptide chain and a short peptide chain, wherein the long peptide chain has at least 90% of the amino acid sequence shown in SEQ ID NO:50. Sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:51.
  • a binding molecule of the present disclosure may comprise a heterotetramer formed of a long peptide chain and a short peptide chain, wherein the long peptide chain has at least 90% of the amino acid sequence shown in SEQ ID NO:64. Sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:51.
  • a binding molecule of the present disclosure may comprise a heterohexamer formed of a long peptide chain, a first short peptide chain, and a second short peptide chain, wherein the long peptide chain has the same composition as that of SEQ ID NO:52.
  • the amino acid sequence shown has at least 90% sequence identity
  • the first short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:54
  • the second short peptide chain has at least 90% sequence identity with SEQ ID NO:
  • the amino acid sequences shown at 53 have at least 90% sequence identity.
  • the binding molecules of the present disclosure may also include a heavy chain constant region, such as CH 1 , CH 2 , CH 3 and/or CH 4 . In some embodiments of the present disclosure, the binding molecules of the present disclosure may further comprise a light chain constant region CL.
  • the present disclosure provides an isolated nucleic acid molecule encoding a binding molecule of the present disclosure or a fragment thereof.
  • the present disclosure provides a vector comprising the nucleic acid molecule described in the present disclosure.
  • the vector is an expression vector that can be transcribed and translated in a host cell to express the binding molecule of the present disclosure or a fragment thereof.
  • the present disclosure provides a host cell comprising a nucleic acid molecule or vector of the present disclosure.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the binding molecule of the present disclosure, the nucleic acid molecule of the present disclosure, the vector of the present disclosure or the host cell of the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of treating or preventing cancer, comprising administering the pharmaceutical composition of the present disclosure to a subject in need thereof.
  • the present disclosure provides the use of a binding molecule of the present disclosure in the manufacture of a medicament for the treatment or prevention of cancer.
  • the cancer is selected from the group consisting of melanoma, glioma, kidney cancer, breast cancer, liver cancer, Wilms tumor, hepatoblastoma, blood cancer, and head and neck cancer.
  • the present disclosure provides the use of the binding molecule of the present disclosure, the nucleic acid molecule of the present disclosure, the vector of the present disclosure, the host cell of the present disclosure or the pharmaceutical composition of the present disclosure in the preparation of a medicament for treating or preventing cancer.
  • the cancer is selected from the group consisting of melanoma, glioma, kidney cancer, breast cancer, liver cancer, Wilms tumor, hepatoblastoma, blood cancer, and head and neck cancer.
  • the 4-1BB activation antibody is constructed in the form of Fab, scFv or single-chain antibody, and the linker sequence composed of flexible amino acids or the inherent linking amino acids of the antibody are fused to form a binding domain of the bispecific antibody, and then the GPC3-specific Sexual antibodies are constructed in the form of Fab, scFv or single-chain antibodies through the fusion of linkers composed of flexible amino acids or inherent linking amino acids of antibodies to form another binding domain of bispecific antibodies.
  • the bispecific binding molecule for 4-1BB and GPC3 of the present disclosure can target and bind GPC3-positive tumor cells and at the same time generate an activation effect of T cells related to the 4-1BB signaling pathway.
  • the bispecific binding molecules of the present disclosure exhibit excellent tumor growth inhibitory effects in mouse tumor models, and the combination of binding molecules targeting 4-1BB and GPC3 has a certain synergistic effect in inhibiting tumor growth.
  • Figure 1 shows the binding of binding molecules of the present disclosure to a CHO cell line stably transfected with human 4-1BB.
  • Figure 1A The binding of the binding molecule of the present disclosure to the CHO cell line highly expressing human 4-1BB;
  • Figure 1B The binding of the binding molecule of the present disclosure to the CHO cell line highly expressing human 4-1BB and the corresponding EC50 value.
  • MFI indicates mean fluorescence intensity.
  • Figure 2 shows the binding of binding molecules of the present disclosure to recombinant human 4-1BB protein.
  • Fig. 2A is the binding of the binding molecule of the present disclosure to the recombinant human 4-1BB protein;
  • Fig. 2B is the binding of the binding molecule of the present disclosure to the recombinant human 4-1BB protein and the corresponding EC50 value.
  • Figure 3 shows the binding of binding molecules of the present disclosure to activated T cells.
  • FIG. 4 shows the ELISA binding of binding molecules of the present disclosure to recombinant human GPC3 protein.
  • FIG. 4A is the binding of the binding molecule of the present disclosure to the recombinant human GPC3 protein;
  • FIG. 4B is the binding of the binding molecule of the present disclosure to the recombinant human GPC3 protein and the corresponding EC50 value.
  • Figure 5 shows the binding of binding molecules to human Huh7 and HepG2 liver cancer cell lines.
  • Fig. 5A the binding of the binding molecule of the present disclosure to the human Huh7 liver cancer cell line;
  • Fig. 5B the binding of the binding molecule of the present disclosure to the human HepG2 liver cancer cell line and the corresponding EC50 value.
  • Figure 6 shows that binding molecules of the present disclosure activate T cells in the presence of GPC3 positive liver cancer cells.
  • a and B) show that the binding molecule of the present disclosure stimulates T cells to secrete interleukins IL2 and IFN ⁇ in the presence of GPC3-positive liver cancer cells Huh7, respectively.
  • C) to (F) respectively show that the binding molecule of the present disclosure stimulates T cells to secrete IL2 and IFN ⁇ in the presence of GPC3-positive liver cancer cell HepG2.
  • G shows that binding molecules of the present disclosure stimulate T cells to secrete IFN ⁇ in the presence of GPC3-positive liver cancer cell Hep3B.
  • Figure 7 shows that binding molecules of the present disclosure inhibit the growth of GPC3 positive tumors.
  • Figure 7A shows the results of inhibition of GPC3-positive tumor growth by 5 mg/kg of binding molecules;
  • Figure 7B shows the inhibition of 20 mg/kg of binding molecules on the growth of GPC3-positive tumors.
  • Figure 8 shows a schematic representation of a binding molecule of the present disclosure.
  • Figure 9 shows that different concentrations of binding molecules of the present disclosure inhibit the growth of GPC3 positive tumors.
  • FIG 10 shows that different concentrations of HEC512-G1D inhibit the growth of GPC3 positive tumors.
  • 4-1BB also known as CD137, TNFRSF9, etc.
  • TNFFRS Tumor Necrosis Factor Receptor Superfamily
  • 4-1BB is a co-stimulatory receptor expressed on various cells of the immune system, especially on CD8+ T cells. Due to its ubiquitous expression, and the ability of 4-1BB to enhance potent and long-lasting immune effects, 4-1BB has become a clinical target for cancer immunotherapy.
  • glycosyl Heparan sulfate proteoglycans anchored by phosphatidylinositol.
  • Glypican regulates the activity of several growth factors including Wnts, Hedgehogs, bone morphogenetic proteins and fibroblast growth factor (FGF).
  • FGF fibroblast growth factor
  • Glypican is characterized by covalent linkages to polysaccharide chains known as heparan sulfate glycosaminoglycans. Glypican participates in cell signaling at the cell-extracellular matrix interface.
  • GPC3 consists of two subunits linked by one or more disulfide bonds. GPC3 is expressed in the developing fetal liver and placenta and is downregulated or silenced in normal adult tissues. GPC3 is highly expressed in various cancers, especially hepatocellular carcinoma (HCC), melanoma, Wilms tumor and hepatoblastoma.
  • HCC hepatocellular carcinoma
  • melanoma melanoma
  • Wilms tumor hepatoblastoma.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, carboxyl, amino and R groups, such as homoserine, norleucine, methionine sulfoxide, Methylsulfonium methionine.
  • Amino acid mimetics are compounds that differ structurally from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.
  • polar amino acid refers to an amino acid comprising a side chain that prefers to reside in an aqueous environment.
  • the polar amino acid is selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, and tyrosine.
  • Polar amino acids can be positively charged, negatively charged, or neutrally charged.
  • non-polar amino acid is selected from the group consisting of alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan acid and valine.
  • substitution with one or more different amino acids means that at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) is replaced by another different “replacement” amino acid residue .
  • Amino acid insertion refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While inserts typically consist of insertions of 1 or 2 amino acid residues, larger “peptide insertions” can also be made, for example insertions of about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residues may be naturally occurring or non-naturally occurring.
  • Amin deletion refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
  • Antibodies suitable for use in the present disclosure may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues.
  • a "conservative amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain.
  • Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (such as alanine, valine , leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chains (e.g.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • an essential or nonessential amino acid residue in an antibody is preferably replaced with another amino acid residue from the same side chain family.
  • amino acid stretches may be replaced with structurally similar stretches that differ in the order and/or composition of side chain family members.
  • mutations may be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants may be incorporated into the binding polypeptides of the invention and directed against these binding polypeptides The ability to bind to the desired target is screened.
  • anti-4-1BB agonistic antibody or “specific agonistic antibody against 4-1BB” means that it specifically binds to 4-1BB and partially or completely promotes, induces, increases and/or activates Antibodies to 4-1BB biological activity, responses and/or downstream pathways mediated by 4-1BB signaling or other 4-1BB mediated functions.
  • Tumor necrosis factor receptor (TNFR) signaling particularly TNFRSF activation, requires receptor clustering and multimerization.
  • 4-1BB is one of the TNFSF receptors known to require clustering to trigger downstream signaling. Formation of 2 or more trimers by cross-linking of 4-1BB has been reported to result in a stronger activated protein.
  • the anti-4-1BB agonistic antibody binds 4-1BB and induces multimerization of 4-1BB into 2 or more trimers.
  • full-length antibody and “intact antibody” of the present invention are used interchangeably herein to refer to an antibody that is substantially similar in structure to a natural antibody.
  • “Native antibody” refers to a naturally occurring immunoglobulin molecule.
  • antibodies of the native IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also called variable heavy domain or heavy chain variable domain) and three constant domains (CH1, CH2 and CH3) (also known as the heavy chain constant region).
  • VH variable region
  • CH1 and CH3 constant domains
  • each light chain has a variable region (VL) (also called variable light domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain domain). chain constant region).
  • the heavy chain of an antibody can be of one of five types, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), which can be further divided into Subtypes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), ⁇ 4 (IgG4), ⁇ 1 (IgA1 ) and ⁇ 2 (IgA2).
  • the light chains of an antibody based on the amino acid sequence of their constant domains, can be of one of two types, kappa and lambda light chains.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • CL light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the binding molecules of the present disclosure are homodimers comprising two identical peptide chains bonded by disulfide bonds.
  • the two identical peptide chains are long peptide chains comprising a first domain and a second domain that specifically bind 4-1BB and GPC3, respectively.
  • the long peptide chain of the binding molecule of the present disclosure may include VL 2 -VH 2 -Fc-VH 1 -VL 1 , VH 2 -VL 2 -Fc-VH 1 -VL 1 , VL 2 -VH 2 - Fc- VL1 - VH1 , VH1 - VL1-Fc-VH2- VL2 , VL1 - VH1 -Fc- VL2 - VH2 , VL1 - VH1 -Fc- VH2 - VL2 , or Other connection methods known to those skilled in the art, wherein each fragment may or may not have the linker sequence disclosed herein.
