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WO2025068449A1 - Protéines à domaine knob - Google Patents

Protéines à domaine knob Download PDF

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Publication number
WO2025068449A1
WO2025068449A1 PCT/EP2024/077193 EP2024077193W WO2025068449A1 WO 2025068449 A1 WO2025068449 A1 WO 2025068449A1 EP 2024077193 W EP2024077193 W EP 2024077193W WO 2025068449 A1 WO2025068449 A1 WO 2025068449A1
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WIPO (PCT)
Prior art keywords
antigen
binding
knob domain
helical
knob
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Inventor
Alastair David Griffiths Lawson
Alexander Macpherson
Mikhail KURAVSKIY
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UCB Biopharma SRL
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UCB Biopharma SRL
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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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/246—IL-2
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

  • the present invention relates to antigen-binding proteins and in particular to antigen-binding proteins comprising knob domains or antigen-binding portions thereof.
  • the present invention also relates to polynucleotides and vectors encoding the antigen-binding proteins, as well as to pharmaceutical compositions comprising the antigen-binding proteins.
  • the present invention further relates to the use of the antigen-binding proteins in therapy and diagnosis.
  • VNAR Very New antigen Receptor
  • the central segment of bovine ultra-long CDR H3 or “knob” can independently bind to antigen with high affinity and specificity and therefore represents the smallest autonomous antibody fragment (3-6 kDa).
  • Macpherson et al. 2020 Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies. PLoS Biol 18(9): e3000821.
  • a stabilising network of disulfide bonds may confer on them high thermostability and resistance to plasma proteolysis.
  • Their extremely small size and the ability to exploit the bovine immune system for targeting a vast range of antigens make knob domains a prospective new class of drugs. Such knob domains are also described in WO 2021/191424.
  • knob domains When produced recombinantly, in particular in mammalian cells, knob domains were found to be more efficiently produced when fused to human Fc or inserted into CDR-H3 of a human Fab via cleavable linkers. Upon separation from fusion partners, the knob domains retain their antigen-binding activity.
  • Another approach described in Macpherson et al. (2021) The Chemical Synthesis of Knob Domain Antibody Fragments. ACS Chem Biol 16(9): 1757-1769, utilises solid-phase peptide synthesis (SPPS) to produce functional knob domains devoid of stalk and any additional fusion tags.
  • SPPS solid-phase peptide synthesis
  • SPPS provides an easy way to incorporate some therapeutically relevant modifications, such as noncanonical amino acids, palmitoylation and head-to-tail cyclisation.
  • some therapeutically relevant modifications such as noncanonical amino acids, palmitoylation and head-to-tail cyclisation.
  • both the approaches are associated with higher costs when scaling up production levels.
  • knob domain production and properties particularly in relation to yield and production costs, as well as the stability of the knob domains, whilst ensuring antigen-binding ability is retained or improved.
  • the present invention provides antigen-binding proteins comprising knob domains, or antigenbinding portions thereof, wherein the knob domains, or antigen-binding portion thereof, are fused at their N-terminus, C-terminus, or both N and C termini, either directly or via a linker, to a helical peptide, wherein the presence of the helical peptide or peptides helps optimise knob domain, or antigen-binding portion thereof, production and improve the properties of the knob domains, or antigen-binding portions thereof, including increasing their stability, compared to a knob domain, or antigen-binding portion thereof, expressed on its own.
  • the antigen-binding protein comprises a knob domain, or antigen-binding portion thereof, that is fused, either directly or via a linker, at its N-terminus to a helical peptide, preferably an a-helical peptide, wherein the knob domain, or antigen-binding portion thereof, is also fused, either directly or via a linker, at its C-terminus to a helical peptide, preferably an a-helical peptide.
  • the two helical peptides are able to form a coiled-coil structure.
  • antigen-binding proteins comprising a knob domain, or antigen-binding portion thereof, in conjunction with a helical peptide fused, either directly or via a linker, at its N-terminus or C-terminus or both termini results in improved properties in comparison to the knob domains, or antigen-binding portions thereof, when expressed recombinantly in host cells on their own.
  • Examples of preferred advantages which may be seen when a knob domain, or antigen-binding portion thereof, is fused to a helical peptide or peptides, compared to knob domains, or antigen-binding portions thereof, on their own, may in particular include improved expression yield and/or improved stability in host cells.
  • the knob domains, or antigen-binding portions thereof when present in antigen-binding proteins of the invention fused to a helical peptide or peptides the knob domains, or antigen-binding portions thereof, retain and/or have improved pharmacokinetic properties (e.g. biodistribution, bioavailability, cell and tissue penetration, clearance) and/or improved biological function (e.g. specificity, binding affinity, neutralisation, cell cytotoxicity).
  • the use of the helical peptides in conjunction with knob domains, or antigen-binding portion thereof also helps facilitate the production of antigen-binding proteins comprising more than one knob domain, or antigen-binding portion thereof, meaning that the antigen-binding proteins have a plurality of antigen-binding sites.
  • the present invention also provides antigen-binding proteins that have different permutations of knob domains, or antigen-binding portions thereof, wherein each knob domain, or antigen binding portion thereof, is fused, either directly or via a linker, at its N terminus, its C -terminus, or at both termini to a helical peptide.
  • Linkers may be also used to join together the different knob domain (or antigen-binding portion thereof) -helical peptide(s) units to give rise to antigen-binding proteins with a plurality of antigen-binding sites.
  • linkers may be used to fuse a knob domain, or an antigen-binding portion thereof, to a helical peptide.
  • a knob domain, or antigen-binding portion thereof may be fused to a helical peptide without such a linker and the knob domain, or antigen-binding portion thereof, is therefore joined to the helical peptide via a direct peptide bond.
  • knob domains, or antigen-binding portions thereof, for employing in the antigen-binding proteins provided are, or are derived from, knob domains, or antigen-binding portions thereof, of bovine ultralong CDR-H3 antibodies and antigen-binding portions thereof.
  • the knob domains, or antigen-binding portions thereof are, or are derived from, the ultralong CDR-H3 of antibodies from animals of the bovini tribe, preferably the bos genus and particularly the bos taurus species within the bovine (bovinae) subfamily.
  • the present invention provides an antigen-binding protein comprising a knob domain or a portion thereof capable of binding antigen, wherein the knob domain or antigen-binding portion thereof is fused, either directly or via a linker, at its N-terminus or C-terminus or both to a helical peptide, preferably an a-helical peptide.
  • the present invention also provides a polynucleotide encoding an antigen-binding protein of the present invention.
  • the present invention further provides a vector comprising a polynucleotide of the present invention.
  • the present invention also provides a host cell comprising a polynucleotide of the present invention or a vector of the present invention.
  • the present invention further provides a method for producing an antigen-binding protein of the present invention comprising expressing an antigen-binding protein from a host of the present invention.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antigenbinding protein of the present invention and a pharmaceutically acceptable excipient, diluent or carrier.
  • the present invention also provides an antigen-binding protein of the present invention or a pharmaceutical composition of the present invention for use in therapy or diagnosis.
