WO2025068449A1

WO2025068449A1 - Knob domain proteins

Info

Publication number: WO2025068449A1
Application number: PCT/EP2024/077193
Authority: WO
Inventors: Alastair David Griffiths Lawson; Alexander Macpherson; Mikhail KURAVSKIY
Original assignee: UCB Biopharma SRL
Current assignee: UCB Biopharma SRL
Priority date: 2023-09-28
Filing date: 2024-09-27
Publication date: 2025-04-03
Anticipated expiration: 2026-03-28
Also published as: TW202521590A; GB202314896D0

Abstract

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 α-helical peptide. The present invention also relates to the production and use of such antigen-binding proteins. The present invention further relations to the antigen-binding proteins for use in therapy.

Description

KNOB DOMAIN PROTEINS

Field of the invention

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.

Background of the invention

The high specificity and affinity of antibodies makes them ideal diagnostic and therapeutic agents. Standard full-length monoclonal antibodies have a size of 150 kDa. Advances in the field of recombinant antibody technology have resulted in the production of antibody fragments, such as Fv, Fab, Fab' and F(ab')₂ fragments. These smaller molecules retain the antigen binding activity of whole antibodies and can also exhibit altered biodistribution, tissue penetration and pharmacokinetic properties in comparison to whole immunoglobulin molecules. Indeed, antibody fragments are proving to be versatile therapeutic agents. To date, the smallest autonomous, naturally occurring, functional antibody fragment reported has been the VHH fragment derived from camelids (Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446-448 (1993)) and the VNAR (Variable New antigen Receptor) fragment from sharks (Greenberg, A. S. et al. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374, 168-173 (1995)), resulting in heavy chain variable region fragments, of some 12-15 kDa.

Whilst such fragments appear to exhibit a number of advantages over whole immunoglobulins, they also suffer from an increased rate of clearance from serum since they lack the Fc domain that imparts a long lifetime in vivo. In addition, the availability of recombinant production methods and systems is limiting on the antibody production and may present technical challenges, e.g. in terms of DNA engineering, productivity in cells, etc. Further miniaturising antibody fragments may allow them to be produced without recombinant antibody expression.

The central segment of bovine ultra-long CDR H3 or “knob” (knob domain peptide or simply knob domain) 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. 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. Compared to recombinant protein technology, SPPS provides an easy way to incorporate some therapeutically relevant modifications, such as noncanonical amino acids, palmitoylation and head-to-tail cyclisation. However, both the approaches are associated with higher costs when scaling up production levels.

Therefore, there remains a need to further optimise 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.

Summary of the invention

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. In a preferred embodiment, 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. In a particularly preferred embodiment, wherein two such helical peptides are present, the two helical peptides are able to form a coiled-coil structure.

The use of 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. Advantageously, 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. Thus, 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.

In one preferred embodiment, linkers may be used to fuse a knob domain, or an antigen-binding portion thereof, to a helical peptide. Alternatively, 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.

Particularly preferred 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. Preferably, 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.

Accordingly, 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 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. Brief Description of the Figures

Figure 1: Structure of bovine ultralong CDR-H3 antibody and examples of antigenbinding proteins of the present invention comprising different knob domain-helical peptide fusions. (A) shows the native conformation of a knob domain when expressed as part of the bovine antibody infrastructure (notably comprising the stalk). (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.

Figure 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).

Figure 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 6: Thermal stability of antigen-binding proteins comprising knob domain-helical peptide fusions. The ability of the antigen-binding proteins comprising particular knob-helical peptide fusions to bind an antigen after a thermal stress was assessed using ELISA to determine antigen-binding ability after incubation for 30 minutes at either 20°C or 90°C.

Figure 7: Antigen-binding proteins with more than one knob domain to provide a plurality of antigen-binding sites. Various antigen-binding proteins were generated comprising more than one knob domain, with each knob-domain fused at their N terminus and C-terminus to a helical peptide. The presence of more than one knob domain meant that a plurality of antigen binding sites is present. The different knob domain-helical peptide units are joined together by linkers. (A) shows examples of bispecific antigen-binding proteins comprising knob domains with two different specificities. (B) shows an example of a bivalent antigen-binding protein where both knob domains bind to the same antigen and epitope. (C) shows an example of an antigen-binding protein that is both bivalent and bispecific. (D) shows an example of a biparatopic antigen-binding protein where the two knob domains bind different epitopes of the same antigen.

Figure 8: Comparison of protein expression of antigen-binding proteins with more than one knob domain. Expi293F cells were transiently transfected with the relevant construct with secreted protein subsequently recovered and enriched, followed by SDS-PAGE analysis under nonreducing conditions. Constructs expressing the different knob domains individually, with fused helical peptides or on their own without any helical peptides were also used as controls.

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.

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 11: Assessment of simultaneous binding to more than one antigen via an SPR bridging assay. A Biacore chip coated with C5 was exposed to the antigen-binding proteins [SNV-N fusion protein (HA) and BECN1 fusion protein (1 IB)], followed by IL-2 to study the ability of the antigen-binding proteins shown to bind to both C5 and IL-2 simultaneously.

Figure 12: Assessment of simultaneous binding to two different epitopes of the same antigen. The ability of biparatopic K8-K57 [SNV-N fusion protein (12A) and BECN1 fusion protein (12B)] to bind both epitopes of C5 recognised by the biparatopic at the same time was measured by Biacore and comparing the proteins binding kinetics to the kinetics of individual knob domains.

Figure 13: Assessment of simultaneous binding to the same epitope on two IL-2 molecules. The ability of an antigen-binding protein comprising two copies of the aIL2_l knob domain and fused helical peptides [SNV-N fusion protein (13 A) and BECN1 fusion protein (13B)] to bind to IL-2 was also studied using Biacore.

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 set of K8-SNV-N proteins with a pair of cysteines at the distal end of the helical peptides, with the helical peptides forming a coiled-coil dimer was generated. (A) shows the different versions generated. (B) shows protein expression of the different versions. (C) shows the antigen binding of the different versions.

Figure 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:

Detailed Description of the Invention

Antigen-binding proteins

In one aspect, 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. In embodiments, where the linkers are peptide linkers, they too may be in the primary amino acid sequence of the antigenbinding protein.