  • the binding molecule of the present disclosure is a heterotetramer, which includes two long peptide chains bonded by disulfide bonds, and two short peptide chains respectively bonded to the corresponding long peptide chains by disulfide bonds.
  • the long peptide chain may include VH 2 -Fc-VH 1 -VL 1 from the N-terminus, and the short peptide chain may include VL 2 from the N-terminus.
  • the long peptide chain may include VH 1 -Fc-VH 2 -VL 2 from the N-terminus, and the short peptide chain may include VL 1 from the N-terminus.
  • the long peptide chain may include VH 1 -Fc-VL 2 -VH 2 from the N-terminus
  • the short peptide chain may include VL 1 from the N-terminus.
  • the functional fragments in the long peptide chain and the short peptide chain can be combined in other ways known to those skilled in the art.
  • the binding molecule of the present disclosure is a heterohexamer comprising two long peptide chains, two first short peptide chains and two second short peptide chains, wherein the two long peptide chains are bonded by disulfide bonds, The first short peptide chain and the second short peptide chain are respectively bonded to corresponding regions of the long peptide chain through disulfide bonds.
  • the long peptide chain may include VH 1 -Fc-VL 2
  • the first short peptide chain includes VL 1
  • the second short peptide chain includes VH 2 .
  • the functional fragments in the long peptide chain and the short peptide chain can be combined in other ways known to those skilled in the art.
  • the terms “specifically bind”, “selectively bind”, “selectively bind” and “specifically bind” as used herein refer to the binding of an antibody or antigen-binding portion thereof to an epitope on a predetermined antigen.
  • the antibody is present at about less than 10 ⁇ 6 M, such as about less than 10 -7 M, 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or even lower equilibrium dissociation constant (K D ) binding
  • binding to the predetermined antigen affinity is binding to other than the predetermined antigen or closely related At least twice the affinity of non-specific antigens (eg, BSA, casein) outside the antigen.
  • subject as used herein includes any human or non-human animal.
  • methods and compositions of the invention can be used to treat a subject with cancer.
  • non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.
  • antibody fragment refers to an antibody that retains binding to a target antigen (e.g., 4-1BB or GPC3) and has the same activity as the full-length antibody. fragment.
  • target antigen e.g. 4-1BB or GPC3
  • fragments include, for example, single chain antibodies, single chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments or F(ab')2 fragments.
  • scFv fragment is a single polypeptide chain that includes the heavy and light chain variable regions of the antibody from which the scFv is derived.
  • intrabodies, minibodies, tribodies and diabodies are included within the definition of antibody and are suitable for use in the methods described herein. See eg Todorovska et al. (2001), J. Immunol. Methods, 248(1): 47-66; Hudson and Kortt, (1999), J. Immunol. Methods, 231(1): 177-189; Poljak, ( 1994), Structure, 2(12):1121-1123; Rondon and Marasco, (1997), Annu.Rev.Microbiol., 51:257-283, the respective disclosures of which are incorporated herein by reference in their entirety middle.
  • Fc fragment refers to the constant region of a full-length immunoglobulin.
  • Fc fragment refers to the last two constant domains (CH2-CH3) of IgA, IgD, IgG, or the last three constant domains (CH2-CH3-CH4) of IgE and IgM, and N-terminal flexible hinges of these domains.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available from http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and length weight 1, 2, 3, 4, 5 or 6 determination.
  • the percent identity between two nucleotide or amino acid sequences can also be calculated using the algorithm of E. Meyers and W.
  • the algorithm has been incorporated into the ALIGN program (version 2.0).
  • the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970) ), using the Blossum 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6 determined, the algorithm has been incorporated into the GCG software package (available from http://www.gcg.com ) in the GAP program.
  • variable region or “variable domain” as used herein refers to the domain of an antibody heavy or light chain that participates in the binding of an antigen-binding molecule to an antigen.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • variable means that certain segments of the variable domains generally differ in sequence among antibodies.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains.
  • HVRs hypervariable regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, mostly in a ⁇ -sheet configuration, connected by three HVRs that form loops connecting and in some cases forming part of the ⁇ -sheet structure.
  • the HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda , MD (1991)).
  • the constant domain is not directly involved in the binding of the antibody to the antigen, but has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.
  • hypervariable region refers to the region of an antibody variable domain region that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops").
  • native four-chain antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).
  • HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)", which have the highest sequence variability and/or are involved in antigen recognition.
  • CDRs complementarity determining regions
  • Exemplary CDRs are located at amino acid residues L26-L32(L1), L50-L52(L2), L91-L96(L3), H26-H32( H1), H52-H56 (H2) and H96-H101 (H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987)).
  • Exemplary CDRs are located at amino acid residues L24-L34(L1), L50-L56(L2), L89-L97(L3), H31-H35( H1), H50-H65 (H2) and H95-H102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).
  • exemplary CDRs are located at amino acid residues L27-L32(L1), L50-L51(L2), L89-L97(L3), H26-H33(H1) , H51-H56(H2) and H93-H102(H3) (Honjo, T. and Alt, FW (1995) Immunoglobulin genes. Academic Press pp. 3-443).
  • Table 1 are listed the corresponding amino acid residues comprising the CDRs defined in the references cited above.
  • CDR of an antibody can be defined in various ways, such as the Kabat definition rule based on sequence variability, the Chothia definition rule based on the position of the structural loop region, and antibody humanization based on CDR grafting A reference tool for design (see J Mol Biol 273:927-48, 1997).
  • FR Framework or "FR” as used herein refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR4.
  • HVR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • a binding molecule of the present disclosure may comprise a Fab arm comprising one heavy chain-light chain pair that specifically binds an antigen.
  • the binding molecules of the present disclosure may include recombinant IgG-like dual targeting molecules, wherein each of the two sides of the molecule contains Fab fragments or parts of Fab fragments of at least two different antibodies; IgG fusion molecules, wherein A full-length IgG antibody fused to an additional Fab fragment or a portion of a Fab fragment; an Fc fusion molecule in which a single chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, an Fc region, or a portion thereof; a Fab fusion molecule in which Different Fab fragments are fused together; scFv and diabodies based heavy chain antibodies (e.g. domain antibodies, nanobodies) where different single chain Fv molecules or different diabodies or different heavy chain antibodies are fused
  • the "class" of an antibody as used herein refers to the type of constant domain or constant region that the heavy chain of the antibody has.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • humanized antibody used herein comprises the amino acid residues from the non-human hypervariable region (HVR) and the constant regions of the heavy chain and light chain from the human antibody spliced to obtain the complete sequence of the humanized antibody.
  • a humanized antibody comprises at least one, usually two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs Corresponds to FRs of human antibodies.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, such as a non-human antibody refers to an antibody that has been humanized.
  • cross-reactivity refers to the ability of an antibody of the present disclosure to bind 4-1BB or GPC3 from a different species.
  • an antibody of the disclosure may bind human 4-1BB while also binding 4-1BB of another species (eg, mouse, rat, cynomolgus monkey, or dog).
  • cross-reactivity is measured by detecting specific reactivity with purified antigen in a binding assay (eg, SPR, ELISA), or with physiologically expressed cells that bind or otherwise functionally interact.
  • having "at least 90% sequence identity" with a sequence compared to a sequence may include at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% %, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • nucleic acid sequence can be directly or indirectly connected to the regulatory elements on the carrier, as long as these regulatory elements It is sufficient that the element can regulate the translation and expression of the nucleic acid molecule.
  • regulatory elements may come directly from the vector itself, or be exogenous, that is, not from the vector itself. That is, a nucleic acid molecule is operably linked to a regulatory element.
  • operably linked refers to linking the exogenous gene to the carrier, so that the regulatory elements in the carrier, such as transcriptional regulatory sequences and translational regulatory sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function.
  • the polynucleotides used to encode the heavy chain and light chain of the antibody can be independently inserted into different vectors, usually inserted into the same vector.
  • Commonly used vectors can be, for example, plasmids, phages and the like.
  • the "cell” or “recombinant cell” used herein may contain an expression vector.
  • Expression vectors can be introduced into mammalian cells to obtain recombinant cells, which are then used to express the antibodies or antigen-binding portions provided in the present disclosure.
  • the corresponding antibody can be obtained by culturing the recombinant cells.
  • These usable mammalian cells are, for example, CHO cells and the like.
  • pharmaceutically acceptable carrier may include any solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof.
  • the pharmaceutical composition may contain isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol) or sodium chloride, etc.
  • pharmaceutically acceptable carriers may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, to prolong the shelf life or potency of the antibody.
  • an antibody of the disclosure, or an antigen-binding portion thereof can be incorporated into a pharmaceutical composition suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration eg, intravenous, subcutaneous, intraperitoneal, intramuscular.
  • These pharmaceutical compositions can be prepared in various forms, such as liquid, semi-solid and solid dosage forms, etc., including but not limited to liquid solutions (such as injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, Powders, liposomes and suppositories.
  • Typical pharmaceutical compositions are in the form of solutions for injection or infusion.
  • Antibodies of the disclosure, or antigen-binding portions thereof can be administered by intravenous infusion, injection, intramuscular or subcutaneous injection.
  • a “therapeutically effective amount” of a drug refers to that amount, in dosage and administration interval and time, effective to achieve the desired therapeutic or prophylactic effect.
  • a therapeutically effective amount of a drug eliminates, alleviates/reduces, delays, minimizes or prevents the adverse effects of a disease.
  • blood cancer includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as cancers of the spleen and lymph nodes.
  • exemplary lymphomas include B-cell lymphoma (B-cell blood cancer) and T-cell lymphoma.
  • B-cell lymphomas include Hodgkin's lymphoma and most non-Hodgkin's lymphomas.
  • B-cell lymphoma include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma (MCL), Burkitt Lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, lymph node marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granuloma.
  • T cell lymphoma examples include extranodal T cell lymphoma, cutaneous T cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma.
  • Hematological malignancies also include leukemias such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia.
  • Hematologic malignancies also include myelomas such as, but not limited to, multiple myeloma and smoldering multiple myeloma. The term hematological malignancies encompasses other blood and/or B or T cell related cancers.
  • Example 1 Transient transfection of HEK293 suspension cells to obtain binding molecules.
  • the peptide chains in Table 2 below were subjected to whole gene synthesis and inserted into the pcDNA3.1 vector respectively.
  • the constructed vectors were transiently transfected into HEK293 cells for protein expression.
  • the vectors inserted with multiple peptide chains are co-transfected into HEK293 cells (for example, peptide chain 1, peptide chain 2 and peptide chain 2 of IOB1-FabIg-S2 are respectively inserted Peptide 3 vector, co-transfected into HEK293 cells).
  • the heavy chain variable region and the light chain variable region of anti-4-1BB are respectively derived from monoclonal antibody HEC511 (the amino acid sequence of the heavy chain is shown in SEQ ID NO: 67, and the amino acid sequence of the light chain is shown in SEQ ID NO: 54) or monoclonal antibody HEC512 (the amino acid sequence of the heavy chain is shown in SEQ ID NO: 68, and the amino acid sequence of the light chain is shown in SEQ ID NO: 66).