  • FIG. B shows examples of antigen-binding proteins of the present invention comprising a knob domain fused to a helical peptide at their N terminus or both their N terminus and C-terminus, the antigen-binding proteins being from left to right an antigen-binding protein with a knob domain fused at both its N-terminus and C-terminus to a helical peptide derived from Sin Nombre orthohantavirus nucleocapsid protein (SNV-N), an antigenbinding protein comprising a knob domain fused at both its N terminus and C terminus to a helical peptide derived from human autophagy -related protein Beclin-1 (BECN1) and an antigen-binding protein comprising a knob domain fused at its N-terminus, but not C-terminus, to a helical peptide derived from human haemoglobin beta subunit (HBB) respectively.
  • SNV-N Sin Nombre orthohantavirus nucleocapsid protein
  • FIG. 2 Comparison of protein expression of knobs domains on their own or fused to SNV-N, BECN1, or HBB derived helical peptides.
  • Expi293F cells were transiently transfected with the relevant construct with secreted protein subsequently recovered and enriched, followed by SDS-PAGE analysis under either reducing conditions (A) or non-reducing conditions (B) and (C).
  • FIG. 3 Molecular weight determination by liquid chromatography-mass spectrometry (LC-MS). Molecular weight determination was performed via LC-MS for the different knob domain - helical peptide fusions shown.
  • Figure 4 Size-exclusion HPLC chromatography of different knob domain - helical peptide fusions. Analysis by SEC -HPLC to confirm monomer formation and the absence of significant levels of high molecular weight aggregates.
  • Figure 5 Assessment of antigen binding measured by ELISA of antigen-binding proteins comprising a knob domain fused to a helical peptide or helical peptides. The ability of antigen-binding proteins comprising different knob domain-helical peptide fusions to bind their target antigen was determined.
  • Figure 9 Molecular weight determination by liquid chromatography-mass spectrometry (LC-MS) of antigen-binding proteins with more than one knob domain and fused helical peptides. Molecular weight determination was performed via LC-MS for the antigen-binding proteins with more than one knob domain to confirm the identity and monomeric status of the expressed proteins.
  • LC-MS liquid chromatography-mass spectrometry
  • Figure 10 Assessment by ELISA of antigen binding by antigen-binding proteins comprising more than one knob domain and fused helical peptides.
  • the ability of antigen-binding proteins comprising more than one knob domain and fused helical peptides was assessed by ELISA with control antigen-binding proteins with a single knob domain and fused helical peptides also assessed.
  • Figure 14 Antigen-binding proteins comprising knob domains fused to helical peptides based on the sequences of Oakley et al and Monera et al. As with other antigen-binding proteins, the ability of antigen-binding proteins comprising two helical peptides forming a coiled-coil based on the sequences of Oakley et al and Monera et al was studied. Expi293F cells were transiently transfected with the relevant construct with secreted protein subsequently recovered and enriched, followed by SDS-PAGE analysis with the results obtained shown in Figure 14A. The ability of the proteins to bind the target antigen C5 was assessed via ELISA with the results shown in Figure 14B.
  • Figure 15 Assessment of the ability of shortened BECN1 helical peptides to increase the expression of knob domains and allow for antigen binding.
  • a number of trimmed versions of K8- BECN 1 lacking one, two or three turns (4, 7 or 11 amino acids) of a-helix from either the distal or proximal end of the helical peptides were produced with sequence substitutions in some of the versions to avoid bulky amino acid residues for positions 1 and 4 of the heptad repeats of the helical peptides.
  • A shows the different trimmed versions produced.
  • B shows protein expression for the trimmed versions.
  • C shows antigen binding as measured by ELISA for the different versions.
  • a trimmed version of K8-BECN1 lacking two turns of a-helix from distal end and one turn of a-helix from proximal end (8 amino acids) was also generated with a number of different knob domains.
  • (D) shows protein expression for the 8 amino acid-long trimmed BECN 1 version.
  • the gel image shows protein expression for the trimmed version with the following knob domains present: K8 (1); K57 (2); aIL2_l (3); aNotchl l (4); aNotchl_2 (5); and aNotchl_3 (6).
  • Figure 16 Assessment of cyclic knob-helical peptide antigen-binding proteins.
  • A) shows the different versions generated.
  • B) shows protein expression of the different versions.
  • C) shows the antigen binding of the different versions.
  • FIG. 17 CDR3 knob domain amino acid numbering.
  • the conserved Cysteine at position 92(Kabat) and the conserved Tryptophan at position 103(Kabat) respectively defines the start and the end of the CDR-H3:
  • the present invention provides antigen-binding proteins comprising a knob domain or a portion thereof capable of binding antigen, wherein the knob domain or antigen-binding portion thereof is fused, either directly or via a linker, at its N-terminus or C-terminus or both termini to a helical peptide, preferably an a-helical peptide.
  • a knob domain or antigen-binding portion thereof may be employed in the antigen-binding proteins of the invention. Where a particular antigen-binding domain is set out herein as comprising knob domain(s), and helical peptide(s), what is set out is in the primary amino acid sequence of the antigen-binding protein unless stated otherwise.
  • the linkers are peptide linkers, they too may be in the primary amino acid sequence of the antigenbinding protein.
  • the knob domain, or antigen-binding portion thereof is bovine, and the helical peptide or helical peptides it is fused to are not, that is they do not naturally occur together.
  • an antigen-binding protein of the present invention does not comprise antiparallel strands fused to a knob domain or antigen-binding portion thereof.
  • knob domains and antigen-binding portions thereof employed in the present invention are not part of a naturally occurring antibody, and in particular not connected or fused to a native stalk region, therefore the knob domain or antigen-binding portion thereof is isolated.
  • an "isolated" knob domain does not comprise its naturally occurring antibody scaffold, and in particular does not comprise a stalk or any portion thereof of an ultralong CDR-H3.
  • An isolated knob domain may be obtained from bovine antibody producing B cells and is optionally engineered to produce any variant according to the invention or may be produced recombinantly using cDNA and/or DNA encoding the knob domain or antigen-binding portion thereof isolated from B cells, such as bovine B cells, or synthetically produced, for example by chemical synthesis, preferably be solid-state peptide synthesis.
  • a helical peptide employed in the present invention is not present in the antibody from which the knob domain is isolated or derived.
  • a helical peptide employed in the present invention does not correspond to a peptide in a bovine antibody having an ultralong CDR-H3.
  • a helical peptide employed by the invention does not correspond to a peptide of a bovine antibody.
  • a helical peptide employed in the present invention may be naturally occurring in another protein, preferably a heterologous protein, i.e. a protein other than the antibody from which the knob domain is derived. It may be a fragment of such naturally occurring polypeptide.
  • a helical peptide employed in the present invention is from a species other than from bovinae, or other than from bovini, particularly other than from bos and preferably other than from bos taunts.
  • a helical peptide employed in the present invention may alternatively be a totally artificial helical peptide, e.g.
  • the antigen-binding proteins of the invention comprise a knob domain, or antigen-binding portion thereof and a helical peptide wherein the combination of (a) the knob domain, or antigen-binding portion thereof and (b) the helical peptide are not normally present within a single naturally occurring protein.