The antigen-binding proteins and their uses are described in more detail below. In one especially preferred embodiment, the antigen-binding proteins comprises a knob domain, or antigen- binding portion thereof, and helical peptide(s) that are not derived from the same naturally occurring protein and preferably are heterologous to each other. For example, in one preferred embodiment the knob domain, or antigen binding portion thereof, is bovine, or is derived from a bovine knob domain or antigen-binding portion thereof, but the helical peptide(s) are not bovine or derived from a bovine protein. Preferably, 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, and the helical peptide(s) are not, or are not 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.

Naturally occurring ultralong CDR-H3 regions comprise a stalk and a knob domain. The inventors have shown that the helical peptide or peptides set out herein can be effectively used to confer distinctive properties on the antigen-binding proteins provided. The helical peptides employed are different to the two antiparallel P-strands found in the stalk domain that is present in naturally occurring ultralong CDR-H3 regions, such as bovine ultralong CDR-H3 regions. The helical peptide or helical peptides therefore do not occur in the same natural protein as the knob domain, or antigen-binding portion thereof, to which they are fused in antigen-binding proteins of the invention, preferably they are heterologous to each other. In one embodiment, 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. In one preferred embodiment, an antigen-binding protein of the present invention does not comprise antiparallel strands fused to a knob domain or antigen-binding portion thereof.

The 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. In particular, in the context of the present disclosure, 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. Preferably, a helical peptide employed in the present invention does not correspond to a peptide in a bovine antibody having an ultralong CDR-H3. In particular, 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. Preferably, 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. a peptide that has not been found in nature. Reference to isolated as used herein means removed from its natural structural context, for example 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. Alternatively, 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. In one embodiment, a linker is employed to fuse the N-terminus of a knob domain, or antigen-binding portion thereof, to a helical peptide. In another embodiment, a linker is employed to fuse the C-terminus of a knob domain, or antigen-binding portion thereof, to a helical peptide. In one preferred embodiment, 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. In another embodiment, 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. Alternatively, it may be 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.

As explained further below, 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. In an alternative embodiment, an antigen-binding protein of the invention comprises a single knob domain or antigen-binding portion thereof. In one embodiment, 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. In one embodiment, 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. In one embodiment, a helical peptide comprises, or is, at least two helical turns in length. In one preferred embodiment, 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.

In a preferred embodiment, where at least two helical peptides are present, they will be able to associate with each other. In an especially preferred embodiment, wherein two helical peptides are present they will form a coiled-coil structure. Preferably, wherein two helical peptides are present, the knob domain, or antigen-binding portion thereof, will be present between them in the linear amino acid sequence (i.e. the primary amino acid sequence) of the polypeptide comprised of the knob domain, or antigen-binding portion thereof, and the two helical peptides with the helical peptides fused to the knob domain, or antigen-binding portion thereof, via a peptide bond or via a linker.

In a further particularly preferred embodiment, the two helical peptides are anti-parallel compared to each other. In one preferred embodiment, the two helical peptides may have charge interactions to promote their association, for instance one helical peptide may have an amino acid with a positively charged side chain and the other with a negatively charged side-chain so that the complementary charges may promote the formation of the coiled-coil structure. In one embodiment, two helical peptides form a dimer of two a-helices aligned alongside each other in anti-parallel orientation. In one embodiment, the two helical peptides form a dimerization interface.

In one embodiment, a linker may be an amino acid sequence of 20 or less amino acids. In one preferred embodiment, the linkers are any of the specific linkers set out herein. In one preferred embodiment where the knob domain, or antigen-binding portion thereof, is joined to two helical peptides it will be joined to each helical peptide via a linker. Hence, for example, their order in the linear amino acid sequence may be helical peptide-linker-knob domain (or antigen-binding portion thereof) -linker-helical peptide. In a preferred embodiment the two helical peptides will form a coiled- coil structure.

In one preferred embodiment, a helical peptide, or a pair of helical peptides, may be from an Orthohantavirus. A particular preferred helical peptide, or pair of helical peptides, is from Sin Nombre orthohantavirus (SNV). In a preferred embodiment, an antigen-binding protein will comprise a knob domain, or antigen binding portion thereof, fused at its N-terminus and C-terminus to a pair of helical peptides from SNV that are able to form a coiled-coil structure with the bovine knob domain, or antigenbinding portion thereof, between the two helical peptides. Variants of the naturally occurring SNV sequences may also be employed, provided that they are still able to form a coiled-coil structure, and the knob domain, or antigen-binding portion thereof, is still able to bind antigen.

In one embodiment, an antigen-binding protein of the present invention may comprise a knob domain, or antigen-binding portion thereof, fused at its N-terminus and C-terminus with a helical peptide so that two helical peptides are able to form a coiled-coil structure, wherein the coiled-coil comprises at least one pair of cysteine residues forming a disulphide bridge. In one embodiment, at least one such disulphide bridge is formed in the distal half of the coiled-coil, preferably between the N-terminus of the first helical peptide and the C-terminus of the second helical peptide.

In one preferred embodiment, an antigen-binding protein of the invention comprises a knob domain, or antigen-binding portion thereof, fused at its N terminus, its C-terminus, or preferably at both termini to a helical peptide derived from SNV-N. Preferably, the knob domain, or antigenbinding portion thereof, is fused at its N-terminus and C-terminus to such a helical peptide with the two able to form a coiled-coil. In one embodiment, a helical peptide comprises the amino acid sequence of SEQ ID NO: 109 or a variant thereof. In one embodiment, a helical peptide comprises the amino acid sequence of SEQ ID NO: 110 or a variant thereof. In one preferred embodiment, the antigen-binding protein comprises a first helical peptide comprising the sequence of SEQ ID NO: 109 or a variant thereof and a second helical peptide comprising the sequence of SEQ ID NO: 110 or a variant thereof, wherein the two helical peptides are each fused to a knob domain, or antigen-binding portion thereof, with one fused at the N-terminus and the other at the C-terminus. In one embodiment, between the two helical peptides in the linear amino acid sequence of the polypeptide is a knob domain, or antigen-binding portion thereof of a bovine ultralong CDR-H3. Preferably the knob domain, or antigen-binding portion thereof, is fused to the helical peptides via a linker. In another preferred embodiment, it is fused to the helical peptides via direct peptide bonds.