  • the heavy chain variable region and the light chain variable region of anti-GPC3 are derived from three monoclonal antibodies HS20 respectively (the sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37) ID NO: 38), 4A6 (the sequence of the heavy chain variable region is shown in SEQ ID NO35, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 36) or GC33 (the sequence of the heavy chain variable region is shown in SEQ ID NO: 36) The sequence is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:40).
  • the expressed binding molecules were purified by ProteinA medium, and the binding molecules IOB1-S, IOB1-M, IOB1-ScFvIg-S, IOB1-FabIg-S2, IOB1-G, IOB1-G-Fab, IOB1-G, IOB1-G-Fab, The structure diagram of IOB1-G-Fab1, IOB1-G-Fab2 and IOB1-G1D-Fab2 is shown in FIG. 8 . These binding molecules obtained were used for the following assay evaluation.
  • Example 2 Binding of the binding molecule of the present disclosure to a CHO cell line stably transfected with human 4-1BB.
  • ELISA was used to detect the binding ability of the binding molecule to human 4-1BB (UniProtKB-Q07011) recombinant protein. Coat the recombinant 4-1BB protein on the ELISA plate overnight at 4°C, wash it three times and then dry it, block it at 37°C for 1 hour, wash it three times and dry it for later use.
  • 4-1BB UniProtKB-Q07011
  • the binding molecule of the present disclosure can effectively bind to the human 4-1BB (UniProtKB-Q07011) recombinant protein.
  • T cells were isolated from human-derived PBMC cells, and CD3 and CD28 antibody-coupled magnetic beads were used to stimulate T cells in vitro for 48-72 hours.
  • the stimulated T cells could highly express 4-1BB protein.
  • the ability of the binding molecule of the present disclosure to bind to the stimulated T cells was detected by flow cytometry. After incubation of the binding molecules with the cells, subsequent binding was performed using a FITC-labeled goat anti-mouse IgG Fc secondary antibody, and an isotype IgG antibody was used as a negative control. The results are shown in FIG. 3 .
  • the binding molecules of the present disclosure can effectively bind to activated human T cells. That is to say, the binding molecules of the present disclosure can effectively bind to the 4-1BB protein expressed by activated T cells.
  • ELISA was used to detect the binding ability of the binding molecule of the present disclosure to human GPC3 (XP_022382036.1) recombinant protein.
  • the recombinant GPC3 protein was coated on the ELISA plate overnight at 4°C, washed three times and then dried, blocked at 37°C for 1 hour, washed three times and then dried for later use.
  • the binding molecule of the present disclosure can effectively bind to the human GPC3 (XP_022382036.1) recombinant protein.
  • GPC3 protein is highly expressed in liver cancer cells.
  • conventionally cultured Huh7 or HepG2 liver cancer cells were digested with trypsin, collected, and flow cytometry was used to detect whether the binding molecules of the present disclosure could bind to human liver cancer cell lines Huh7 or HepG2. Binding capacity of HepG2. After incubating the binding molecule of the present disclosure with the cells, FITC-labeled goat anti-mouse IgG Fc secondary antibody was used to bind, the isotype IgG antibody was used as a negative control, and the corresponding antibodies GC33 and HEC511 used to construct the fusion protein were used as control samples. The results are shown in FIG. 5 .
  • the binding molecule of the present disclosure can effectively bind to the GPC3 protein expressed by the human liver cancer cell line.
  • Example 7 Affinity of binding molecules of the present disclosure to human 4-1BB recombinant protein.
  • the binding affinity of the binding molecules of the present disclosure to 4-1BB was determined by using the biofilm light interference technique (Bio-Layer Interferometry, BLI).
  • BLI Bio-Layer Interferometry
  • the human 4-1BB (UniProtKB-Q07011) protein was immobilized on the sensor for 300s, then the sensor was put into the PBST solution to balance the baseline for 180s, and then the probe was immersed in the fusion protein solution for 600s , and then the probe was placed in PBST solution to dissociate for 1200s, and finally the probe was placed in the regeneration solution to complete the regeneration and preservation of the probe.
  • the binding affinity of the binding molecule of the present disclosure to the human recombinant protein 4-1BB was detected by BLI detection. The results are shown in Table 3.
  • the binding affinity of the binding molecule of the present disclosure to GPC3 was measured by using bio-layer interferometry (Bio-Layer Interferometry, BLI).
  • BLI Bio-Layer Interferometry
  • the human GPC3 (XP_022382036.1) protein was immobilized on the sensor for 300s, then the sensor was put into the PBST solution to balance the Baseline180s, then the probe was immersed in the fusion protein solution for 600s, and then The probe was placed in PBST solution for 1200s, and finally the probe was placed in the regeneration solution to complete the regeneration and preservation of the probe.
  • BLI detection the binding affinity of the binding molecule of the present disclosure to the human recombinant protein GPC3 was determined. The results are shown in Table 4.
  • Binding molecules of the present disclosure activate T cells in a GPC3 positive liver cancer cell dependent manner.
  • human T cells After human T cells are isolated, the T cells and the binding molecule of the present disclosure are added to a microtiter plate, and the expression level of IL2 in the supernatant is detected after three days, or the expression level of IFN- ⁇ in the supernatant is detected after five days.
  • HEC511, HS20, 4A6 or isotype IgG antibodies were used as controls. The result is shown in Figure 6.
  • Figures 6A and 6B show that binding molecules of the present disclosure can stimulate T cells to secrete interleukins IL2 and IFN ⁇ in the presence of GPC3-positive liver cancer cells Huh7.
  • 6C-6F show that the binding molecules of the present disclosure can stimulate T cells to secrete IL2 and detect IFN ⁇ in the presence of GPC3-positive liver cancer cells HepG2.
  • FIG. 6G shows that the binding molecules of the present disclosure can stimulate T cells to secrete IFN ⁇ in the presence of GPC3-positive liver cancer cells Hep3B.
  • Example 10 Verification of the tumor suppressive effect of the binding molecules of the present disclosure in the tumor-bearing humanized mouse model of MC38 high-expression GPC3 stably transfected cell line
  • the MC38 tumor cell line (MC38-hGPC3 cells) resuspended in PBS with high expression of human GPC3 protein was inoculated in B- h4-1BB /h4-1BBL human-derived Subcutaneously on the right side of the mouse.
  • the appropriate mice were selected according to the tumor volume and body weight of the mice, and were evenly distributed into 3 experimental groups, with 6 mice in each group.
  • the samples of each group were intraperitoneally administered at a dose of 5 mg/kg (the control group was given PBS), twice a week. After grouping, the tumor volume was measured twice a week with a vernier caliper.
  • the tumor volume was measured, and the long and short diameters of the tumor were measured.
  • the results are shown in Figure 7A. It can be seen that the bispecific antibody of the present disclosure can significantly inhibit the growth of tumor after administration; and at the same dose, the onset time of bispecific antibody inhibition of tumor is earlier than that of anti-4-1BB monoclonal Anti-41BB monoclonal HEC512 also significantly earlier eliminated tumors.
  • mice The right side of 41BB/41BBL double humanized C57BL/66 mice was subcutaneously inoculated with 5 ⁇ 10 5 cells/0.1 mL/mouse, and the tumor cell line MC38 highly expressing human GPC3 protein was inoculated.
  • the average tumor volume reaches 100-150mm 3
  • select suitable mice according to their body weight and tumor volume, inject the test antibody IOB1-G-Fab2 into the mice at a dose of 20 mg/kg intraperitoneally, and measure the tumor regularly Size, tumor growth or inhibition was observed.
  • FIG. 7B showing that the tested antibody has the effect of significantly inhibiting the growth of GPC3-positive tumors.
  • Example 12 Anti-tumor activity evaluation of the tested antibody in the tumor-bearing humanized mouse model of MC38 high expression GPC3 stably transfected cell line
  • mice The right side of 41BB/41BBL double humanized C57BL/66 mice was subcutaneously inoculated with 5 ⁇ 10 5 cells/0.1 mL/mouse, and the tumor cell line MC38 highly expressing human GPC3 protein was inoculated.
  • test antibody IOB1-G1D-Fab2 and HEC512-G1D (the amino acid sequence of the heavy chain is as SEQ ID NO: 65, the amino acid sequence of the light chain is shown in SEQ ID NO: 66) according to 20mg/kg, 5mg/kg and 1mg/kg doses of intraperitoneal injection into mice, regularly measure the size of the tumor, observe the growth or inhibition of the tumor .
  • the results are shown in Figures 9 and 10, showing that the tested antibodies significantly inhibited the growth of GPC3-positive tumors, and the inhibitory effect of IOB1-G1D-Fab2 was better than that of HEC512-G1D.