  • the antigen-binding proteins of the invention comprise a knob domain, or antigen-binding portion thereof, fused, either directly or via a linker, to a helical peptide at either their N-terminus, their C -terminus, or both termini.
  • the fusion between a knob domain, or antigen-binding portion thereof, and a helical peptide may be a direct peptide bond between the two.
  • the two may be fused by a linker.
  • the linker or linkers employed are preferably peptide linkers, but other non-peptide linkers may also be employed.
  • a linker is employed to fuse the N-terminus of a knob domain, or antigen-binding portion thereof, to a helical peptide.
  • a linker is employed to fuse the C-terminus of a knob domain, or antigen-binding portion thereof, to a helical peptide.
  • a linker is employed to fuse the N-terminus of a knob domain, or antigen-binding portion thereof, to a helical peptide and a linker is also employed to fuse the C- terminus of a knob domain, or antigen-binding portion thereof, to a helical peptide.
  • the fusion at the N-terminus is via a linker, but the fusion at the C-terminus is a direct fusion via a peptide bond between the knob domain or antigen-binding portion thereof and the helical peptide.
  • the fusion at the N-terminus is a direct fusion between the knob domain, or antigen-binding portion thereof, and the helical peptide via a peptide bond, but the fusion at the C-terminus between the knob domain, or antigen-binding portion thereof, and the helical peptide is via a linker.
  • antigen-binding proteins of the invention may comprise a plurality of knob domains, or antigen-binding portions thereof, with each knob domain, or antigen-binding portion thereof, fused at either or both of its N and C termini either directly or via a linker.
  • an antigen-binding protein of the invention comprises a single knob domain or antigen-binding portion thereof.
  • an antigen-binding protein of the invention comprises at least one knob domain, or antigen-binding portion thereof. In one particularly, preferred embodiment it comprises two knob domains or antigen-binding portions thereof. In a further particularly preferred embodiment, it comprises three knob domains or antigen-binding portions thereof.
  • the presence of the helical peptide or helical peptides fused to the knob domain or antigenbinding portion thereof in the antigen-binding protein typically improves the properties of the knob domain, or antigen-binding portion thereof, of the antigen-binding protein and/or the production the antigen-binding protein in comparison to the knob domain, or antigen-binding portion thereof, on its own expressed without the fused helical peptide or helical peptides.
  • a particularly preferred helical peptide for use in the present invention is an alpha-helical peptide.
  • a helical peptide may be of any suitable length.
  • at least one helical peptide is 4 amino acids in length or more, 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, and up to 25 amino acids in length.
  • a helical peptide comprises, or is, at least two helical turns in length.
  • a helical peptide is from 2 to 5 turns in length. In one preferred embodiment, it is from 5 to 25 amino acids in length. In another particularly preferred embodiment, it is from 10 to 25 amino acids in length. In one embodiment, it is from 10 to 20 amino acids in length. In another embodiment, it is from 15 to 20 amino acids in length. In another embodiment a helical peptide is from 2 to 6 turns in length. In one embodiment, where more than one helical peptide is present the helical peptides will be of the same length. In another embodiment, they are not the same length.
  • An antigen-binding protein of the present invention does not comprise the stalk domain of the bovine ultralong CDR-H3 unless it is an antigen-binding protein with a plurality of antigen-binding sites wherein at least one antigen-binding site comprises a knob domain, or antigen-binding portion thereof, fused at its N-terminus, or C-terminus, or both termini, either directly or via a linker, to a helical peptide as defined herein, wherein one or more of the other antigen-binding regions comprise a stalk region.
  • the protein does not comprise such a stalk region at all.
  • the germline encoded VHBUL D H 2 J H 1 has the following sequence (SEQ ID NO: 2): CTTVHQSCPDGYSYGYGCGYGYGCSGYDCYGYGGYGGYGGYGYSSYSYSYTYEYYVDA GQG LLVTVSS
  • Kabat numbering system may be used for heavy-chain residues 1 to 100 and 101 to 228 but residues between 100 and 101 (corresponding to residues encoded by D H 2 and JHI genes) do not accommodate to the Kabat numbering system and may be numbered differently, for example sequentially with a D identifier, as described in Stanfield et al. (supra), with the conserved Cysteine residue at the start of D H 2 being “D2”, followed by D3, D4 etc).
  • Figure 17 indicates identifiers D2, D10, D20, D30 and D40 within the D m segment.
  • the common motif TTVHQ (SEQ ID NO: 3)(positions 93-97 in the germline VHBUL, according to Kabat) starts the ascending strand of the P-stalk region of the CDR-H3.
  • the length between the end of the VHBUL and the “CPD” conserved motif in D H 2 is variable due to differences in junctional diversity formed through V-D recombination.
  • those junctional residues are referred as “a,b,c” following H100 residue, depending on the length (for example, as illustrated in Figure 17, the bovine CDR-H3 BLV1H12 comprises 3 residues following H100, referred as a, b and c).
  • the D H 2 region has been characterised to encode the knob domain and part of the descending strand of the stalk region.
  • D H 2 begins with a conserved Cysteine which is part of a conserved “CPD” motif in the germline sequence, which characterises the beginning of the knob domain.
  • the knob domain terminates at the beginning of the descending strand of the P-stalk region.
  • the descending strand of the P-stalk region has been characterised by alternating aromatic-aliphatic residues in some ultralong CDR-H3.
  • the descending strand of the P-stalk region ends with the residues encoded by the genetic J region, followed by residue Hl 01, Hl 02 according to Kabat.
  • the minimal sequence that may define a knob domain corresponds to the portion of the ultralong CDR-H3 encapsulated by disulphide bonds, more particularly the minimal knob domain sequence starts from the first cysteine residue of an ultralong CDR-H3 and ends with the last cysteine residue of the ultralong CDR-H3. Therefore, a minimal knob domain typically comprises at least two cysteines. In one embodiment, the knob domain sequence starts from one residue preceding the first cysteine residue of an ultralong CDR-H3 and ends after the residue subsequent to the last cysteine residue of the ultralong CDR-H3.
  • the knob domain of this sequence may therefore be defined as the following sequence (SEQ ID NO: 5): SCPDGYRERSDCSNRPACGTSDCCRVSVFGNCL
  • a knob domain that may be defined according to the present application for this sequence is in bold, starting from one residue preceding the first cysteine residue of the ultralong CDR-H3 and ending after the residue subsequent to the last cysteine residue of the ultralong CDR-H3.
  • a knob domain of a bovine ultralong CDR-H3 i.e. is a full-length knob domain, notably comprised between the ascending stalk and the descending stalk of the ultralong CDR- H3.
  • the knob domain comprises or consists of a portion of the knob domain of a bovine ultralong CDR-H3 which binds to an antigen of interest.
  • the knob domain or antigen-binding portion thereof comprises at least two, or at least four, or at least six, or at least eight, or at least ten cysteine residues. In one embodiment, the knob domain or antigen-binding portion thereof comprises at least two cysteine residues. In one embodiment, the knob domain or antigen-binding portion thereof comprises at least four cysteine residues. In one embodiment, the knob domain or antigen-binding portion thereof comprises at least six cysteine residues. In one embodiment, the knob domain or antigen-binding portion thereof comprises at least eight cysteine residues. In one embodiment, the knob domain or antigen-binding portion thereof comprises at least ten cysteine residues.