In one embodiment, rather than the specific sequences of SEQ ID NOs: 109 and/or 110, a variant helical peptide or helical peptides is employed. In one embodiment, a variant helical peptide has one, two, three, four, or five amino acid sequence changes in total compared to the specific sequences of SEQ ID NOs: 109 and/or 110. In one embodiment, a variant peptide has at most three amino acid sequence changes in total compared to the specific sequences of SEQ ID NOs: 109 and/or 110, with the sequence changes selected from amino acid substitutions, insertions, and deletions. In one embodiment, a variant helical peptide has at least 90% amino acid sequence identity to the specific sequence of SEQ ID NOs: 109 and/or 110. In another embodiment, it has at least 95% amino acid sequence identity. In another embodiment, it has at least 99% amino acid sequence identity. Preferably, a variant helical peptide when used in a pair of helical peptides will still be able to form a coiled-coil structure with the knob domain, or antigen-binding portion thereof still able to bind antigen.

In one embodiment, the antigen-binding protein comprises a first and second helical peptide derived from SNV-N, wherein each peptide has at least one cysteine residue that forms a disulphide bridge with a cysteine in the other helical peptide. In such embodiments, one helical peptide is fused to the N-terminus of a knob domain (or antigen-binding portion thereof), with the other helical peptide fused to the C-terminus. The fusion for either or both may be direct via peptide bonds or via a linker. Examples of preferred pairs of helical peptides present in an antigen-binding protein include those indicated in Table 6. Hence, one preferred pair of helical peptides is the pair of helical peptides of SEQ ID NOs 143/144. Another preferred pair of helical peptides is those of SEQ ID NOs 146/147. Another preferred pair of helical peptides is those of SEQ ID NOs 149/150. Another preferred pair of helical peptides is those of SEQ ID NOs 152/153. Variant helical peptides may be employed, for example variant helical peptides that have at most a total of three amino acid sequence changes per helical peptide, wherein the changes are not of the cysteine residues that form disulphide bonds and still allow the two helical peptides to form a coiled-coil structure, with the antigen-binding protein comprising the knob domain, or antigen-binding portion thereof, present between the two helical peptides, fused to them, still able to bind antigen. In another embodiment, a variant helical peptide has at least 95% amino acid sequence identity to one of the specific helical peptides. In another embodiment, it has at least 99% amino acid sequence identity. Preferably, a variant helical peptide or variant peptides when used in a pair of helical peptides will still be able to form a coiled-coil structure with the knob domain, or antigen-binding portion thereof still able to bind antigen.

In one embodiment, an antigen-binding protein of the present invention comprises at least one helical peptide derived from human beclin-1 protein (BECN-1). Preferably in such embodiments, the antigen-binding protein comprises two such helical peptides, wherein they form a coiled-coil. In one embodiment, the antigen-binding protein comprises a helical peptide having the amino acid sequence of SEQ ID NO: 111 or a variant thereof. In one embodiment, the antigen-binding protein comprises a helical peptide having the amino acid sequence of SEQ ID NO: 112 or a variant thereof. In one preferred embodiment, the antigen-binding protein comprises a first helical peptide having the sequence of SEQ ID NO: 111 and a second helical peptide having the sequence of SEQ ID NO: 112, preferably wherein between the two helical peptides in the linear amino acid sequence of the polypeptide is a knob domain or an antigen-binding portion thereof either fused directly to the peptides or via linkers. In one embodiment, rather than the specific sequences of SEQ ID NOs: 111 and/or 112, a variant helical peptide is employed. In one embodiment, a variant helical peptide has at most three amino acid sequence changes per helical peptide compared to the specific sequences of SEQ ID NOs: 111 and/or 112, with the sequence changes selected from amino acid substitutions, insertions, and deletions. In another embodiment, it has at least 90% amino acid sequence identity to one of the specific sequence helical peptides of SEQ ID NOs: 111 and/or 112. In another embodiment, it has at least 95% amino acid sequence identity to one of the specific sequence helical peptides. In another embodiment, it has at least 99% amino acid sequence identity. Preferably, a variant helical peptide when used in a pair of helical peptides will still be able to form a coiled-coil structure with the knob domain, or antigen-binding portion thereof still able to bind antigen.

In one embodiment, an antigen-binding protein comprises two helical peptides derived from human beclin-1 protein (BECN-1), but rather than having the helical peptides having the amino acid sequences of SEQ ID Nos: 111 and 112, the helical peptides comprise a portion of those sequences which still allows the formation of a coiled-coil and for a knob domain, or antigen-binding portion thereof, present in the linear amino acid sequence of the antigen-binding protein between the helical peptides to still bind antigen. In a preferred embodiment, the pair of helical peptides will each comprise three or more helical turns. One embodiment of such a preferred pair of helical peptides are those of SEQ ID NOs: 122/123. One embodiment of such a preferred pair of helical peptides are those of SEQ ID NOs: 125/126. One embodiment of such a preferred pair of helical peptides are those of SEQ ID NOs: 128/129. In another embodiment the pair of helical peptides have the amino acid sequences of SEQ ID NOs: 131/132. In a further preferred embodiment, the pair of helical peptides have the amino acid sequences of SEQ ID Nos: 140/141. As well as those pairs of specific helical peptide sequences, variants of them may be employed, for example those with a maximum of three amino acid sequence changes compared to the specific sequences per helical peptide, wherein the amino acid sequence changes are selected from amino acid substitutions, insertions and/or deletions. In one embodiment, a variant helical peptide has at least 90% amino acid sequence identity to one of the specific helical peptides. In another embodiment, a variant helical peptide has at least 95% amino acid sequence identity to one of the specific helical peptides. In another embodiment, it has at least 99% amino acid sequence identity. Preferably, a variant helical peptide, when used in a pair of helical peptides, will still be able to form a coiled-coil structure with the knob domain, or antigen-binding portion thereof still able to bind antigen.

In one embodiment, an antigen-binding protein comprises at least one helical peptide comprising, or having the sequence of, SEQ ID NO: 116. In one embodiment, an antigen-binding protein comprises at least one helical peptide comprising, or having the sequence of, SEQ ID NO: 117. In one preferred embodiment, an antigen-binding protein comprises a first helical peptide having the sequence of SEQ ID NO: 116 and a second helical peptide having the sequence of SEQ ID NO: 117, preferably wherein between the two helical peptides in the linear amino acid sequence of the polypeptide is a knob domain or antigen-binding portion thereof. Preferably the antigen-binding domain is either directly joined to each helical peptide or is joined via a linker. In one embodiment, rather than the specific sequences of SEQ ID NOs: 116 and/or 117, a variant helical peptide is employed. In one embodiment, a variant helical peptide has at most three amino acid sequence changes per helical peptide compared to the specific sequences of SEQ ID NOs: 116 and/or 117, with the sequence changes selected from amino acid substitutions, insertions, and deletions. Preferably, the variant peptides will still be able to form a coiled-coil structure with the knob domain, or antigenbinding portion thereof, still able to bind antigen. In one embodiment, a variant has at least 95% amino acid sequence identity to one of the specific helical peptides. In another embodiment, a variant has at least 95% amino acid sequence identity to one of the specific helical peptides. In another embodiment, it has at least 99% amino acid sequence identity. Preferably, a variant helical peptide, when used in a pair of helical peptides, will still be able to form a coiled-coil structure with the knob domain, or antigen-binding portion thereof, still able to bind antigen.