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Abstract

提供了抗癌结合分子及其应用，具体提供了4-1BB和GPC3的双特异性结合分子，该双特异性结合分子包括特异性结合4-1BB的第一结构域，和特异性结合GPC3的第二结构域。还提供了该双特异性结合分子治疗或预防癌症的应用。

Description

抗癌结合分子及其应用 技术领域
本公开属于生物技术领域，具体涉及特异性结合4-1BB和磷脂酰肌醇蛋白聚糖3(GPC3)的结合分子及其应用。
背景技术
4-1BB(也称为CD137、TNFRSF9等)是肿瘤坏死因子受体超家族(TNFRS)的成员。针对4-1BB的抗体具备激活4-1BB信号通路的能力，对肿瘤治疗、抗感染、抗自体免疫疾病等具有潜在医学价值。磷脂酰肌醇蛋白聚糖3(GPC3)属于磷脂酰肌醇蛋白聚糖家族的癌胚抗原，高表达于多种癌症细胞，特别是肝细胞癌(HCC)、黑素瘤、维尔姆斯瘤和肝母细胞瘤中。因此，在临床上需要能够在靶向肿瘤细胞的同时，激活4-1BB通路来治疗肿瘤的以4-1BB激活抗体为基础的新型蛋白药物。
发明内容
为了解决现有技术中存在的上述技术问题之一，本公开提供了靶向肿瘤相关抗原GPC3(磷脂酰肌醇蛋白聚糖3)，同时激活4-1BB信号通路相关免疫途径的新方法，可有效解决4-1BB激活抗体的副作用。
在本公开的一个方面，提供了一种结合分子，所述结合分子包括：特异性结合4-1BB或其片段的第一结构域，和特异性结合磷脂酰肌醇蛋白聚糖3(GPC3)或其片段的第二结构域。
根据本公开的一些实施方式，所述结合分子还包括第三结构域，其包括免疫球蛋白的Fc片段。
根据本公开的一些实施方式，所述结合分子还包括第三结构域，其包括免疫球蛋白的Fc片段。
根据本公开的一些实施方式，所述免疫球蛋白选自IgA、IgG、IgM、IgD和IgE。在本公开的具体实施方式中，所述免疫球蛋白为IgG，例如为IgG1、IgG2、IgG3或IgG4。
根据本公开的一些实施方式，所述Fc片段具有L234F、L235E、P331S、D356E和L358M中的一个或多个突变，其中所述Fc片段按照Kabat的EU索引进行编号。
在本公开的具体实施方式中，所述第三结构域具有如SEQ ID NO:55、62或69所示的氨基酸序列或其变体。
在本公开的一些实施方式中，所述结合分子可以包括，与所述Fc片段的N端连接的重链恒定区CH 1。
根据本公开的一些实施方式，所述第一结构域包括第一重链可变区VH 1和第一轻链可变 区VL 1。根据本公开的一些实施方式，所述第一结构域的结构选自Fab、Fab’、F(ab’) 2、Fv或scFv。对于具有Fv或scFv结构的所述第一结构域，所述VH 1肽链的C末端与所述VL 1肽链的N末端直接连接或者通过连接子连接，反之亦然。
在本公开的一些具体实施方式中，所述VH 1包括：(a)包括分别如SEQ ID NO:1、2和3所示的氨基酸序列的HCDR1、HCDR2和HCDR3；或者，(b)包括分别如SEQ ID NO:7、8和9所示的氨基酸序列的HCDR1、HCDR2和HCDR3。在本公开的一些具体实施方式中，所述VL 1包括：(c)包括分别如SEQ ID NO:4、5和6所示的氨基酸序列的LCDR1、LCDR2和LCDR3；或者，(d)包括分别如SEQ ID NO:10、11和12所示的氨基酸序列的LCDR1、LCDR2和LCDR3。
根据本公开的一些具体实施方式，所述第一结构域包括：包括如SEQ ID NO:1所示的氨基酸序列的HCDR1，包括如SEQ ID NO:2所示的氨基酸序列的HCDR2，包括如SEQ ID NO:3所示的氨基酸序列的HCDR3，包括如SEQ ID NO:4所示的氨基酸序列的LCDR1，包括如SEQ ID NO:5所示的氨基酸序列的LCDR2，和包括如SEQ ID NO:6所示的氨基酸序列的LCDR3。
根据本公开的一些具体实施方式，所述第一结构域包括：包括如SEQ ID NO:7所示的氨基酸序列的HCDR1，包括如SEQ ID NO:8所示的氨基酸序列的HCDR2，包括如SEQ ID NO:9所示的氨基酸序列的HCDR3，包括如SEQ ID NO:10所示的氨基酸序列的LCDR1，包括如SEQ ID NO:11所示的氨基酸序列的LCDR2，和包括如SEQ ID NO:12所示的氨基酸序列的LCDR3。
根据本公开的一些实施方式，本公开结合分子的第一结构域包括VH 1和VL 1。所述VH 1包括与如SEQ ID NO:31或33所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。所述VL 1包括与如SEQ ID NO:32或34所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
在本公开的具体实施方式中，本公开结合分子的VH 1包括与如SEQ ID NO:31所示的氨基酸序列具有至少90％序列同一性的氨基酸序列，且VL 1包括与如SEQ ID NO:32所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
在本公开的具体实施方式中，本公开结合分子的VH 1包括与如SEQ ID NO:33所示的氨基酸序列具有至少90％序列同一性的氨基酸序列，且VL 1包括与如SEQ ID NO:34所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
根据本公开的一些实施方式，本公开结合分子的第二结构域包括第二重链可变区VH 2和第二轻链可变区VL 2。所述第二结构域的结构选自Fab、Fab’、F(ab’) 2、Fv或scFv。对于具有Fv或scFv结构的所述第一结构域，所述VH 2肽链的C末端与所述VL 2肽链的N末端直接连接或者通过连接子连接，反之亦然。
在本公开的一些具体实施方式中，所述VH 2包括：(e)包括分别如SEQ ID NO:13、14和15所示的氨基酸序列的HCDR1、HCDR2和HCDR3；(f)包括分别如SEQ ID NO:19、20 和21所示的氨基酸序列的HCDR1、HCDR2和HCDR3；或，(g)包括分别如SEQ ID NO:25、26和27所示的氨基酸序列的HCDR1、HCDR2和HCDR3。在本公开的一些具体实施方式中，所述VL 2包括：(h)包括分别如SEQ ID NO:16、17和18所示的氨基酸序列的LCDR1、LCDR2和LCDR3；(i)包括分别如SEQ ID NO:22、23和24所示的氨基酸序列的LCDR1、LCDR2和LCDR3；或，(j)包括分别如SEQ ID NO:28、29和30所示的氨基酸序列的LCDR1、LCDR2和LCDR3。
根据本公开的一些具体实施方式，所述第二结构域包括：包括如SEQ ID NO:13所示的氨基酸序列的HCDR1，包括如SEQ ID NO:14所示的氨基酸序列的HCDR2，包括如SEQ ID NO:15所示的氨基酸序列的HCDR3，包括如SEQ ID NO:16所示的氨基酸序列的LCDR1，包括如SEQ ID NO:17所示的氨基酸序列的LCDR2，和包括如SEQ ID NO:18所示的氨基酸序列的LCDR3。
根据本公开的一些具体实施方式，所述第二结构域包括：包括如SEQ ID NO:19所示的氨基酸序列的HCDR1，包括如SEQ ID NO:20所示的氨基酸序列的HCDR2，包括如SEQ ID NO:21所示的氨基酸序列的HCDR3，包括如SEQ ID NO:22所示的氨基酸序列的LCDR1，包括如SEQ ID NO:23所示的氨基酸序列的LCDR2，和包括如SEQ ID NO:24所示的氨基酸序列的LCDR3。
根据本公开的一些具体实施方式，所述第二结构域包括：包括如SEQ ID NO:25所示的氨基酸序列的HCDR1，包括如SEQ ID NO:26所示的氨基酸序列的HCDR2，包括如SEQ ID NO:27所示的氨基酸序列的HCDR3，包括如SEQ ID NO:28所示的氨基酸序列的LCDR1，包括如SEQ ID NO:29所示的氨基酸序列的LCDR2，和包括如SEQ ID NO:30所示的氨基酸序列的LCDR3。
根据本公开的一些实施方式，本公开结合分子的第二结构域包括VH 2和VL 2。所述VH 2包括与如SEQ ID NO:35、37或39所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。所述VL 2包括与如SEQ ID NO:36、38或40所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
在本公开的具体实施方式中，本公开结合分子的VH 2包括与如SEQ ID NO:35所示的氨基酸序列具有至少90％序列同一性的氨基酸序列，且VL 2包括与如SEQ ID NO:36所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
在本公开的具体实施方式中，本公开结合分子的VH 2包括与如SEQ ID NO:37所示的氨基酸序列具有至少90％序列同一性的氨基酸序列，且VL 2包括与如SEQ ID NO:38所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
在本公开的具体实施方式中，本公开结合分子的VH 2包括与如SEQ ID NO:39所示的氨基酸序列具有至少90％序列同一性的氨基酸序列，且VL 2包括与如SEQ ID NO:40所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
根据本公开的一些实施方式，本公开的结合分子中，所述第一结构域、所述第二结构域 和/或第三结构域直接连接或者通过连接子连接。根据本公开的一些实施方式，所述第一结构域的第一重链可变区和第一轻链可变区可以直接连接或者通过连接子连接。根据本公开的一些实施方式，所述第二结构域的第二重链可变区和第二轻链可变区直接连接或者通过连接子连接。
在本公开的一些实施方式中，本公开结合分子所使用的连接子具有如(G nS) z所示的序列，其中n和z各自独立地为1～4的整数。例如，本公开所使用的连接子序列可以是GS、GSGS(SEQ ID NO:57)、GGGGS(SEQ ID NO:58)、GGGGSGS(SEQ ID NO:59)、GGGGSGGGGSGGGGS(SEQ ID NO:60)或GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:61)所示的氨基酸序列。
在本公开的一些实施方式中，本公开结合分子包括以如下顺序连接的第一结构域、第二结构域和第三结构域：第一结构域-第三结构域-第二结构域；第二结构域-第三结构域-第一结构域；第一结构域-第二结构域-第三结构域；或，第二结构域-第一结构域-第三结构域。
根据本公开的一些实施方式，本公开的结合分子可以包括两条相同的肽链，这两条相同的肽链形成四价同源二聚体。在一些具体实施方式中，本公开结合分子的一条肽链具有与SEQ ID NO:41、42或43所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
在一些具体实施方式中，本公开的结合分子可以包括长肽链和短肽链形成的异源四聚体，其中该长肽链具有与SEQ ID NO:44所示的氨基酸序列具有至少90％序列同一性，该短肽链具有与SEQ ID NO:45所示的氨基酸序列具有至少90％序列同一性。
在一些具体实施方式中，本公开的结合分子包括可以包括长肽链和短肽链形成的异源四聚体，其中该长肽链具有与SEQ ID NO:46所示的氨基酸序列具有至少90％序列同一性，该短肽链具有与SEQ ID NO:47所示的氨基酸序列具有至少90％序列同一性。