  • a knob domain or antigen-binding portion thereof present in an antigenbinding protein of the present invention comprises at least one, or at least two, or at least three, or at least four, or a at least five disulphide bonds.
  • a knob domain or antigen-binding portion thereof comprises one, two, three, four, five, six, or seven disulphide bonds.
  • a knob domain or antigen-binding portion thereof comprises between one disulphide bond and five disulphide bonds.
  • a knob domain, or antigen-binding portion thereof comprises between two disulphide bonds and four disulphide bonds.
  • a knob domain, or antigen-binding portion thereof, present in an antigenbinding protein comprises a (Zi) Xi C X 2 motif at its N-terminal extremity, wherein: a. Zi is present or absent, and when Zi is present, Zi represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and, b. Xi is any amino acid residue; and, c. C is cysteine; and, d. X 2 is an amino acid selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine.
  • Zi as defined in the present invention represents any amino acid or any sequence of 2, 3, 4, or 5 independently selected amino acids that may be the same or different.
  • Zi is 1 amino acid.
  • Zi is 2 amino acids, which may be the same or different.
  • Zi is 3 amino acids, which may be the same or different.
  • Zi is 4 amino acids, which may be the same or different.
  • Zi is 5 amino acids, which may be the same or different.
  • Xi is selected from the list consisting of Serine, Threonine, Asparagine, Alanine, Glycine, Proline, Histidine, Lysine, Valine, Arginine, Isoleucine, Leucine, Phenylalanine and Aspartic acid.
  • a knob domain or antigen-binding portion thereof present in an antigen-binding protein of the present invention comprises a (Zi) Xi C X 2 motif at its N-terminal extremity, wherein: a. Zi is present or absent, and when Zi is present, Zi represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and, b.
  • Xi is any amino acid residue, preferably selected from the list consisting of Serine, Threonine, Asparagine, Alanine, Glycine, Proline, Histidine, Lysine, Valine, Arginine, Isoleucine, Leucine, Phenylalanine and Aspartic acid; and, c. C is cysteine; and, d.
  • X 2 is an amino acid selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine.
  • the N-terminal extremity of a knob domain or antigen-binding portion thereof present in an antigen-binding protein of the present invention comprises is initiated by a motif selected in the list consisting of (Zi)SCP, (Zi)TCP, (Zi)NCP, (Zi)ACP, (Zi)GCP, (Zi)HCP, (Zi)KCP, (Zi)VCP, (Zi)RCP, (Zi)ICP, (Zi)DCP, wherein Zi is present or absent, and when Zi is present, Zi represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids.
  • a knob domain or antigen-binding portion thereof present in an antigenbinding protein of the present invention comprises a motif of 2-8 amino acids which is rich in aromatic and/or aliphatic amino acids.
  • the knob domain or antigen-binding portion thereof comprises a motif of 2-8 amino acids which comprises at least 2, or at least 3 or at least 4, or at least 5 amino acids selected from the group consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H).
  • knob domain or antigen-binding portion thereof present in an antigenbinding protein of the present invention comprises 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length or more.
  • a knob domain or antigen-binding portion thereof present in an antigen-binding protein of the present invention is up to 50 amino acids in length or up to 55 amino acids in length.
  • the knob domain or antigen-binding portion thereof is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length or more, and is up to 55 amino acids in length.
  • an antigen binding protein of the present invention comprises an antigen-binding portion of a knob domain of a bovine ultralong CDR-H3 which is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids in length.
  • the antigenbinding portion of the knob domain is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.
  • the antigen-binding portion of a knob domain of a bovine ultralong CDR-H3 which is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.
  • the knob domain, or antigen-binding portion thereof, of the ultralong CDR- H3 when expressed on its own, binds to an antigen of interest with a binding affinity which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of that of the ultralong CDR-H3 which comprises said knob domain or antigen-binding portion thereof, e.g. when the knob domain, or antigenbinding portion thereof, of the ultralong CDR-H3 is expressed or synthesised as part of an entire ultralong CDR-H3.
  • an antigen-binding protein comprises a knob domain, or an antigen-binding portion thereof, which binds an antigen of interest, where the knob domain or antigen-binding portion thereof comprises or consisting of the sequence of formula (I):
  • C represents one cysteine residue
  • Zi is present or absent, and when Zi is present, Zi represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and,
  • Z 2 represents any amino acid or any sequence of 2, 3, 4, or 5 independently selected amino acids that may be the same or different.
  • Z 2 is 1 amino acid.
  • Z 2 is 2 amino acids, which may be the same or different.
  • Z 2 is 3 amino acids, which may be the same or different.
  • Z 2 is 4 amino acids, which may be the same or different.
  • Z 2 is 5 amino acids, which may be the same or different.
  • the knob domain, or antigen-binding portion thereof comprises 2 Cysteine residues. Therefore, in one particular aspect, the knob domain, or antigen-binding portion thereof, comprises a peptide which binds an antigen of interest comprising or consisting of the sequence of formula (II):
  • m 2, 3, 4, 5, 6, 7, 8 or 9.
  • n 3 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • m 2, 3, 4, 5, 6, 7, 8, or 9.
  • n 7 l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • n 9 l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.
  • the peptide has the sequence of formula (IVc):
  • the peptide has the sequence of formula (IVd):
  • the peptide has the sequence of formula (IVe):
  • the peptide has the sequence of formula (IVf): (Zi) (Xi) C X 2 (Y)m C C C (Y)n7 C (Y)n9 C (Xs) (Z2) (IVf) wherein Zi, Xi, C, X 2 , Y, n 1. n 7 , n 9 , X 3 , and Z 2 are defined as above, and wherein the peptide is up to 55 amino acids in length.
  • the peptide has the sequence of formula (IVg):
  • knob domain or antigen-binding portion thereof has the sequence of formula (IVh):
  • the knob domain or antigen-binding portion thereof which binds an antigen of interest comprising or consisting of the sequence of formula (IV), (IVa), (IVb), (IVc), (IVd), (IVe), (IVf), (IVg), or (IVh), is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length.
  • the knob domain or antigen-binding portion thereof which binds an antigen of interest comprising or consisting of the sequence of formula (IV), (IVa), (IVb), (IVc), (IVd), (IVe), (IVf), (IVg), or (IVh), is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.
  • the peptide comprises 8 Cysteine residues. Therefore, in one particular aspect, the invention provides a knob domain or antigen-binding portion thereof which binds an antigen of interest comprising or consisting of the sequence of formula (V):
  • the knob domain or antigen-binding portion thereof which binds an antigen of interest comprising or consisting of the sequence of formula (V) is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length.
  • the peptide which binds an antigen of interest comprising or consisting of the sequence of formula (V) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.