In one embodiment, an antigen-binding protein comprises at least one helical peptide comprising, or having the sequence of, SEQ ID NO: 119. In one embodiment, the antigen-binding protein comprises at least one helical peptide comprising, or having the sequence of, SEQ ID NO: 120. In one preferred embodiment, the antigen-binding protein region comprises a first helical peptide having the sequence of SEQ ID NO: 119 and a second helical peptide having the sequence of SEQ ID NO: 120, preferably wherein between the two helical peptides in the linear amino acid sequence of the polypeptide is a knob domain or antigen-binding portion thereof. Preferably the antigen-binding domain is either directly joined to each helical peptide or is joined via a linker. In one embodiment, rather than the specific sequences of SEQ ID NOs: 119 and/or 120, a variant helical peptide or helical peptides is employed. In one embodiment, a variant helical peptide has at most three amino acid sequence changes per helical peptide compared to the specific sequences of SEQ ID NOs: 119 and/or 120, with the sequence changes selected from amino acid substitutions, insertions, and deletions. Preferably, the variant helical peptides will still be able to form a coiled-coil structure with the knob domain, or antigen-binding portion thereof, still able to bind antigen.

In one embodiment, an antigen-binding protein of the present invention comprises at least one helical peptide derived from human haemoglobin B (HHB). In one preferred embodiment, a single such helical peptide from HBB is fused to the knob domain or antigen-binding portion thereof. In one preferred embodiment, the helical peptide is present joined to the N terminus of the knob domain or antigen-binding portion thereof. An example of a preferred HBB helical peptide is provided in SEQ ID NO: 113. In one embodiment, rather than SEQ ID NO: 113 a variant is employed with at most three amino acid sequence changes in total selected from amino acid substitutions, insertions and deletions. In one embodiment, a variant has at least 90% amino acid sequence identity to one of the specific helical peptide of SEQ ID NO: 113. In another embodiment, a variant helical peptide is employed which has at least 95% amino acid sequence identity to SEQ ID NO: 113. In another embodiment, it has at least 99% amino acid sequence identity. Where such a variant helical peptide is employed the knob domain, or antigen-binding portion thereof, it is fused to will still able to bind antigen.

In one embodiment, the HBB peptide is glycosylated. In a preferred embodiment, the HBB peptide is glycosylated with O-linked glycosylation. In an even more preferred embodiment, the O- linked glycosylation is O-linked tetrasaccharide -GalNAc(-NeuNAc)-Gal-NeuNAc. The presence of the helical peptide or peptides fused to a knob domain in an antigen-binding protein of the invention results in a number of advantages. In one preferred embodiment, the presence of the helical peptide or peptide may increase the production level of the antigen-binding protein compared to that of the knob domain, or antigen-binding portion thereof, expressed on its own in the same system. Any suitable expression system may be used to perform the comparison. In one embodiment, the comparison is performed using transient transfection of Expi293F cells, with cell supernatants to be enriched by IMAC and analysed by SDS-PAGE, in particular the comparison may be performed as described in the Examples of the present application.

In another preferred embodiment, the presence of the helical peptide or peptides fused to the knob domain, or antigen-binding portion thereof may increase stability of the antigen-binding protein. Preferably thermal stability of the antigen-binding protein is increased compared to that of a knob domain, or antigen-binding portion thereof, expressed on its own. In one embodiment, identical samples of a test protein are incubated at a lower and higher temperature for a set period and then ability to bind antigen is measured and compared between the two samples. For example, the different temperatures may be about 25°C and about 90°C with incubation for about thirty minutes, followed by assessment of antigen binding. In one preferred embodiment, the methods used in the Examples are employed to compare thermal stability.

Antibodies and knob domains

An antigen-binding protein of the present invention comprises at least one knob domain or antigen-binding portion thereof. In a preferred embodiment, a knob domain, or antigen-binding portion thereof, from a bovine ultralong CDR-H3 may be employed or a portion thereof capable of binding antigen. A knob domain, or antigen-binding portion thereof, present in an antigen-binding protein of the present invention will lack the bovine stalk region found in native bovine ultralong CDR-H3 regions and/or any other part of the bovine antibody from which the knob domain is derived. The knob domain or antigen-binding portion thereof may be therefore thought of as isolated.

In one preferred embodiment the whole knob domains of a bovine ultralong CDR-H3 is employed in an antigen-binding protein of the present invention. In an alternative embodiment, rather than the whole knob domain and any portion thereof, notably any functionally active portion thereof (i.e., any portion of a knob domain of a bovine ultralong CDR-H3 that contains an antigen binding region that specifically binds an antigen of interest) may be employed.

Prior to considering bovine ultralong CDR-H3 regions and the knob domains from them, it is useful to consider the structure of conventional antibodies in general and hence how the bovine ultralong CDR-H3 regions and knob domains differ from them. Whole antibodies also known as “immunoglobulins (Ig)” generally relate to intact or full-length antibodies i.e. comprising the elements of two heavy chains and two light chains, inter-connected by disulphide bonds, which assemble to define a characteristic Y-shaped three-dimensional structure. Classical natural whole antibodies are monospecific in that they bind one antigen type, and bivalent in that they have two independent antigen binding domains. The terms “intact antibody”, “full-length antibody” and “whole antibody” are used interchangeably to refer to a monospecific bivalent antibody having a structure similar to a native antibody structure, including an Fc region as defined herein.

Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Each heavy chain is comprised of a heavy variable region (abbreviated herein as VH) and a heavy chain constant region (CH) constituted of three constant domains CHI , CH2 and CHS, or four constant domains CHI , CH2, CH3 and CH4, depending on the Ig class. The “class” of an Ig or antibody refers to the type of constant region and includes IgA, IgD, IgE, IgG and IgM and several of them can be further divided into subclasses, e.g. IgGl, IgG2, IgG3, IgG4. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term “constant domain(s)”, “constant region”, as used herein are used interchangeably to refer to the domain(s) of an antibody which is outside the variable regions. The constant domains are identical in all antibodies of the same isotype but are different from one isotype to another. Typically, the constant region of a heavy chain is formed, from N to C terminal, by CHl-hinge-CH2-CH3- optionnaly CH4, comprising three or four constant domains.

“Fc”, “Fc fragment”, “Fc region” are used interchangeably to refer to the C-terminal region of an antibody comprising the constant region of an antibody excluding the first constant region domain. Thus, Fc refers to the last two constant domains, CH2 and CH3, of IgA, IgD, and IgG, or the last three constant domains of IgE and IgM, and the flexible hinge N-terminal to these domains.

The VH and VL regions of a whole antibody can be further subdivided into regions of hypervariability (or “hypervariable regions”) determining the recognition of the antigen, termed complementarity determining regions (CDR), interspersed with regions that are more structurally conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and the FR together form a variable region. By convention, the CDRs in the heavy chain variable region of an antibody or antigen-binding fragment thereof are referred as CDR- Hl, CDR-H2 and CDR-H3 and in the light chain variable region as CDR-L1, CDR-L2 and CDR-L3. They are numbered sequentially in the direction from the N-terminus to the C-terminus of each chain.

CDRs are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al. (supra)”). This numbering system is used in the present specification except where otherwise indicated. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence.

The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 93-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, A.M. J. Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless indicated otherwise ‘CDR- Hl’ as employed herein is intended to refer to residues 26 to 35, as described by a combination of the Kabat numbering system and Chothia’s topological loop definition. The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system. Based on the alignment of sequences of different members of the immunoglobulin family, numbering schemes have been proposed and are for example described in Kabat et al., 1991, and Dondelinger et al., Frontiers in Immunology, Vol 9, article 2278 (2018).

Different species exhibit a diversity of CDR-H3 lengths. Some bovine antibodies have been characterized by unusually long CDR-H3 (so called “bovine ultralong CDR-H3”) with lengths of up to 69 residues, representing 1-15 % of the bovine repertoire, whereas more conventional bovine antibodies have CDR-H3 of around 23 residues. CDR-H3 of camelid single chain antibodies have up to 24 residues and CDR-H3 of shark IgNAR antibodies have up to 27 residues. The CDR-H3 are too long to be accommodated by any of these numbering schemes, but alternative systems have been used, as the one discussed in Stanfield et al. (supra).

The term “heterologous”, as used herein means that it is derived from different species. Two proteins, peptides or other biological molecules are heterologous to each other if they are derived from different species.

The term “naturally occurring” or “natural protein” as used herein refers to a protein which exists in nature, e.g. which is expressed by a biological organism. “Naturally occurring” or “natural protein” can be considered the opposite of artificial.

“Bovine CDR-H3” as used herein encompasses all CDR-H3 found in bovines, including bovine regular CDR-H3 and bovine ultralong CDR-H3. The term “Bovine ultralong CDR-H3” refers to the subset of CDR-H3 which has the features of characterized ultralong CDR-H3 as defined hereinafter, notably comprising a duplication of the IGHVI-7 gene segment. The ultralong CDR-H3 has been found in bovine IgG of all classes.

Bovine ultralong CDR-H3 have been characterized by a very unusual tridimensional structure comprising a “stalk domain” and a “knob domain”. The stalk domain is composed of two antiparallel strands (each strand generally corresponding to about 12 residues). The knob domain is a disulfide rich domain which comprises a loop motif and sits atop of the stalk, which serves as a bridge to link the knob domain with the main bovine antibody scaffold.

The CDR-H3 is derived from DNA rearrangement of variable (V), diversity (D), and joining (J) gene segments. The ultralong CDR-H3 are encoded by the VHBUL (Bovine Ultra Long), D_H2, and JHI gene segments, and their length is due to an unusually long D_H2 segment. Ultralong CDR-H3 have been characterized by a duplication of the IGHVI-7 gene segment.

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. In an especially preferred embodiment though, the protein does not comprise such a stalk region at all.

Stalk and knob domain have been defined previously, notably in Macpherson et al., 2020 (supra). The “stalk domain” of bovine ultralong CDR-H3 has been characterised by its structure notably. The skilled person will appreciate that the definition of a “stalk domain” may rely on crystal structure analysis and/or sequencing information, notably as he will understand that the stalk domain position and structure may vary slightly from one ultralong CDR-H3 to another, e.g. in terms of size. The term “stalk domain” will be generally appreciated by the skilled person to correspond to the antiparallel P strands that bridge the knob domain with the main bovine antibody scaffold. The length of the stalk strands can differ, notably from long P strands (12 or more residues) to shorter P strands.

The skilled person will appreciate that the definition of a knob domain may rely on crystal structure analysis and/or sequencing information, notably as the skilled person will understand that the knob domain position and structure may vary slightly from one ultralong CDR-H3 to another, e.g. in terms of size, cysteine content, disulphide bond content. In particular, the sequence of ultralong CDR- H3 can be determined by well-known sequencing methods, and the skilled person will be able to identify the minimal sequence which define a knob domain, based for example on a comparative analysis, with well characterised ultralong CDR-H3 as well as stalk and knob domains thereof, e.g. by alignment with well-known and/or standard nucleic and/or amino acid sequences, and/or based on crystal structure analysis. An antigen-binding portion of a knob domain may be defined as any portion of a knob domain that is able to still bind to the same antigen. Preferably, the antigen-binding portion will still have the same or similar affinity for the antigen as the knob domain that it is a portion of.

As mentioned above, the ultralong CDR-H3 are too long to be accommodated by any of existing numbering scheme, but alternative systems have been used, as the one discussed in Stanfield et al. (supra). Structural analysis has also been provided for example by Wang et al. (Wang, F. et al. Reshaping antibody diversity. Cell 153, 1379-1393 (2013)). 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, as illustrated in Figure 17. The germline encoded VHBUL D_H2 J_H1 has the following sequence (SEQ ID NO: 2): CTTVHQSCPDGYSYGYGCGYGYGCSGYDCYGYGGYGGYGGYGYSSYSYSYTYEYYVDA GQG LLVTVSS

(VHBUL; followed by D_H2 gene region in bold; followed by JHI gene region underlined; The sequence coding the CDR-H3 is in italic, between positions 92 and 103 according to Kabat).