在一些具体实施方式中，本公开的结合分子可以包括长肽链和短肽链形成的异源四聚体，其中该长肽链具有与SEQ ID NO:48所示的氨基酸序列具有至少90％序列同一性，该短肽链具有与SEQ ID NO:49所示的氨基酸序列具有至少90％序列同一性。
在一些具体实施方式中，本公开的结合分子可以包括长肽链和短肽链形成的异源四聚体，其中该长肽链具有与SEQ ID NO:50所示的氨基酸序列具有至少90％序列同一性，该短肽链具有与SEQ ID NO:51所示的氨基酸序列具有至少90％序列同一性。
在一些具体实施方式中，本公开的结合分子可以包括长肽链和短肽链形成的异源四聚体，其中该长肽链具有与SEQ ID NO:64所示的氨基酸序列具有至少90％序列同一性，该短肽链具有与SEQ ID NO:51所示的氨基酸序列具有至少90％序列同一性。
在一些具体实施方式中，本公开的结合分子可以包括长肽链、第一短肽链和第二短肽链形成的异源六聚体，其中该长肽链具有与SEQ ID NO:52所示的氨基酸序列具有至少90％序列同一性，该第一短肽链具有与SEQ ID NO:54所示的氨基酸序列具有至少90％序列同一性，该第二短肽链具有与SEQ ID NO:53所示的氨基酸序列具有至少90％序列同一性。
在本公开的一些实施方式中，本公开的结合分子还可以包括重链恒定区，例如CH 1、CH 2、CH 3和/或CH 4。在本公开的一些实施方式中，本公开的结合分子还可以包括轻链恒定区CL。
本领域技术人员可以根据常规技术，调整上述各功能片段(例如，VH 1、VL 1、Fc、VH 2、VL 1等)的位置和连接顺序，同时保持其各自的结合特性。
在另一方面，本公开提供了一种分离的核酸分子，其编码本公开所述的结合分子或其片段。
在又一方面，本公开提供了一种载体，其包括本公开所述的核酸分子。根据本公开的一些实施方式，所述载体是表达载体，可在宿主细胞中进行转录和翻译，以表达本公开的结合分子或其片段。
在又一方面，本公开提供了一种宿主细胞，其包括本公开的核酸分子或载体。
在又一方面，本公开提供了一种药物组合物，其包括本公开的结合分子、本公开的核酸分子、本公开的载体或本公开的宿主细胞和药学上可接受的载剂。
在又一方面，本公开提供了一种治疗或预防癌症的方法，其包括向有需要的受试者施用本公开的药物组合物。
在又一方面，本公开提供了本公开的结合分子在制备用于治疗或预防癌症的药物中的应用。
在本公开的一些实施方式，所述癌症选自由黑色素瘤、神经胶质瘤、肾癌、乳腺癌、肝癌、维尔姆斯瘤、肝母细胞瘤、血液癌症以及头颈癌。
在又一方面，本公开提供了本公开的结合分子、本公开的核酸分子、本公开的载体、本公开的宿主细胞或本公开的药物组合物在制备治疗或预防癌症的药物中的应用。
在本公开的一些实施方式，所述癌症选自由黑色素瘤、神经胶质瘤、肾癌、乳腺癌、肝癌、维尔姆斯瘤、肝母细胞瘤、血液癌症以及头颈癌。
本公开将4-1BB激活抗体构建为Fab、scFv或单链抗体形式，通过柔性氨基酸组成的连接子序列或者抗体固有的链接氨基酸融合，形成双特异性抗体的一结合结构域，再将GPC3特异性抗体构建为Fab、scFv或单链抗体形式通过柔性氨基酸组成的连接子或者抗体固有的链接氨基酸融合，形成双特异性抗体的另一结合结构域。本公开的针对4-1BB和GPC3的双特异性结合分子，能够在靶向结合GPC3阳性肿瘤细胞的同时，产生4-1BB信号通路相关的T细胞的激活效应。另外，本公开的双特异性结合分子在小鼠肿瘤模型中显示出优异的抑制肿瘤生长效果，且针对4-1BB和GPC3的结合分子组合在抑制肿瘤生长方面具有一定的协同效果。
下面的描述中阐述了本发明的一个或多个实施方案的细节。本公开的其它特征或优点将通过以下附图和对几个实施例的详细描述并且还通过所附权利要求书变得显而易见。
附图说明
为了使本公开的内容更容易被清楚的理解，下面根据本公开的具体实施例并结合附图，对本公开作进一步详细的说明，其中
图1示出了本公开的结合分子与稳定转染有人源4-1BB的CHO细胞株的结合。图1A本 公开的结合分子与高表达人源4-1BB的CHO细胞株的结合；图1B本公开的结合分子与高表达人源4-1BB的CHO细胞株的结合以及对应的EC50值。MFI表示平均荧光强度。
图2示出了本公开的结合分子与重组人源4-1BB蛋白的结合。图2A本公开的结合分子与重组人源4-1BB蛋白的结合；图2B本公开的结合分子与重组人源4-1BB蛋白的结合及对应的EC50值。
图3示出了本公开的结合分子与激活后的T细胞的结合。
图4示出了本公开的结合分子与重组人源GPC3蛋白ELISA结合。图4A本公开的结合分子与重组人源GPC3蛋白的结合；图4B本公开的结合分子与重组人源GPC3蛋白的结合及对应的EC50值。
图5示出了结合分子与人Huh7和HepG2肝癌细胞株的结合。图5A本公开的结合分子与人Huh7肝癌细胞株的结合；图5B本公开的结合分子与人HepG2肝癌细胞株的结合及对应的EC50值。
图6示出了本公开的结合分子在GPC3阳性肝癌细胞的存在下激活T细胞。(A)和(B)分别示出本公开的结合分子在GPC3阳性肝癌细胞Huh7存在下，刺激T细胞分泌白介素IL2和IFNγ。(C)～(F)分别示出本公开的结合分子在GPC3阳性肝癌细胞HepG2的存在下，刺激T细胞分泌IL2和IFNγ检测。(G)示出本公开的结合分子在GPC3阳性肝癌细胞Hep3B的存在下刺激T细胞分泌IFNγ。
图7示出了本公开的结合分子抑制GPC3阳性肿瘤的生长。图7A示出了5mg/kg剂量的结合分子对GPC3阳性肿瘤生长的抑制结果；图7B示出了20mg/kg剂量的结合分子对GPC3阳性肿瘤生长的抑制结果。
图8示出了本公开的结合分子的示意图。
图9示出了本公开的不同浓度的结合分子抑制GPC3阳性肿瘤的生长。
图10示出了不同浓度的HEC512-G1D抑制GPC3阳性肿瘤的生长。
具体实施方式
为使本公开的目的、技术方案及优点更加清楚明白，以下结合实施例，对本公开进行进一步的详细说明。此处所描述的具体实施例仅用于解释本公开，并不用于构成对本公开的任何限制。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本公开的概念。
定义
除非另外定义，否则在此使用的所有术语(包括技术和科学术语)具有本领域技术人员通常所理解的含义。应注意，这里使用的术语应解释为具有与本说明书的上下文相一致的含义，而不应以理想化或过于刻板的方式来解释。
本文所使用的表述“约”是如本领域的普通技术人员所理解的，并且根据其使用的背景而在一定范围内变化。如果本领域的普通技术人员根据其使用的上下文对该术语的使用不了 解，则“约”将意味着特定值至多加上或减去10％。
本文所用的“4-1BB”，也称为CD137、TNFRSF9等，是肿瘤坏死因子受体超家族(TNFRS)的成员。4-1BB是在免疫系统的多种细胞，特别是在CD8+T细胞上表达的共刺激受体。由于其广泛的表达，以及4-1BB的增强强效和持久免疫效应的能力，使4-1BB成为癌症免疫治疗的临床靶标。
本文所用的“磷脂酰肌醇蛋白聚糖3”或“GPC3”可互换使用，是属于磷脂酰肌醇蛋白聚糖家族的癌胚抗原，其中磷脂酰肌醇蛋白聚糖家族是由糖基磷脂酰肌醇锚接的硫酸肝素蛋白聚糖组成的。磷脂酰肌醇蛋白聚糖调节若干生长因子的活性，这些生长因子包括Wnts、刺猬因子(Hedgehogs)、骨形态发生蛋白和成纤维细胞生长因子(FGF)。磷脂酰肌醇蛋白聚糖的特征是与被称为硫酸肝素糖胺聚糖的多糖链的共价连接。磷脂酰肌醇蛋白聚糖参与细胞-胞外基质界面的细胞信号转导。迄今为止，已经鉴定了人磷脂酰肌醇蛋白聚糖家族的六个不同成员。细胞膜结合的GPC3由通过一个或多个二硫键连接的两个亚基组成。GPC3在发育过程中的胎肝和胎盘中表达，并且在正常的成人组织中被下调或沉默。GPC3高表达于各种癌症，特别是肝细胞癌(HCC)、黑素瘤、维尔姆斯瘤和肝母细胞瘤中。
本文所使用的术语“氨基酸”是指天然存在的和合成的氨基酸，以及以与天然存在的氨基酸类似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的氨基酸，以及经修饰的氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指具有与天然存在的氨基酸相同的基本化学结构的化合物，即，碳结合至氢、羧基、氨基和R基团，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍类。这些类似物具有修饰的R基团(例如正亮氨酸)或修饰的肽主链，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指结构不同于氨基酸的一般化学结构，但以类似于天然存在的氨基酸的方式起作用的化合物。
氨基酸在本文中可由其通常已知的三字母符号或由IUPAC-IUB生物化学命名委员会推荐的单字母符号提及。同样，核苷酸也可以由其公认的单字母代码提及。如本文所使用的，“极性氨基酸”是指包含偏好驻留在水环境中的侧链的氨基酸。在一些实施方案中，极性氨基酸选自精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、组氨酸、赖氨酸、丝氨酸、苏氨酸以及酪氨酸。极性氨基酸可以带正电、带负电或呈电中性。如本文所使用的，“非极性氨基酸”选自丙氨酸、半胱氨酸、甘氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、色氨酸以及缬氨酸。
如本文所使用的“被一个或多个不同的氨基酸取代”是指预定氨基酸序列(起始多肽的氨基酸序列)中的至少一个现有氨基酸残基被另一个不同的“置换”氨基酸残基置换。“氨基酸插入”是指将至少一个额外氨基酸掺入预定氨基酸序列中。虽然插入物通常由插入1或2个氨基酸残基组成，但也可以制备较大的“肽插入物”，例如插入约3至5个或甚至最多约10、15或20个氨基酸残基。如以上所公开，插入的残基可以是天然存在或非天然存在的。“氨基酸缺失”是指从预定氨基酸序列去除至少一个氨基酸残基。
适用于本公开的抗体可在一个或多个氨基酸残基处，例如在必需或非必需氨基酸残基处 包含保守氨基酸取代。“保守氨基酸取代”是氨基酸残基被具有类似侧链的氨基酸残基置换的氨基酸取代。本领域中已定义具有类似侧链的氨基酸残基的家族，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支的侧链(例如苏氨酸、缬氨酸、异亮氨酸)以及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，抗体中的必需或非必需氨基酸残基优选地用来自同一侧链家族的另一个氨基酸残基置换。在某些实施方式中，氨基酸链段可以用结构类似且侧链家族成员的次序和/或组成不同的链段置换。或者，在某些实施方式中，可沿编码序列的全部或一部分随机引入突变，如通过饱和诱变引入，并且由此得到的突变体可掺入本发明的结合多肽中，并针对这些结合多肽结合至所希望的靶的能力进行筛选。
本文所使用的术语“抗4-1BB激动型抗体”或“针对4-1BB的特异性激动型抗体”是指特异性结合4-1BB，并且部分或完全地促进、诱导、增加和/或激活4-1BB生物活性、应答和/或由4-1BB信号传导介导的下游通路或其它4-1BB介导的功能的抗体。肿瘤坏死因子受体(TNFR)信号传导，特别是TNFRSF激活需要受体聚簇和多聚化。4-1BB是已知需要聚簇以触发下游信号传导的TNFSF受体之一。据报道，4-1BB通过交联形成2个或更多个三聚体会产生较强激活的蛋白质。在一些实施方案中，抗4-1BB激动型抗体结合4-1BB，诱导4-1BB发生2个或更多个三聚体的多聚化。