  • the knob domain or antigen-binding portion thereof comprises 10 Cysteine residues. Therefore, in one particular aspect, the invention provides a peptide which binds an antigen of interest comprising or consisting of the sequence of formula (VI):
  • the knob domain or antigen-binding portion thereof which binds an antigen of interest comprising or consisting of the sequence of formula (VI) is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length. In one embodiment, the knob domain or antigen-binding portion thereof which binds an antigen of interest comprising or consisting of the sequence of formula (VI) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.
  • radioisotopes of interest are alpha emitting radioisotopes, in particular short-lived alpha-emitting isotopes such as Astatine isotopes.
  • the effector molecule is Astatine 211.
  • Astatine 211 may be advantageously used for targeted alpha-particle therapy (TAT) in particular in cancer treatment, with a potential to deliver radiation in a highly localised and toxic manner, while having advantageously having a low half-life of 7,2 hours. Radiochemical methodologies using coupling agents have been described.
  • Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases.
  • Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a protein such as insulin, oc-interferon, -interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g.
  • angiostatin or endostatin or, a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor and immunoglobulins.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • NGF nerve growth factor
  • effector molecules may include detectable substances useful for example in diagnosis.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions.
  • the effector molecule may increase the half-life of the antigen-binding protein in vivo, and/or reduce immunogenicity of the antigen-binding protein and/or enhance the delivery of an antigen-binding protein across an epithelial barrier to the immune system.
  • suitable effector molecules of this type include Fc fragments, polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in WO05/117984.
  • the effector molecule is palmitic acid. Palmitic acid has the advantageous property to bind albumin and improve interaction with cells.
  • the effector molecule is an activated form of palmitic acid such as palmitoyl.
  • the effector molecule is a polymer it may be, in general, a synthetic or a naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. a homo- or hetero- polysaccharide.
  • synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or derivatives thereof.
  • Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
  • Derivatives as used herein is intended to include reactive derivatives, for example thiolselective reactive groups such as maleimides and the like.
  • the reactive group may be linked directly or through a linker segment to the polymer. It will be appreciated that the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.
  • the size of the polymer may be varied as desired, but will generally be in an average molecular weight range from 500Da to 50000Da, for example from 5000 to 40000Da such as from 20000 to 40000Da.
  • Suitable polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 15000Da to about 40000Da.
  • antigen-binding proteins for use in the present invention are attached to poly(ethyleneglycol) (PEG) moieties.
  • the PEG molecules may be attached through any available amino acid side-chain or terminal amino acid functional group located in the antigen-binding protein, for example any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may occur naturally in the antigen-binding proteinor may be engineered into the fragment using recombinant DNA methods.
  • PEG molecules are covalently linked through a thiol group of at least one cysteine residue located in the antigen-binding protein.
  • an antigen-binding protein of the present invention may be modified by the addition of one or more conjugate groups and so comprise such a group or be said to be a conjugate.
  • conjugates refers to any molecule or moiety appended to another molecule.
  • conjugates may be polypeptide (amino acid) based or not.
  • Conjugates may comprise lipids, small molecules, RNA, DNA, polypeptides, polymers, or combinations thereof. Functionally, conjugates may serve as targeting molecules or may serve as payload to be delivered to a cell, organ or tissue.
  • Conjugates are typically covalent modifications introduced by reacting targeted amino acid residues or the termini of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
  • the conjugation process may involve PEGylation, lipidation, albumination, biotinylation, desthiobiotinylation, the addition of other polypeptide tails, or grafting onto antibody Fc domains, CDR regions of intact antibodies, or antibody domains produced by any number of means.
  • the conjugate may include anchors including cholesterol oleate moiety, cholesteryl laurate moiety, an a-tocopherol moiety, a phytol moiety, an oleate moiety, or an unsaturated cholesterol-ester moiety or a lipophilic compound selected from acetanilides, anilides, aminoquinolines, benzhydryl compounds, benzodiazepines, benzofurans, cannabinoids, cyclic polypeptides, dibenzazepines, digitalis glycosides, ergot alkaloids, flavonoids, imidazoles, quinolines, macrolides, naphthalenes, opiates (such as, but not limited to, morphinans or other psychoactive drugs), oxazines, oxazoles, phenylalkylamines, piperidines, polycyclic aromatic hydrocarbons, pyrrolidines, pyrrolidinones, stilbenes, sulfonylureas, sul
  • the effector molecule is albumin. In one embodiment, the effector molecule is human serum albumin. In one embodiment, the effector molecule is rat serum albumin.
  • the antigen-binding protein may comprise at its N- and/or C-terminal extremity albumin. In one embodiment, an antigen-binding protein of the present invention is inserted into albumin. In such embodiment, the insertion is preferably at a position distal to the albumin interaction site with FcRn. In one embodiment, the antigen-binding protein is inserted into human serum albumin.
  • Residues on albumin, distal to the interaction with FcRn may be selected as sites for inserting the knob domain, or antigen-binding portion thereof, and fused helical peptide(s), for example Alanine 59, Alanine 171, Alanine 364, Aspartic acid 562 on human serum albumin.
  • the antigen-binding protein is inserted into albumin, optionally via one or more, for example two, linker(s).
  • they may be inserted into albumin via two linkers, one linker at the N-terminal extremity of the antigenbinding protein and the other linker at the C- terminal extremity of the antigen-binding protein.
  • a suitable linker may be a flexible linker as described herein.
  • the linker or at least one of the linkers is SGGGS (SEQ ID NO: 7).
  • an antigen-binding protein of the present invention has one knob domain or antigen-binding portion thereof. In a further preferred embodiment, it comprises at least two knob domains or antigen-binding portions thereof.
  • an antigen-binding protein of the present invention may be bispecific, i.e. bind two different epitopes whether that be on the same or different antigens.
  • an antigen-binding protein of the present invention may be multi-specific, i.e. bind multiple different epitopes whether they be on the same or different antigens.
  • an antigen-binding protein of the present invention may be biparatopic, that is it binds two different epitopes of the same antigen.
  • an antigen-binding protein of the present invention may be multi-paratopic, that is it binds multiple different epitopes of the same antigen.
  • an antigen-binding protein of the present invention has a valency of one for a given antigen, that is it has one binding site specific for the antigen.
  • it is bivalent for a given antigen, that is it has two binding sites specific for a given antigen.
  • an antigen-binding protein will be multi-valent, that is have multiple valencies for a given antigen.
  • the antigen-binding protein of the present invention further comprises at least one further knob domain or antigen-binding portion thereof fused at its N-terminus, C-terminus, or both, optionally via a linker, to a helical peptide.
  • the combination of a knob domain, or antigenbinding portion thereof, fused at its N-terminus, C-terminus, or both termini to a helical peptide may be simply referred to as a unit.
  • the overall, antigen-binding protein may comprise at least two such units. In one embodiment, it may comprise at least three such units. In one embodiment, it may comprise from one to five such units. In one embodiment, where at least two such units are present they may be joined together by a linker, for example comprising a sequence as presented above, such as any of those described herein. In another embodiment, the units may be directly joined together.