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_H2 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_H2 being “D2”, followed by D3, D4 etc...). For illustration purposes, Figure 17 indicates identifiers D2, D10, D20, D30 and D40 within the D_m segment.

Following Cys H92, 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_H2 is variable due to differences in junctional diversity formed through V-D recombination. In Stanfield, 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_H2 region has been characterised to encode the knob domain and part of the descending strand of the stalk region. D_H2 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.

In the context of the present invention, 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. Additional amino acids may be present in the N-terminal extremity and/or in the C-terminal extremity of the knob domain sequence, preferably up to 10, more particularly up to 5, additional amino acids may be present in the N-terminal and/or in the C-terminal extremity. In some embodiments, the number of amino acids present in the C-terminal extremity corresponds to the number of amino acids between the start of CDR-H3 and the first cysteine. As an example, the sequence of the bovine ultralong CDR-H3 BLV1H12 is in italic in the following sequence (SEQ ID NO: 4) comprising VHBUL, D_H2 (underlined), JHI (Cys92 and Trpl03 Kabat are in bold):

CTSVHQ ETKKYQ SCPDGYRERSDCSNRPACGTSDCCR VSVFGNCLTTLP VSYSYTYNYEW

HFDFWGQGLLVTVSS

The knob domain of this sequence may therefore be defined as the following sequence (SEQ ID NO: 5): SCPDGYRERSDCSNRPACGTSDCCRVSVFGNCL

(i.e. from one residue preceding the first cysteine to the residue subsequent to the last cysteine residue of the ultralong CDR-H3; cysteine residues in bold):

Another example is provided below with the K149 ultralong CDR-H3 (SEQ ID NO:1 of the present patent application):

TSEL2S7XP2FSCPDGFSYRSWDDFCCPMVGRCK4P TT7TEF77EA

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.

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, the knob domain or antigen-binding portion thereof comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen cysteine residues. In one embodiment, the knob domain or antigen-binding portion thereof comprises between two cysteine residues and ten cysteine residues. In one embodiment, the knob domain or antigen-binding portion thereof comprises between four cysteine residues and eight cysteine residues.

Two cysteine residues may bridge together to form a disulphide bond within the knob domain or antigen-binding portion thereof. In one embodiment, 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. In one embodiment, a knob domain or antigen-binding portion thereof comprises one, two, three, four, five, six, or seven disulphide bonds. In one embodiment, a knob domain or antigen-binding portion thereof comprises between one disulphide bond and five disulphide bonds. In one embodiment, a knob domain, or antigen-binding portion thereof, comprises between two disulphide bonds and four disulphide bonds.

It will be appreciated that an increased content in cysteine residues will increase the possibility to form disulphide bonds within the knob domain or antigen-binding portion thereof. Such disulphide bonds contribute to form a loop motif, which may be advantageous to increase the stability, and/or rigidity and/or binding specificity and/or binding affinity of the knob domain or antigen-binding portion thereof.

In one embodiment, a knob domain, or antigen-binding portion thereof, present in an antigenbinding protein comprises a (Zi) Xi C X₂ 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. X₂ 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. In one embodiment, Zi is 1 amino acid. In another embodiment, Zi is 2 amino acids, which may be the same or different. In another embodiment, Zi is 3 amino acids, which may be the same or different. In another embodiment, Zi is 4 amino acids, which may be the same or different. In another embodiment, Zi is 5 amino acids, which may be the same or different.

In one embodiment, Xi is selected from the list consisting of Serine, Threonine, Asparagine, Alanine, Glycine, Proline, Histidine, Lysine, Valine, Arginine, Isoleucine, Leucine, Phenylalanine and Aspartic acid. Thus, in one aspect, a knob domain or antigen-binding portion thereof present in an antigen-binding protein of the present invention comprises a (Zi) Xi C X₂ 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. X₂ is an amino acid selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine.

In one embodiment, a knob domain, or antigen-binding portion thereof, present in an antigenbinding protein of the present invention comprises a (Zi)Xi C X₂ motif at its N-terminal extremity, wherein C is cysteine; and Xi is selected in the list consisting of Serine (S), Threonine (T), Asparagine (N), Alanine (A), Glycine (G), Proline (P), Histidine (H), Lysine (K), Valine (V), Arginine (R), Isoleucine (I), Leucine (L), Phenylalanine (F) and Aspartic acid (D), and X₂ is selected from the list consisting of Proline (P), Arginine (R), Histidine (H), Lysine (K), Glycine (G) and Serine (S), and 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.

In one embodiment, the N-terminal extremity of a knob domain or antigen-binding portion thereof present in an antigen-binding protein of the present invention comprises a motif which comprises 3 amino acid residues, corresponding to a XiC X₂ motif, selected in the list consisting of SCP, TCP, NCP, ACP, GCP, PCR, HCP, SCR, KCP, VCP, TCH, RCP, ICP, ICR, HCR, LCR, SCK, SCG, NCP, TCS, DCP and FCR.

Preferably, 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.

In one embodiment, the knob domain or antigen-binding portion thereof present in an antigenbinding protein of the present invention comprises a (AB)n and/or (BA)n motif, wherein A is any amino acid residue, B is an aromatic amino acid selected from the group consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and wherein n is 1, 2, 3 or 4.

In one embodiment, A is an aliphatic amino acid residue. An aliphatic amino acid is an amino acid containing an aliphatic side chain functional group. Aliphatic amino acid residues include Alanine, isoleucine, leucine, proline, and valine.

In one embodiment, 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. In one embodiment, 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).

In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, rather than comprise a full knob domain, 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. In one embodiment, 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. In one embodiment, 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.

In one embodiment, 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.