本发明的术语“全长抗体”和“完整抗体”在本文中可互换使用，指代与天然抗体结构基本上相似的抗体。“天然抗体”是指天然存在的免疫球蛋白分子。例如，天然IgG类抗体是约150,000道尔顿的异源四聚体糖蛋白，由二硫键键合的两条轻链和两条重链组成。从N末端到C末端，每条重链具有可变区(VH)(也称为可变重链结构域或重链可变结构域)和三个恒定结构域(CH1、CH2和CH3)(也称为重链恒定区)。从N末端到C末端，每条轻链具有可变区(VL)(也称为可变轻链结构域或轻链可变结构域)和轻链恒定结构域(CL)(也称为轻链恒定区)。抗体的重链可以是五种类型中的一种，所述五种类型为α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM)，还可以进一步分为亚型，例如γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、γ4(IgG4)、α1(IgA1)和α2(IgA2)。抗体的轻链基于其恒定结构域的氨基酸序列，可以是两种类型中的一种，所述两种类型为κ轻链和λ轻链。
在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。
本公开的结合分子为同源二聚体，其包括由二硫键键合的两条相同肽链。在一些实施方式中，这两条相同的肽链是长肽链，其包括分别特异性结合4-1BB和GPC3的第一结构域和 第二结构域。本公开结合分子的该长肽链中，自N末端可以包括VL 2-VH 2-Fc-VH 1-VL 1、VH 2-VL 2-Fc-VH 1-VL 1、VL 2-VH 2-Fc-VL 1-VH 1、VH 1-VL 1-Fc-VH 2-VL 2、VL 1-VH 1-Fc-VL 2-VH 2、VL 1-VH 1-Fc-VH 2-VL 2或者本领域技术人员知晓的其他连接方式，其中每一片段之间都可以有或没有本公开的连接子序列。
本公开的结合分子为异源四聚体，其包括由二硫键键合的两条长肽链，以及分别与相应长肽链通过二硫键键合的两条短肽链。在一些实施方式中，该长肽链自N末端可以包括VH 2-Fc-VH 1-VL 1，且该短肽链自N末端可以包括VL 2。在一些实施方式中，该长肽链自N末端可以包括VH 1-Fc-VH 2-VL 2，该短肽链自N末端可以包括VL 1。在一些实施方式中，该长肽链自N末端可以包括VH 1-Fc-VL 2-VH 2，该短肽链自N末端可以包括VL 1。或者，长肽链和短肽链中各功能片段可以通过本领域技术人员知晓其他的方式进行组合。
本公开的结合分子为异源六聚体，其包括由两条长肽链、两条第一短肽链和两条第二短肽链，其中两条长肽链通过二硫键键合，第一短肽链和第二短肽链分别与长肽链的相应区域通过二硫键键合。在一些实施方式中，该长肽链可以包括VH 1-Fc-VL 2，第一短肽链包括VL 1，第二短肽链包括VH 2。或者，长肽链和短肽链中各功能片段可以通过本领域技术人员知晓其他的方式进行组合。
本文所使用的术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体或其抗原结合部分结合至预定抗原上的表位。典型地，当通过表面等离子体共振(SPR)技术，在BIACORE 2000仪器中使用重组人4-1BB作为分析物且抗体作为配体测定时，该抗体以大约小于10 -6M，如大约小于10 -7M、10 -8M、10 -9M或10 -10M甚至更低的平衡解离常数(K D)结合，并且结合至预定抗原的亲和力是结合至除该预定抗原或密切相关的抗原外的非特异性抗原(例如BSA、酪蛋白)的亲和力的至少两倍。
本文所使用的术语“受试者”包括任何人或非人动物。例如，本发明的方法和组合物可用于治疗患有癌症的受试者。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，如非人灵长类动物、绵羊、狗、牛、鸡、两栖动物、爬行动物等。
本文所使用的术语“抗体片段”、“抗原结合片段”、“抗原结合部分”或类似术语是指抗体中保留结合至靶抗原(例如4-1BB或GPC3)并具有与抗体全长相同活性的片段。此类片段包括例如单链抗体、单链Fv片段(scFv)、Fd片段、Fab片段、Fab'片段或F(ab')2片段。scFv片段是单一多肽链，该片段包括作为scFv来源的抗体的重链和轻链可变区。此外，内抗体、微型抗体、三功能抗体和双功能抗体也包括在抗体的定义内并且适用于本文所述的方法中。参见例如Todorovska等人(2001),J.Immunol.Methods,248(1):47-66；Hudson和Kortt,(1999),J.Immunol.Methods,231(1):177-189；Poljak,(1994),Structure,2(12):1121-1123；Rondon和Marasco,(1997),Annu.Rev.Microbiol.,51:257-283，所述文献各自的公开内容以全文引用的方式并入本文中。
本文所使用的术语“Fc片段”是指全长免疫球蛋白的恒定区。在一些实施方式中，“Fc片段”是指IgA、IgD、IgG的后两个恒定结构域(CH2-CH3)，或者IgE和IgM的后三个恒定结构域(CH2-CH3-CH4)，以及这些结构域的N末端柔性铰链。
两个序列之间的同一性百分比随这些序列共有的相同位置的数量而变(即，同一性％＝相同位置的数量/位置总数×100)，其中要考虑为最佳地使这两个序列对齐而需要引入的空位的数量和每个空位的长度。两个核苷酸序列之间的同一性百分比可使用GCG软件包(可得自http://www.gcg.com)中的GAP程序，使用NWSgapdna.CMP矩阵以及空位权重40、50、60、70或80和长度权重1、2、3、4、5或6测定。两个核苷酸或氨基酸序列之间的同一性百分比也可使用E.Meyers和W.Miller(CABIOS，4:11-17(1989))的算法，使用PAM120权重残基表、空位长度罚分12和空位罚分4测定，该算法已被并入到ALIGN程序(2.0版)中。另外，两个氨基酸序列之间的同一性百分比可使用Needleman和Wunsch(J.Mol.Biol.(48):444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵，以及空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6测定，该算法已被并入到GCG软件包(可得自http://www.gcg.com)中的GAP程序。
本文所使用的术语“可变区”或“可变结构域”是指参与抗原结合分子与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域(分别为VH和VL)通常具有相似的结构，其中每个结构域包含四个保守框架区(FR)和三个高变区(HVR)。单个VH或VL结构域可足以赋予抗原结合特异性。
本文所使用的术语“可变”是指可变域的某些区段在抗体之间在序列上普遍不同。V结构域介导抗原结合并限定特定抗体对于其特定抗原的特异性。然而，可变性在整个可变域并非均匀分布的。相反，集中于轻链与重链可变域内三个称为高变区(HVR)的区段中。可变域的更高度保守部分被称作框架区(FR)。天然重链与轻链的可变域各自包含四个FR区，大部分采用β-折叠构型，由三个HVR连接，其形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的HVR通过FR区紧密保持在一起，并且与其它链的HVR一起促成抗体的抗原结合位点的形成(参见Kabat等,Sequences of Immunological Interest,第5版,National Institute of Health,Bethesda,MD(1991))。恒定域不直接参与抗体与抗原的结合，具有其他效应功能，例如参与抗体的抗体依赖性细胞毒性。
本文所使用的术语“高变区”或“HVR”是指在抗体可变结构域区域中序列上高变和/或形成结构上限定的环(“高变环”)的区域。通常，天然四链抗体包含六个HVR：三个存在于VH中(H1、H2、H3)，三个存在于VL中(L1、L2、L3)。HVR通常包含来自高变环和/或来自“互补决定区(CDR)”的氨基酸残基，来自CDR的氨基酸残基具有最高的序列可变性和/或参与抗原识别。基于Chothia定义规则，示例性CDR(LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3)位于氨基酸残基L26-L32(L1)、L50-L52(L2)、L91-L96(L3)、H26-H32(H1)、H52-H56(H2)和H96-H101(H3)处(Chothia等人,J.Mol.Biol.196:901-917(1987))。基于Kabat定义规则，示例性CDR(LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3)位于氨基酸残基L24-L34(L1)、L50-L56(L2)、L89-L97(L3)、H31-H35(H1)、H50-H65(H2)和H95-H102(H3)处(Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991))。基于
Figure PCTCN2022116450-appb-000001
定义规则，示例性CDR(LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3)位于氨基酸 残基L27-L32(L1)、L50-L51(L2)、L89-L97(L3)、H26-H33(H1)、H51-H56(H2)和H93-H102(H3)处(Honjo,T.和Alt,F.W.(1995)Immunoglobulin genes.Academic Press pp.3-443)。作为比较，在表1中列出了包含上文引用的参考文献中所定义的CDR的相应氨基酸残基。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则、基于结构环区域位置的Chothia定义规则和基于CDR移植的抗体人源化设计的参考工具(参见J Mol Biol 273:927-48,1997)。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应理解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本公开请求保护的范围是基于Kabat定义规则所示出的序列，但是根据其他CDR定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
表1.各CDR区的氨基酸编号系统(可参见http://bioinf.org.uk/abs/)
CDR\编号体系 Kabat Chothia IMGT
LCDR1 24-34 26-32 27-32
LCDR2 50-56 50-52 50-51
LCDR3 89-97 91-96 89-97
HCDR1 31-35 26-32 26-33
HCDR2 50-65 52-56 51-56
HDCR3 95-102 96-101 93-102
本文所使用的“框架”或“FR”是指除高变区(HVR)残基之外的可变结构域残基。可变结构域的FR通常由以下四个FR结构域组成：FR1、FR2、FR3和FR4。因此，HVR和FR序列通常在VH(或VL)中以如下序列出现：FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。
根据本公开的一些实施方式，本公开的结合分子可以包括Fab臂，其包括特异性结合抗原的一个重链-轻链对。根据本公开的一些实施方式，本公开的结合分子可以包括重组IgG样双靶向分子，其中该分子的两侧各自含有至少两种不同抗体的Fab片段或Fab片段的一部分；IgG融合分子，其中全长IgG抗体与额外的Fab片段或Fab片段的部分融合；Fc融合分子，其中单链Fv分子或稳定的双体抗体与重链恒定结构域、Fc区或其部分融合；Fab融合分子，其中不同的Fab片段融合在一起；基于scFv和双体抗体的重链抗体(例如结构域抗体、纳米抗体)，其中不同的单链Fv分子或不同的双体抗体或不同的重链抗体彼此融合或与另一蛋白或载体分子融合。