  • an antigen-binding protein of the invention comprises a plurality of units, wherein each unit comprises a knob domain, or antigen-binding portion thereof, fused, optionally via a linker, at its N-terminus to a helical peptide, at its C-terminus to a helical peptide, or at both termini to a helical peptide.
  • the antigen-binding protein comprises from two to ten such units. In one embodiment, it comprises from 2 to 5 such units. In another embodiment, it comprises 2 such units. In one embodiment, it comprises three such units. In one preferred embodiment, at least two of the knob domains, or antigen-binding portions thereof have a different specificity. In another embodiment, all of the knob domains or antigen-binding portions thereof, have the same specificity.
  • the invention provides an antigen-binding protein comprising knob domains, or antigen-binding portions thereof, and fused helical peptides as described herein, wherein the knob domains, or antigen-binding portions thereof, bind to the same antigen.
  • each of the three binds to a different epitope of the same antigen.
  • each bind to the same epitope of the same antigen.
  • each bind to a different antigen, i.e. bind to three different and distinct antigens.
  • two of the three bind to the same antigen (including binding to the same epitope on that antigen or binding to different epitopes on the antigen) and the third bind to a distinct antigen.
  • the antigen-binding proteins of the present invention may be monospecific, bispecific or multi-valent. In one particularly preferred embodiment, they are bispecific.
  • valency of an antigen-binding protein as used herein denotes how knob domains, or antigen-binding portions thereof, an antigen-binding molecule of the present invention comprises.
  • the “specificity” of an antigen-binding protein as used herein in the sense of monospecific, bispecific or multi-specific denotes how many different epitopes overall an antigen-binding molecule of the present invention binds.
  • “Monospecific” as employed herein refers to an antigen-binding protein wherein all of the knob domains, or antigen-binding portions thereof, present bind the same epitope.
  • “Bispecific polypeptide” as employed herein refers to an antigen-binding protein wherein the knob domains, or antigen-binding portions thereof, present bind two different epitopes. In one embodiment, wherein the two on the same antigen. In another embodiment, wherein they are present on different antigens. “Bispecific polypeptide” as employed herein refers to a polypeptide with two antigen specificities.
  • the antigen-binding protein comprises two knob domains, or antigen-binding portions thereof, each associated with a helical peptide or peptides, wherein one knob domain or antigen-binding portion thereof binds ANTIGEN 1 and the other knob domain, or antigenbinding portion thereof, binds ANTIGEN 2, i.e. each knob domain, or antigen-binding portion thereof, is monovalent for each antigen.
  • the antibody is a tetravalent bispecific polypeptide, i-e the polypeptide comprises four knob domains, or antigen-binding portions thereof, wherein for example two bind ANTIGEN 1 and the other two bind ANTIGEN 2.
  • the antigenbinding protein is a trivalent bispecific antigen-binding protein.
  • Biparatopic refers to an antigen-binding protein, wherein the knob domains, or antigen-binding portions thereof, present bind two different epitopes on the same antigen.
  • Multi-specific polypeptide refers to an antigen-binding protein wherein the knob domains, or antigen-binding portions thereof present bind at least two different epitopes. In some embodiments, all of the epitopes may be on the same antigen. In an alternative embodiment, they are on different antigens. Multi-specific proteins may be monovalent for each specificity (antigen). Multi-specific polypeptides described herein encompass monovalent and multivalent, e.g.
  • bivalent, trivalent, tetravalent multi-specific polypeptides as well as multi-specific polypeptides having different valences for different epitopes (e.g, a multi-specific polypeptide which is monovalent for a first antigen specificity and bivalent for a second antigen specificity which is different from the first one).
  • the antigen-binding protein is monospecific and bivalent. In another embodiment, the antigen-binding protein is bispecific. In one embodiment, antigen-binding protein is a tri-specific polypeptide. “Trispecific or Trispecific polypeptide” as employed herein refers to a polypeptide with knob domains, or antigen-binding portions thereof, cumulatively recognising three specificities, so recognising three different epitopes. For example, the polypeptide is a polypeptide with three knob domains, or antigen-binding portions thereof (trivalent), which independently bind three different antigens or three different epitopes on the same antigen, i.e. each binding region is monovalent for each antigen.
  • An antigen-binding protein of the invention may be a multi-paratopic polypeptide.
  • “Multiparatopic polypeptide” as employed herein refers to a polypeptide as described herein which comprises two or more knob domains, or antigen-binding portions thereof which comprising distinct paratopes, which interact with different epitopes either from the same antigen or from two different antigens.
  • Multi-paratopic antigen-binding proteins described herein may be, for example, biparatopic, triparatopic, tetraparatopic.
  • preferred formats comprise two units of a knob domain, or antigen-binding portion thereof, with fused helical polypeptide(s), wherein the two units are joined together by any of the linkers set out herein.
  • a GSG linker is employed to joined together the units.
  • the two units are joined together via a G4P linker.
  • the two units are joined together by a G4P linker.
  • a longer linker is employed, for example the 127 amino acid sequence linker used in Figure 7D is employed.
  • an antigen-binding protein of the present invention comprising at least one knob domain of a bovine ultralong CDR-H3 or a portion thereof capable of binding antigen and a fused helical peptide or peptides, it may also comprise a bovine ultralong CDR-H3 which includes the native stalk region. In a particularly preferred embodiment, an antigen-binding protein of the present invention does not comprise a stalk region from a bovine ultralong CDR-H3.
  • a linker may be used to join together units of knob domain (or antigenbinding portions thereol) and fused helical peptide(s).
  • the polypeptide comprising at least two knob domains, or antigen-binding portions thereof, and fused helical peptide(s) units is cyclised.
  • the polypeptide comprises at least one bridging moiety between two amino acids.
  • polypeptide When the polypeptide is cyclic and does not have end-amino acids, it may be referred to as a macrocycle.
  • bridging moiety described above in connection with cyclised antibody fragments also apply to the cyclised polypeptides of the present disclosure.
  • the bridging moiety may be a disulphide bond.
  • an antigen-binding protein of the present invention shows enhanced protein levels when expressed in comparison to expression of a knob domain, or antigenbinding portion thereof, from the protein on its own.
  • an antigen-binding protein of the present invention shows thermal stability.
  • the protein shows thermal stability as evidenced by the absence of any significant drop in antigen binding activity for antigen following incubation for 30 minutes at 90°C in comparison for a sample incubated for the same time at 20°C.
  • the antigen-binding protein provided does not have an antigen-binding site specific for bovine leukaemia virus.
  • an antigen-binding protein of the invention does not comprise a Fab region.
  • the knob domain(s), or antigen-binding portions thereof, in an antigen-binding protein of the invention and fused helical peptide(s) are not present within an antibody or any fragment thereof.
  • an antigen-binding protein of the invention does not comprise a conventional antibody variable region or any part thereof such as a conventional antibody framework region.
  • an antigen-binding protein of the present invention does not comprise an antibody constant region.
  • the antigen-binding protein comprising the knob domain, or antigen-binding portion thereof does not comprise the stalk region or regions of the antibody from which the knob domain, or antigen-binding portion is isolated, and preferably does not comprise the stalk region or regions of a naturally occurring bovine antibody with ultra-long CDR H3.