In one aspect, 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):

(Zi) (Xl) C X₂ (Y)ni (C)n2 (Y)n3 (C)_n4 (Y)_n5 (C)n6 (Y)_n7 (C)n8 (Y)n9 (C)_n10 (Y)_nll (C)_n12 (Y)_n13 (C)nl4 (Y)nl5 (C)_n16 (Y)_n17 C (X₃) (Z₂) (I) wherein:

C represents one cysteine residue; and,

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,

Xi is present or absent, and when Xi is present, 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,

X₂ is selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine; and, Z₂ is present or absent, and when Z₂ is present, Z₂ represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and, n2, n4, n6, n8, nlO, nl2, nl4 and nl6 are independently 0 or 1; and, Y represents any amino acid or any sequence of amino acids that may be the same or different; and, nl, n3, n5, n7, n9, nl 1, n!3, n!5 and n!7 represent the number of amino acids in Y, and are independently selected from 0 to 22, preferably from 1 to 15; and, at least one of nl, n3, n5, n7, n9, nil, nl3, nl5 and nl7 is not equal to 0; and,

X₃ is present or absent, and when X₃ is present, X₃ represents any amino acid, preferably selected from the list consisting of Leucine, Serine, Glycine, Threonine, Tryptophan, Asparagine, Tyrosine, Arginine, Isoleucine, aspartic acid, Histidine, Glutamic acid, Valine, Lysine, Proline; and, wherein the peptide is up to 55 amino acids in length.

Zi represents any amino acid or any sequence of 2, 3, 4, or 5 independently selected amino acids that may be the same or different. In one embodiment, Zi is 1 amino acid. In another embodiment, Zi is 2 amino acids, which may be the same or different. In another embodiment, Zi is 3 amino acids, which may be the same or different. In another embodiment, Zi is 4 amino acids, which may be the same or different. In another embodiment, Zi is 5 amino acids, which may be the same or different.

Z₂ represents any amino acid or any sequence of 2, 3, 4, or 5 independently selected amino acids that may be the same or different. In one embodiment, Z₂ is 1 amino acid. In another embodiment, Z₂ is 2 amino acids, which may be the same or different. In another embodiment, Z₂ is 3 amino acids, which may be the same or different. In another embodiment, Z₂ is 4 amino acids, which may be the same or different. In another embodiment, Z₂ is 5 amino acids, which may be the same or different.

Zi and Z₂ may comprise any amino acid as long as the properties of the peptide otherwise defined is retained, e.g. binding capability to an antigen of interest.

In one embodiment, the knob domain or antigen-binding potion thereof comprises or consists of the sequence of formula (I) 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 peptide which binds an antigen of interest comprising or consisting of the sequence of formula (I) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

Brackets are generally used for optional residues or sequences. For example, (C) generally indicates an optional Cysteine residue, in the context of the present disclosure.

In one embodiment, 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):

(Zi) (Xi) C X₂ (Y)m C (Xs) (Z₂) (II) wherein Zi, Xi, C, X₂, Y, _ni, X₃, and Z₂ are defined as above, and wherein the peptide is up to 55 amino acids in length. In such embodiment, may be comprised between 1 and 20 amino acids. In one embodiment, nl is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

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 (II) 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 peptide which binds an antigen of interest comprising or consisting of the sequence of formula (II) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

In another embodiment, the knob domain, or antigen-binding portion thereof comprises 4 Cysteine residues. Therefore, in one particular aspect, the knob domain, or antigen-binding portion thereof comprises or consisting of the sequence of formula (III) :

(Zi) (Xi) C X₂ (Y)m C (Y)n3 C (Y)n5 C (Xs) (Z₂) (III) wherein Zi, Xi, C, X₂, Y, _ni, n3, ns, X₃, and Z₂ are defined as above, and wherein the knob domain or antigen-binding portion thereof is up to 55 amino acids in length.

In one embodiment, nl is comprised between 3 and 15 and/or n3 is comprised between 4 and 12 and/or n5 is comprised between 1 and 14. In one embodiment, nl is 3, 5, 7, 8, 10, 11, 14, or 15. In one embodiment, n3 is 4, 5, 6, 8, 10, 11 or 12. In one embodiment, n5 is 3, 4, 5, 6, 7, 9, 10, 11, or 14.

In another embodiment, nl and/or n3 and/or n5 is equal to 0 and two or three Cysteine residues are contiguous.

In one embodiment, the knob domain or antigen-binding portion thereof has the sequence of formula (Illa):

(Zi) (Xi) C X₂ C C (Y)n5 C (Xs) (Za) (Illa) wherein Zi, Xi, C, X₂, Y, _ns, X₃, and Z₂ are defined as above and wherein the peptide is up to 55 amino acids in length.

In one embodiment, the peptide has the sequence of formula (Illb):

(Zi) (Xi) C X₂ (Y)m C C (Y)n5 C (Xs) (Z₂) (Illb) wherein Zi, Xi, C, X₂, Y, ni.n5.X3, and Z₂ are defined as above and wherein the peptide is up to 55 amino acids in length.

In one embodiment, the peptide has the sequence of formula (IIIc):

(Zi) (Xi) C X₂ (Y)nl C (Y)n3 C C (Xs) (Z2) (IIIc) wherein Zi, Xi, C, X₂, Y, _ni, n3, X₃, and Z₂ are defined as above and wherein the peptide is up to 55 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 (III) , (Illa), (Illb), or (IIIc) 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 peptide which binds an antigen of interest comprising or consisting of the sequence of formula (III) , (Illa), (IHb), or (IIIc) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

In another embodiment, the knob domain or antigen-binding portion thereof comprises 6 Cysteine residues. Therefore, in one particular aspect, the knob domain or antigen-binding portion thereof which binds an antigen of interest comprises or consists of the sequence of formula (IV): (Zi) (Xi) C X₂ (Y)m C (Y)n3 C (Y)n5 C (Y)_n7 C (Y)_n9 C (Xs) (Z2) (IV) wherein Zi, Xi, C, X₂, Y, , n₃, n₅, n₇, n₉, X₃, and Z₂ are defined as above, and wherein the peptide is up to 55 amino acids in length.

In one embodiment, ni=2 to 9 and/or n₃=l to 10 and/or n₅= 2 to 9 and/or n₇= 1 to 15 and/or n₉= 1 to 14. In one embodiment, m=2, 3, 4, 5, 6, 7, 8 or 9. In one embodiment, n₃ =1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, m=2, 3, 4, 5, 6, 7, 8, or 9. In one embodiment, n₇=l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In one embodiment, n₉=l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

In one embodiment, ni=0 and/or n₃ =0 and/or n₅=0 and/or n₇ =0 and/or n₉=0 and two or three Cysteine residues are contiguous. In one embodiment, the knob domain or antigen-binding portion thereof has the sequence of formula (IVa):

(Zi) (Xi) C X₂ C C (Y)n5 C (Y)_n7 C (Y)^ C (Xs) (Z₂) (IVa) wherein Zi, Xi, C, X₂, Y, n₅, n₇, n₉, X₃, and Z₂ are defined as above, and wherein the knob domain or antigen-binding portion thereof is up to 55 amino acids in length.