本文所使用的抗体的“类别”是指抗体的重链所具有的恒定结构域或恒定区的类型。存在五类抗体：IgA、IgD、IgE、IgG和IgM，并且这些类别中的若干可以进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类别的免疫球蛋白的重链恒定结构域分别称为α、δ、ε、γ和μ。
本文所使用的“人源化抗体”包含来自非人源高变区(HVR)的氨基酸残基和来自人源抗体的重链及轻链的恒定区进行拼接，得到人源化抗体完整序列。在某些实施例中，人源化抗体包含至少一个，通常两个可变结构域，其中所有或基本上所有HVR(例如CDR)对应于非人抗体的HVR，并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。“人源化形式”的抗体，例如非人抗体，是指已 经进行了人源化的抗体。
本文所使用的“交叉反应”是指本公开的抗体结合来自不同物种的4-1BB或GPC3的能力。例如，本公开的抗体可以结合人4-1BB，同时也可以结合另一物种(例如小鼠、大鼠、食蟹猴或狗)的4-1BB。如本文所使用，交叉反应性是通过检测在结合分析(例如SPR、ELISA)中与纯化抗原的特异性反应性，或与生理学上表达)的细胞结合或以其它方式功能上相互作用来测量。
相比于一条序列，本文所使用的与该序列具有“至少90％序列同一性”可以包括与该序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的序列同一性。
本文所使用的“表达载体”和“表达构建体”可互换使用，在将上述分离的核酸分子连接到载体上时，可以将核酸序列与载体上的调控元件直接或者间接相连，只要这些调控元件能够调控该核酸分子的翻译和表达等即可。当然这些调控元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。也就是说，核酸分子与调控元件可操作地连接。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的调控元件，例如转录调控序列和翻译调控序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。当然用来编码抗体重链和轻链的多核苷酸，可以分别独立的插入到不同的载体上，常见的是插入到同一载体上。常用的载体例如可以为质粒、噬菌体等等。
本文所使用的“细胞”或“重组细胞”中可包含有表达载体。可以将表达载体导入到哺乳动物细胞中，获得重组细胞，然后利用这些重组细胞表达本公开提供的抗体或者抗原结合部分。通过该重组细胞进行培养，即可以获得相应抗体。这些可用的哺乳动物细胞例如可以为CHO细胞等。
本文所使用的“药学上可接受的载剂”可以包括生理学上相容的任何溶剂、分散介质、包衣、抗细菌剂、抗真菌剂、等渗剂和延迟吸收剂等等。具体实例可以是水、盐水、磷酸盐缓冲盐水、葡萄糖、甘油、乙醇等，以及它们的组合中的一种或多种。有许多情况下，药物组合物中可以包括等渗剂，例如糖类、多元醇(如甘露醇、山梨醇)或氯化钠等。当然药学上可接受的载剂还可包括微量的辅助物质，例如润湿剂或乳化剂、防腐剂或缓冲剂，用来延长抗体的保存限期或效力。
例如，本公开的抗体或其抗原结合部分可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物组合物中。这些药物组合物可以被制备成各种形式，例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。典型的药物组合物为注射溶液或输注溶液形式。本公开的抗体或其抗原结合部分可通过静脉输注、注射、肌肉内或皮下注射来施用。
药物(例如药用组合物)的“治疗有效量”指在剂量和给药间隔和时间上有效实现想要的治疗或预防效果必需的量。例如，治疗有效量的药物消除、减轻/减少、延迟、最小化或预防疾病的不利影响。
本文所使用的术语“血液癌症”包括淋巴瘤、白血病、骨髓瘤或淋巴系恶性疾病，以及 脾和淋巴结的癌症。示例性淋巴瘤包括B细胞淋巴瘤(B细胞血液癌症)和T细胞淋巴瘤。B细胞淋巴瘤包括霍奇金氏淋巴瘤和大多数的非霍奇金氏淋巴瘤。B细胞淋巴瘤的非限制性实例包括弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、粘膜相关淋巴组织淋巴瘤、小细胞淋巴细胞性淋巴瘤、套细胞淋巴瘤(MCL)、伯基特氏淋巴瘤、纵隔大B细胞淋巴瘤、瓦尔登斯特仑氏巨球蛋白血症、淋巴结边缘区B细胞淋巴瘤、脾边缘区淋巴瘤、血管内大B细胞淋巴瘤、原发性渗出性淋巴瘤、淋巴瘤样肉芽肿。T细胞淋巴瘤的非限制性实例包括结外T细胞淋巴瘤、皮肤T细胞淋巴瘤、间变性大细胞淋巴瘤和血管免疫母细胞性T细胞淋巴瘤。血液恶性疾病还包括白血病，如但不限于继发性白血病、慢性淋巴细胞性白血病、急性髓性白血病、慢性髓性白血病和急性淋巴母细胞性白血病。血液恶性疾病还包括骨髓瘤，如但不限于多发性骨髓瘤和冒烟性多发性骨髓瘤。术语血液恶性疾病涵盖其它血液和/或B细胞或T细胞相关癌症。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。在以下说明中，省略了对公知技术的描述，以避免不必要地混淆本公开的概念。这样的技术在许多出版物，例如《分子克隆实验指南(第四版)》(冷泉港实验室科学出版社)中进行了描述。
实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1.瞬时转染HEK293悬浮细胞来获得结合分子。
使用全基因合成手段，将下表2中的肽链进行全基因合成，分别插入到pcDNA3.1载体中。将构建好的载体，分别瞬时转染到HEK293细胞中，进行蛋白表达。另外，对于包含多条肽链的结合分子，将分别插入了多条肽链的载体，共转染到HEK293细胞中(例如将分别插入了IOB1-FabIg-S2的肽链1、肽链2和肽链3的载体，共转染到HEK293细胞中)。
表2.各多肽序列对应的结构及其序列
Figure PCTCN2022116450-appb-000002
注：抗4-1BB的重链可变区和轻链可变区分别来源于单克隆抗体HEC511(重链的氨基 酸序列如SEQ ID NO:67所示，轻链的氨基酸序列如SEQ ID NO:54所示)或单克隆抗体HEC512(重链的氨基酸序列如SEQ ID NO:68所示，轻链的氨基酸序列如SEQ ID NO:66所示)。抗GPC3的重链可变区和轻链可变区分别来源于三个单克隆抗体HS20(重链可变区的序列如SEQ ID NO:37所示，轻链可变区的氨基酸序列如SEQ ID NO:38所示)、4A6(重链可变区的序列如SEQ ID NO35所示，轻链可变区的氨基酸序列如SEQ ID NO:36所示)或GC33(重链可变区的序列如SEQ ID NO:39所示，轻链可变区的氨基酸序列如SEQ ID NO:40所示)。
通过ProteinA介质的纯化表达出的结合分子，分别获取纯度超过95％的结合分子IOB1-S、IOB1-M、IOB1-ScFvIg-S、IOB1-FabIg-S2、IOB1-G、IOB1-G-Fab、IOB1-G-Fab1、IOB1-G-Fab2和IOB1-G1D-Fab2，其结构示意图示于图8中。获得的这些结合分子用于下面的检测评估。
测定了每100mL悬浮细胞培养液中，获得的上述结合分子的表达滴度，结果示于表2。在表2中，Isotype(同种型)IgG作为阴性对照。
表2.每100mL悬浮细胞培养液中各结合分子的滴度。
Figure PCTCN2022116450-appb-000003
实施例2.本公开的结合分子与稳定转染人源4-1BB的CHO细胞株的结合。
本实施例使用流式细胞仪，检测了结合分子对稳定转染有人源4-1BB的CHO细胞株的结合能力。结合分子与细胞孵育后，随后使用FITC标记的山羊抗鼠IgG Fc二抗(Abcam：ab97023)结合，使用GC33和同种型IgG抗体作为阴性对照，并使用构建结合分子对应的抗体HEC511(重链的氨基酸序列如SEQ ID NO:67所示，轻链的氨基酸序列如SEQ ID NO:54所示)和抗体HEC512(重链的氨基酸序列如SEQ ID NO:68所示，轻链的氨基酸序列如SEQ ID NO:66所示)作为阳性对照样品。结果示于图1中。从图1可以看出，结合分子可以与人源4-1BB(UniProtKB-Q07011)膜表达的蛋白有效结合。
实施例3.本公开的结合分子与人源4-1BB重组蛋白的结合
本实施例使用ELISA，检测了结合分子对人源4-1BB(UniProtKB-Q07011)重组蛋白的结合能力。将重组4-1BB蛋白包被于酶标板4℃过夜，3次洗净后甩干，37℃封闭1小时，3次洗净后甩干备用。将本公开的结合分子与上述备用酶标板孵育4℃2小时，3次洗净后甩干，随后使用HRP标记的山羊抗鼠IgG-Fc二抗结合，使用同种型IgG抗体作为阴性对照，并使用于构建结合分子对应的抗体HEC511、HEC512作为对照样品。结果示于图2中。
从图2可以看出，本公开的结合分子可以与人源4-1BB(UniProtKB-Q07011)重组蛋白有效结合。
实施例4.本公开的结合分子与人血体外分离并激活的T细胞的结合
从人体来源PBMC细胞群中分离出来T细胞，并使用CD3和CD28抗体耦联磁珠体外刺激T细胞48～72小时，经刺激的T细胞可高表达4-1BB蛋白。然后，使用流式细胞仪检测本公开的结合分子与刺激后T细胞的结合能力。将结合分子与细胞孵育后，随后使用FITC标记的山羊抗鼠IgG Fc二抗结合，使用同种型IgG抗体作为阴性对照。结果示于图3中。
从图3可以看出，本公开的结合分子可以有效与经激活的人源T细胞结合。也就是说，本公开的结合分子可以有效与经激活的T细胞所表达的4-1BB蛋白结合。
实施例5.本公开的结合分子与人源GPC3重组蛋白的结合
本实施例使用ELISA，检测了本公开的结合分子对人源GPC3(XP_022382036.1)重组蛋白的结合能力。将重组GPC3蛋白包被于酶标板4℃过夜，3次洗净后甩干，37℃封闭1小时，3次洗净后甩干备用。将抗体与上述备用酶标板孵育4℃2小时，3次洗净后甩干，随后使用HRP标记的山羊抗鼠IgGFc二抗结合，使用抗体HEC511、同种型IgG抗体作为阴性对照，并使用于构建融合蛋白对应的抗体HS20和GC33作为对照样品。结果示于图4中。
从图4可以看出，本公开的结合分子可以与人源GPC3(XP_022382036.1)重组蛋白有效结合。
实施例6.本公开的结合分子与人肝癌细胞株的结合
已知GPC3蛋白在肝癌细胞中高表达。为了检测本公开的结合分子是否能与肝癌细胞结合，将常规培养的Huh7或HepG2肝癌细胞用胰酶消化后，收集起来，使用流式细胞仪检测本公开的结合分子对人肝癌细胞株Huh7或HepG2的结合能力。将本公开的结合分子与细胞孵育后，随后使用FITC标记的山羊抗鼠IgGFc二抗结合，使用同种型IgG抗体作为阴性对照，并使用于构建融合蛋白对应的抗体GC33、HEC511作为对照样品。结果示于图5中。
从图5可以看出，本公开的结合分子可以有效与人肝癌细胞株表达的GPC3蛋白有效结合。
实施例7.本公开的结合分子与人源4-1BB重组蛋白的亲和力。
本实施例使用生物膜光干涉技术(Bio-LayerInterferometry，BLI)，测定了本公开的结合分子与4-1BB的结合亲和力。简单来讲，将人源4-1BB(UniProtKB-Q07011)蛋白固定在传感器上持续时间300s，再将传感器进入到PBST溶液中平衡基线180s，随后将探针浸入到融合蛋白溶液中结合持续时间600s，再将探针置于PBST溶液中解离持续时间1200s，最后将探针置入再生液中完成探针的再生及保存。使用BLI检测，检测本公开的结合分子与人源重组蛋白4-1BB的结合亲和力。结果如表3所示。
表3.本公开的结合分子与人源重组4-1BB蛋白的结合亲和力
Figure PCTCN2022116450-appb-000004