  • the helical peptide or peptides which are fused to the knob domain(s), or antigen-binding portion(s) thereof, in the antigen-binding protein may also be a shorter portion of a longer naturally occurring protein, but are isolated from that longer naturally occurring protein.
  • An antigen-binding protein of the invention may be produced advantageously by recombinant expression.
  • the present invention also provides a polynucleotide encoding an antigen-binding protein of the present invention.
  • the polynucleotide (i.e. DNA sequence) of the present invention may comprise synthetic DNA, for instance produced by chemical processing, cDNA, genomic DNA or any combination thereof.
  • polypeptide comprising at least two knob domains, or antigen-binding portions thereof, and fused helical peptide(s)
  • they are preferably encoded by a single polynucleotide.
  • each may be expressed separately and the two subsequently conjugated or linked together after expression.
  • a particularly preferred embodiment though is either for each unit of knob domain, or antigen-binding portion thereof, and fused helical peptide(s) to either be joined to another unit in the same linear amino acid sequence or via a linker, such as one of the linkers set out herein.
  • the present invention also provides a vector encoding an antigen-binding protein of the present invention.
  • the present invention provides a cloning or expression vector comprising one or more polynucleotides of the present invention. General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art.
  • a host cell comprising one or more vectors of the present invention. Further, provided is a host cell comprising one or more polynucleotides of the present invention. Any suitable host cell/vector system may be used for expression. Bacterial, for example E. coll, and other microbial systems may be used or eukaryotic, for example mammalian, host cell expression systems may also be used. Suitable mammalian host cells include HEK, CHO, myeloma or hybridoma cells.
  • Suitable types of Chinese Hamster Ovary (CHO cells) for use in the present invention may include CHO and CHO- K1 cells including dhfr- CHO cells, such as CHO-DG44 cells and CH0-DXB11 cells, which may be used with a DHFR selectable marker or CHOK1-SV cells which may be used with a glutamine synthetase selectable marker.
  • Other cell types of use in expressing antibodies include lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells.
  • a process for producing an antigen-binding protein of the present invention comprising expressing such a protein from a host cell of the present invention.
  • the method further comprises recovering the protein.
  • the method may comprise cleavage and/or purification steps.
  • the method may further comprise formulating the antigen-binding protein into a pharmaceutical composition.
  • knob domains, or antigen-binding portions thereof, present in the antigen-binding regions of the present invention will preferably be originally obtained from bovine ultralong CDR-H3 regions.
  • Sequences coding for producing bovine ultralong CDR-H3 and hence knob domains may be obtained, for example by the methods described in WO 2021/191424 which is incorporated by reference in its entirety as well as specifically in relation to such methods for the generation of knob domains and antigen-binding portions thereof.
  • the method may in particular comprise the following steps: a) immunising a bovine with an immunogenic composition, and; b) isolating total RNA from PBMC or secondary lymphoid organ, or antigen-specific memory B-cells, and; c) amplifying the cDNA of the ultralong CDR-H3, and; d) sequencing an ultralong CDR-H3 or portion thereof; wherein the immunogenic composition comprises an antigen of interest or immunogenic portions thereof, or DNA encoding the same.
  • an “immunogenic composition” refers to a composition which is able to generate an immune response in bovine administered with said composition.
  • An immunogenic composition typically allows the expression of an immunogenic antigen of interest in the administered bovine, against which bovine antibodies may be raised as part of the immune response.
  • Protein immunisation refers to the technique of administration of an immunogenic protein comprising an antigen of interest, or immunogenic portion of said protein, comprising said antigen of interest or immunogenic portion thereof.
  • the immunogenic composition comprises a full-length protein. In another embodiment, the immunogenic composition comprises an immunogenic portion of a protein.
  • DNA immunisation refers to the technique of direct administration into the cells of the bovine of a genetically engineered nucleic acid molecule encoding a full-length protein or an immunogenic portion thereof comprising an antigen of interest (also referred to as nucleic acid vaccine or DNA vaccine herein) to produce an immunological response in said cells, against said antigen of interest.
  • DNA immunisation uses the host cellular machinery for expressing peptide(s) corresponding to the administered nucleic acid molecule and/or achieving the expected effect, in particular antigen expression at the cellular level, and furthermore immunotherapeutic effect(s) at the cellular level or within the host organism.
  • Cell immunisation refers to the technique of administration of cells naturally expressing or transfected with an immunogenic protein comprising an antigen of interest, or immunogenic portion of said protein, comprising said antigen of interest or immunogenic portion thereof.
  • the immunisation at step a) is performed using cell immunisation with fibroblasts transfected with an immunogenic protein comprising an antigen of interest, or immunogenic portion of said protein, comprising said antigen of interest or immunogenic portion thereof.
  • Immunogenic portion it is meant a portion of the protein or antigen of interest which retains the capacity of inducing an immune response in the bovine animal administered with said portion of the protein or antigen of interest or DNA encoding the same, in order to enable the production of knob domains, or antigen-binding portions thereof, to be employed in the present invention.
  • the immunisation step a) may be performed using protein immunisation, DNA immunisation, or cell immunisation or any combination thereof.
  • the immunisation step a) may be performed using a prime-boost immunisation protocol implying a first administration (prime immunisation or prime administration) of the immunogenic composition, and then at least one further administration (boost immunisation or boost administration) that is separated in time from the first administration within the course of the immunisation protocol.
  • Boost immunisations encompass one, two, three or more administrations.
  • the immunisation step a) is performed using a prime-boost immunisation protocol comprising a prime immunisation with an antigen of interest in presence of a first adjuvant, then at least one boost immunisation with said antigen of interest in presence of a second adjuvant.
  • Adjuvant refers to an immune stimulator.
  • Adjuvants are substances well known in the art.
  • the adjuvant may be a Freund's adjuvant, a Montanide adjuvant, or a Fama adjuvant.
  • Step b) isolating total RNA from PBMC or secondary lymphoid organ, or antigen-specific memory B- cells
  • step c) generally comprises a first step of obtaining cDNA from the total RNA obtained at step b), using RT-PCR.
  • a method for amplifying directly the cDNA of ultralong CDR-H3 and discriminate from standard CDR-H3 may be used.
  • the method may comprise a primary polymerase chain reaction (PCR) with primers flanking CDR-H3, annealing to the conserved framework 3 and framework 4 of the VH, to amplify all CDR-H3 sequences, irrespective of their length or amino acid sequence.
  • the method may additionally comprise a second round of PCR with stalk primers to specifically amplify ultralong sequences from the primary PCR.
  • the method for amplifying the cDNA of CDR-H3 comprises:
  • the primers used at step 1) comprise or consist of SEQ ID NO: 154 and SEQ ID NO: 311.
  • the primers used at step 2) are selected from the group consisting of SEQ ID NO:312 to SEQ ID NO:315. It will be appreciated that the primers used at step 2) comprise one ascending primer and one descending primer, i.e. the primers may comprise one ascending primer of any one of SEQ ID NO: 312 to SED ID NO: 313, and one descending primer of any one of SEQ ID NO: 314 to SEQ ID NO:315.
  • Step d sequencing an ultralong CDR-H3 or portion thereof
  • Step d) comprises sequencing the cDNA of CDR-H3 or portion thereof in order to identify the knob domain peptide of the ultralong CDR-H3 or portions thereof.
  • Step d) may be performed according to methods well known in the art such as direct nucleotide sequencing.
  • the knob domain may be defined as described herein and its sequence isolated.
  • the method may optionally further comprise a screening step. It may be ultralong CDR-H3 regions, knob domains (or antigen-binding portions thereof) on their own, or the knob domains (or antigen-binding portions thereol) fused to helical peptide(s) as described herein may be screened. Preferably, they may be screened in vitro for binding to the antigen of interest. In one alternative embodiment, rather than being fused to a helical peptide(s), knob domains (or antigen-binding portions thereof) may be joined to a carrier for screening.
  • knob domain (or an antigen-binding portion thereof) of the ultralong CDR- H3 may be expressed after step d) and screened for binding to the antigen of interest before step d) optionally after a step of reformatting the ultralong CDR-H3 into a screening format as described herein.
  • the carrier is an Fc polypeptide.
  • An “Fc polypeptide” as used herein is a polypeptide comprising a Fc fragment.
  • the Fc polypeptide is a scFc.
  • Single-chain Fc polypeptide” or “scFc” as employed herein refers to a single chain polypeptide comprising two CH2 domains and two CH3 domains characterized in that said CH2 and CH3 domains form a functional Fc domain within the chain.
  • the functional Fc domain in the single-chain polypeptides of the present invention is not formed by dimerisation of two chains i.e.
  • the two CH2 domains and two CH3 domains are present in a single chain and form a functional Fc domain within the single chain.
  • the term ‘functional’ as used herein refers to the ability of the Fc domain formed within the single chain polypeptide to provide one or more effector functions usually associated with Fc domains although it will be appreciated that other functions may be engineered into such domains.
  • the carrier is a scFc and comprises the sequence SEQ ID NO: 155. In one embodiment, the carrier is a scFc and the fusion protein comprises a linker, wherein the linker comprises a TEV protease cleavage site and a Gly-Ser linker. In one embodiment, the carrier is a scFc and the fusion protein comprises the sequence SEQ ID NO: 156.
  • scFc sequences and variants useful in the context of the present disclosure have been described in W02008/012543.
  • a carrier may be used for the screening step prior to reformatting into an antigenbinding protein format as described herein, preferably the screening may be performed with knob domains, or antigen-binding portions thereof fused to helical peptide(s).
  • Methods for producing proteins according to the invention may be performed according to well- known methods to express polypeptides, notably by using cloning, expression vectors, and host cells as described above, using sequences of knob domains of bovine ultralong CDR-H3 (or portions thereof which bind to an antigen of interest) discovered according to the methods described above, or from sequences previously published.
  • sequences coding knob domains, and antigen-binding portions thereof, for use in the present invention may be derived from libraries, as described for example in WO 2021/191424 which is incorporated by reference in its entirety, as well as specifically in relation to such methods.
  • Libraries may be immune libraries or naive libraries of knob domains, or antigen-binding portions thereof, for use in the context of the invention, prepared from animals which have not been administered an immunogen.
  • Phage display libraries of knob domains, or antigen-binding portions thereof may be used, wherein the knob domains, or antigen-binding portions thereof, may be expressed directly at the surface of phages using any suitable method.
  • Libraries of ultralong CDR-H3 sequences, i-e libraries of knob domains, or antigen-binding portions thereof, when expressed as part of the full sequence of CDR-H3 (i.e. comprising the knob and stalk domains) may be screened.
  • the libraries are naive libraries. In one embodiment, the naive libraries are prepared from cattle. In another embodiment, the libraries are immune libraries. In one embodiment, the libraries are prepared from immunised cattle.
  • the phage display library is a M13 phage display library. In one embodiment, the knob domains, or antigen-binding portions thereof, are optionally displayed within the full sequences of CDR-H3, are fused directly to the pill coat protein of the M13 phage.
  • the knob domains or antigen-binding portions thereof are fused to the pill coat protein of the M13 phage via a linker (or “spacer”).
  • a suitable linker may be a linker which allows to separate the cysteine-rich domain from the cysteines of the pill, notably to ensure that the pill and the knob domain peptide, or antigen-binding portion thereof, folds independently and correctly.
  • Methods for producing a phage display library are well known. Phagemid vectors have for example been described in Hoogenboom HR at al. (Hoogenboom HR, Multi-subunit proteins on the surface of fdamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 1991 ; 19(15):4133-4137).
  • the invention provides a phage display library, comprising a plurality of recombinant phages; each of the plurality of recombinant phages comprising an M13-derived expression vector, wherein the M13-derived expression vector comprises a polynucleotide sequence encoding an antigen-binding protein as set out herein.
  • a library may comprise the knob domain, or antigen-binding portion thereof, optionally displayed within the full sequence of ultralong CDR-H3, where once preferred knob domains, or antigen-binding portions thereof, have been identified they are fused to a helical peptide or peptides as described herein.
  • the knob domain optionally displayed within the full sequence of ultralong CDR-H3, is fused to the sequence encoding the pill coat protein of the M13 phage, directly or via a spacer.
  • the invention provides methods for generating phage display libraries of ultralong CDR-H3 sequences, i-e libraries of knob domains, or antigen-binding portion thereof, displayed within the full sequence of CDR-H3.
  • a method for generating an immune phage display library of ultralong CDR-H3 sequences comprising: a) immunising a bovine with an immunogenic composition, and; b) isolating total RNA from PBMC or secondary lymphoid organ, and; c) amplifying the sequences of the ultralong CDR-H3, and; d) fusing the sequences obtained in c) to the sequence coding for the pill protein of a M 13 phage within a phagemid vector, and; e) transforming host bacteria with the phagemid vector obtained at step d) in combination with a helper phage co-infection, and; f) culturing the bacteria obtained at step e), and; g) recovering the phages from the culture medium of the bacteria, wherein the immunogenic composition comprises an antigen of interest or immunogenic portions thereof, or DNA encoding the same.
  • Steps a) to g) are methods well known in the art.
  • the method for amplifying the cDNA of CDR-H3 comprises:

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Abstract

La présente invention concerne une protéine de liaison à l'antigène comprenant un domaine knob ou une partie de celui-ci capable de se lier à l'antigène, le domaine knob ou la partie de liaison à l'antigène de celui-ci étant fusionné, soit directement, soit par l'intermédiaire d'un lieur, à son extrémité N-terminale ou C-terminale ou à la fois à un peptide hélicoïdal, de préférence un peptide α-hélicoïdal. La présente invention concerne également la production et l'utilisation de telles protéines de liaison à l'antigène. La présente invention concerne en outre les protéines de liaison à l'antigène pour une utilisation en thérapie.
PCT/EP2024/077193 2023-09-28 2024-09-27 Protéines à domaine knob Pending WO2025068449A1 (fr)

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WO2005117984A2 (fr) 2004-06-01 2005-12-15 Celltech R & D Limited Composes liant l'albumine
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