实施例8.本公开的结合分子与人源GPC3重组蛋白的结合亲和力
本实施例使用生物膜光干涉技术(Bio-LayerInterferometry，BLI)，测定了本公开的结合分子与GPC3的结合亲和力。简单来讲，将人源GPC3(XP_022382036.1)蛋白固定在传感器上持续时间300s，再将传感器进入到PBST溶液中平衡Baseline180s，随后将探针浸入到融合蛋白溶液中结合持续时间600s，再将探针置于PBST溶液中解离持续时间1200s，最后将探针置入再生液中完成探针的再生及保存。使用BLI检测，测定了本公开的结合分子与人源重组蛋白GPC3的结合亲和力。结果如表4所示。
表4.本公开的结合分子与人源重组GPC3蛋白的结合亲和力
Figure PCTCN2022116450-appb-000005
实施例9.本公开的结合分子以GPC3阳性肝癌细胞依赖的方式激活T细胞。
将CD3抗体包被在酶标板上，均匀覆盖一定密度的肿瘤细胞。分离人体T细胞后，将T细胞和本公开的结合分子添加到酶标板中，三天后检测上清中IL2的表达量，或五天后检测上清中IFN-γ的表达量。使用HEC511、HS20、4A6或同种型IgG抗体作为对照。结果如图6所示。
图6A和6B示出了，本公开的结合分子能够在GPC3阳性肝癌细胞Huh7存在下，刺激T细胞分泌白介素IL2和IFNγ。图6C～6F示出了，本公开的结合分子能够在GPC3阳性肝癌细胞HepG2的存在下，刺激T细胞分泌IL2和IFNγ检测。图6G示出了，本公开的结合分子能够在GPC3阳性肝癌细胞Hep3B的存在下，刺激T细胞分泌IFNγ。
实施例10.在MC38高表达GPC3稳转细胞系荷瘤人源化小鼠模型中，对本公开的结合分子的肿瘤抑制效果的验证
将PBS重悬的MC38高表达人源GPC3蛋白的肿瘤细胞系(MC38-hGPC3细胞)，以5×10 5个/0.1mL/只的量，接种于B-h4-1BB/h4-1BBL人源化小鼠的右侧皮下。当平均肿瘤体积达到100～150mm 3时，根据小鼠肿瘤体积和体重选择合适的小鼠入组，平均分配到3个实 验组中，每组6只。各组样品以5mg/kg剂量腹腔给药(对照组给予PBS)，每周两次。分组后每周使用游标卡尺对肿瘤体积进行2次测量，安乐死前测量肿瘤体积，测量肿瘤的长径和短径，其体积计算公式为：肿瘤体积＝0.5×长径×短径 2。结果见图7A，可以看出本公开的双特异性抗体可以在给药后明显抑制肿瘤的生长；且同剂量下，双特异性抗体抑制肿瘤的起效时间早于anti-4-1BB单克隆体，消除肿瘤的时间也明显早于anti-41BB单克隆体HEC512。
实施例11 MC38高表达GPC3稳转细胞系荷瘤人源化小鼠模型中受试抗体的抗肿瘤活性评价
在41BB/41BBL双人源化C57BL/66小鼠的右侧皮下，以5×10 5个/0.1mL/只的量，接种MC38高表达人源GPC3蛋白的肿瘤细胞系。当平均肿瘤体积达到100-150mm 3时，根据小鼠体重和肿瘤体积选择合适的小鼠入组，将受试抗体IOB1-G-Fab2按照20mg/kg剂量腹腔注射到小鼠体内，定期测量肿瘤大小，观察肿瘤的生长或抑制情况。结果如图7B所示，显示受试抗体有显著抑制GPC3阳性肿瘤生长的效果。
实施例12.MC38高表达GPC3稳转细胞系荷瘤人源化小鼠模型中受试抗体的抗肿瘤活性评价
在41BB/41BBL双人源化C57BL/66小鼠的右侧皮下，以5×10 5个/0.1mL/只的量，接种MC38高表达人源GPC3蛋白的肿瘤细胞系。当平均肿瘤体积达到100-150mm 3时，根据小鼠体重和肿瘤体积选择合适的小鼠入组，将受试抗体IOB1-G1D-Fab2和HEC512-G1D(重链的氨基酸序列如SEQ ID NO:65所示，轻链的氨基酸序列如SEQ ID NO:66所示)按照20mg/kg、5mg/kg和1mg/kg剂量腹腔注射到小鼠体内，定期测量肿瘤大小，观察肿瘤的生长或抑制情况。结果如图9和10所示，显示受试抗体有显著抑制GPC3阳性肿瘤生长的效果，IOB1-G1D-Fab2的抑制效果优于HEC512-G1D。
本公开的技术方案不限于上述具体实施例的限制，凡是根据本公开的技术方案做出的技术变形，均落入本公开的保护范围之内。

Claims (18)

  1. 一种结合分子，其特征在于，所述结合分子包括：
    第一结构域，其中所述第一结构域特异性结合4-1BB或其片段，和
    第二结构域，所述第二结构域特异性结合磷脂酰肌醇蛋白聚糖3(GPC3)或其片段。
  2. 根据权利要求1所述的结合分子，其特征在于，所述结合分子还包括第三结构域，所述第三结构域包括免疫球蛋白的Fc片段，
    优选地，所述免疫球蛋白选自IgA、IgG、IgM、IgD和IgE，优选为IgG，更优选为IgG1、IgG2、IgG3或IgG4；
    优选地，所述Fc片段具有L234F、L235E、P331S、D356E和L358M中的一个或多个突变，其中所述Fc片段按照Kabat的EU索引进行编号；
    更优选地，所述第三结构域包括如SEQ ID NO:55、62或69所示的氨基酸序列或其变体。
  3. 根据权利要求1或2所述的结合分子，其特征在于，所述第一结构域包括第一重链可变区VH 1和第一轻链可变区VL 1，其中：
    所述VH 1包括：
    (a)包括分别如SEQ ID NO:1、2和3所示的氨基酸序列的HCDR1、HCDR2和HCDR3；或
    (b)包括分别如SEQ ID NO:7、8和9所示的氨基酸序列的HCDR1、HCDR2和HCDR3，
    所述VL 1包括：
    (c)包括分别如SEQ ID NO:4、5和6所示的氨基酸序列的LCDR1、LCDR2和LCDR3；或者
    (d)包括分别如SEQ ID NO:10、11和12所示的氨基酸序列的LCDR1、LCDR2和LCDR3。
  4. 根据权利要求1至3任意一项所述的结合分子，其特征在于，
    所述VH 1包括与如SEQ ID NO:31或33所示的氨基酸序列具有至少90％序列同一性的氨基酸序列，和/或
    所述VL 1包括与如SEQ ID NO:32或34所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
  5. 根据权利要求1至4任意一项所述的结合分子，其特征在于，所述第二结构域包括第二重链可变区VH 2和第二轻链可变区VL 2，其中
    所述VH 2包括：
    (e)包括分别如SEQ ID NO:13、14和15所示的氨基酸序列的HCDR1、HCDR2和HCDR3；
    (f)包括分别如SEQ ID NO:19、20和21所示的氨基酸序列的HCDR1、HCDR2和 HCDR3；或
    (g)包括分别如SEQ ID NO:25、26和27所示的氨基酸序列的HCDR1、HCDR2和HCDR3，
    所述VL 2包括：
    (h)包括分别如SEQ ID NO:16、17和18所示的氨基酸序列的LCDR1、LCDR2和LCDR3；
    (i)包括分别如SEQ ID NO:22、23和24所示的氨基酸序列的LCDR1、LCDR2和LCDR3；或
    (j)包括分别如SEQ ID NO:28、29和30所示的氨基酸序列的LCDR1、LCDR2和LCDR3。
  6. 根据权利要求1至5任意一项所述的结合分子，其特征在于，
    所述VH 2包括与如SEQ ID NO:35、37或39所示的氨基酸序列具有至少90％序列同一性的氨基酸序列，和/或
    所述VL 2包括与如SEQ ID NO:36、38或40所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
  7. 根据权利要求1至6任意一项所述的结合分子，其特征在于，所述第一结构域和/或所述第二结构域的结构选自Fab、Fab’、F(ab’) 2、Fv或scFv。
  8. 根据权利要求1至7任意一项所述的结合分子，其特征在于，所述第一结构域、所述第二结构域和/或第三结构域直接连接或者通过连接子连接，
    所述第一结构域的第一重链可变区和第一轻链可变区直接连接或者通过连接子连接，和/或
    所述第二结构域的第二重链可变区和第二轻链可变区直接连接或者通过连接子连接。
  9. 根据权利要求1至8任意一项所述的结合分子，其特征在于，所述连接子具有如(G nS) z所示的序列，其中n和z各自独立地为1～4的整数。
  10. 根据权利要求1至9任意一项所述的结合分子，其特征在于，所述结合分子包括以如下顺序连接的第一结构域、第二结构域和第三结构域：
    第一结构域-第三结构域-第二结构域；
    第二结构域-第三结构域-第一结构域；
    第一结构域-第二结构域-第三结构域；或，
    第二结构域-第一结构域-第三结构域。
  11. 根据权利要求1至10任意一项所述的结合分子，其特征在于，所述结合分子包括一条肽链形成的同源二聚体，
    优选地，所述肽链具有与SEQ ID NO:41、42或43所示的氨基酸序列具有至少90％序列同一性的氨基酸序列。
  12. 根据权利要求1至10中任意一项所述的结合分子，其特征在于，所述结合分子包括 长肽链和短肽链形成的异源四聚体，其中
    (i)长肽链具有与SEQ ID NO:44所示的氨基酸序列具有至少90％序列同一性，且
    短肽链具有与SEQ ID NO:45所示的氨基酸序列具有至少90％序列同一性；
    (ii)长肽链具有与SEQ ID NO:46所示的氨基酸序列具有至少90％序列同一性，且
    短肽链具有与SEQ ID NO:47所示的氨基酸序列具有至少90％序列同一性；
    (iii)长肽链具有与SEQ ID NO:48所示的氨基酸序列具有至少90％序列同一性，且
    短肽链具有与SEQ ID NO:49所示的氨基酸序列具有至少90％序列同一性；或
    (iv)长肽链具有与SEQ ID NO:50所示的氨基酸序列具有至少90％序列同一性，且
    短肽链具有与SEQ ID NO:51所示的氨基酸序列具有至少90％序列同一性，或
    (v)长肽链具有与SEQ ID NO:64所示的氨基酸序列具有至少90％序列同一性，且
    短肽链具有与SEQ ID NO:51所示的氨基酸序列具有至少90％序列同一性。
  13. 根据权利要求1至10任意一项所述的结合分子，其特征在于，所述结合分子包括长肽链、第一短肽链和第二短肽链形成的异源六聚体，其中
    长肽链具有与SEQ ID NO:52所示的氨基酸序列具有至少90％序列同一性，
    第一短肽链具有与SEQ ID NO:54所示的氨基酸序列具有至少90％序列同一性，和
    第二短肽链具有与SEQ ID NO:53所示的氨基酸序列具有至少90％序列同一性。
  14. 一种分离的核酸分子，其特征在于，所述核酸分子编码如权利要求1至13任意一项所述的结合分子。
  15. 一种载体，其特征在于，所述载体包括如权利要求14所述的核酸分子，所述载体优选是表达载体。
  16. 一种宿主细胞，其特征在于，所述宿主细胞包括如权利要求14所述的核酸分子或如权利要求15所述的载体。
  17. 一种药物组合物，其特征在于，所述药物组合物包括如权利要求1至13任意一项所述的结合分子、如权利要求14所述的核酸分子、如权利要求15所述的载体或如权利要求16所述的宿主细胞和药学上可接受的载剂。
  18. 如权利要求1至13任意一项所述的结合分子、如权利要求14所述的核酸分子、如权利要求15所述的载体、如权利要求16所述的宿主细胞或如权利要求17所述的药物组合物在制备治疗或预防癌症的药物中的应用，优选地，所述癌症选自由黑色素瘤、神经胶质瘤、肾癌、乳腺癌、肝癌、维尔姆斯瘤、肝母细胞瘤、血液癌症以及头颈癌。
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