In one embodiment, the peptide has the sequence of formula (IVb):

(Z (Xi) C X₂ (Y)m C C (Y)n5 C (Y)_n7 C (Y)_n9 C (Xs) (Z₂) (IVb) wherein Zi, Xi, C, X₂, Y, , n₅, n₇, n₉, X₃, and Z₂ are defined as above, and wherein the knob domain or antigen-binding portion thereof is up to 55 amino acids in length.

In one embodiment, the peptide has the sequence of formula (IVc):

(Zi) (Xi) C X₂ (Y)m C (Y)_n3 C C (Y)_n7 C (Y)_n9 C (X₃) (Z₂) (IVc) wherein Zi, Xi, C, X₂, Y, , n₃, n₇, n₉, X₃, and Z₂ are defined as above, and wherein the knob domain or antigen-binding portion thereof is up to 55 amino acids in length.

In one embodiment, the peptide has the sequence of formula (IVd):

(Zi) (Xi) C X₂ (Y)m C (Y)_n3 C (Y)n5 C C (Y)_n9 C (X₃) (Z₂) (IVd) wherein Zi, Xi, C, X₂, Y, , n₃, n₅, n₉, X₃, and Z₂ are defined as above, and wherein the knob domain or antigen-binding portion thereof is up to 55 amino acids in length.

In one embodiment, the peptide has the sequence of formula (IVe):

(Zi) (Xi) C X₂ (Y)m C (Y)„3 C (Y)n5 C (Y)_n7 C C (Xs) (Z₂) (IVe) wherein Zi, Xi, C, X₂, Y, ui. n₃, n₅, n₇, X₃, and Z₂ are defined as above, and wherein the knob domain or antigen-binding portion thereof is up to 55 amino acids in length.

In one embodiment, the peptide has the sequence of formula (IVf): (Zi) (Xi) C X₂ (Y)m C C C (Y)n7 C (Y)n9 C (Xs) (Z2) (IVf) wherein Zi, Xi, C, X₂, Y, n 1. n₇, n₉, X₃, and Z₂ are defined as above, and wherein the peptide is up to 55 amino acids in length.

In one embodiment, the peptide has the sequence of formula (IVg):

(Zi) (Xi) C X₂ (Y)m C (Y)n3 C C C (Y)„9 C (XS) (Z2) (IVg) wherein Zi, Xi, C, X₂, Y, m. n₃, n₉, X₃, and Z₂ are defined as above, and wherein the peptide is up to 55 amino acids in length.

In one embodiment, the knob domain or antigen-binding portion thereof has the sequence of formula (IVh):

(Zi) (Xi) C X₂ (Y)m C (Y)_n3 C (Y)n5 C C C (Xs) (Z2) (IVh) wherein Zi, Xi, C, X₂, Y, m. n₃, n₅, X₃, and Z₂ are defined as above, and wherein the peptide is up to 55 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 (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. 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 (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.

In another embodiment, 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):

(Z (Xi) C X₂ (Y)m C (Y)_n3 C (Y)n5 C (Y)_n7 C (Y)_n9 C (Y)_nn C (Y)m₃ C (Xs) (Z₂) (V) wherein Zi, Xi, C, X₂, Y, , n₃, n₅, n₇, n₉, nn, _3> X₃, and Z₂ are defined as above, and wherein the peptide is up to 55 amino acids in length.

In one embodiment, =0 and/or n₃=0 and/or n₅=0 and/or n₇=0 and/or n₉=0 and/or nn=0, and/or ₃=0 and two or three Cysteine residues are contiguous.

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 (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. In one embodiment, 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. In another embodiment, 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):

(Zi) (Xi) C X₂ (Y)m C (Y)n3 C (Y)n5 C (Y)_n7 C (Y)„9 C (Y)nll C (Y)nl3 C (Y)_n15 C (Y)m₇ C

(Xs) (Z₂) (VI) wherein Zi, Xi, C, X₂, Y, , n₃, n₅, n₇, n₉,nn, ni₃, ni5,ni_7>X₃, and Z₂ are defined as above, and wherein the peptide is up to 55 amino acids in length.

In one embodiment, ni=0 and/or n₃=0 and/or n₅=0 and/or n₇=0 and/or n₉=0 and/or nn=0, and/or ni₃=0 and/or ni₅=0 and/or ₇=0 and two or three Cysteine residues are contiguous.

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 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.

In one embodiment, rather than a full-length knob domain an antigen-binding region will comprise, or consist of, an antigen-binding portion of a knob domain. Variant knob domains and antigen-binding portions thereof may also be employed in antigen-binding proteins of the present invention. For example, knob domains, or antigen-binding portions thereof, of bovine ultralong CDR- H3 or portions thereof, which comprise sequences which are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similar or identical to a specific knob domain sequence given herein may be employed in antigen-binding proteins of the present invention.

In one embodiment, knob domains, antigen-binding portions thereof, or the overall antigenbinding proteins are processed to provide variants with improved affinity for a target antigen or antigens. Such variants can be obtained by a number of affinity maturation protocols including chain shuffling, use of mutator strains of E. coli, DNA shuffling, phage display and sexual PCR. Vaughan et al (Nature Biotechnology, 16, 535-539, 1998) discusses these methods of affinity maturation. Another method useful in the context of the present invention to improve binding of an antigen-binding protein to antigen is a method as described in WO 2014/198951. Improved affinity as employed herein in this context refers to an improvement over the starting knob domain, antigen-binding portion thereof, or overall antigen-binding protein. Affinity can be measured as described herein.

In one embodiment, a variant knob domain, antigen-binding portion thereof, or overall antigenbinding protein is a variant which has an affinity which is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the affinity of the original knob domain, antigen-binding portion thereof, or overall original antigen-binding protein as measured, for example, by Biacore. In one embodiment, a variant or portion of a knob domain, or antigen-binding portion thereof, may have at least an increase in affinity of such percentage amounts compared to the original binding domain.

“Truncated variants" are those with one or more amino acids in the native or starting amino acid sequence removed from either terminus of the polypeptide.

In some embodiments, the knob domain, or antigen-binding portion thereof, is a variant which has been engineered to comprise a disulfide bond which is in a non-naturally occurring position. This may be engineered into the molecule by introducing cysteine(s) into the amino acid chain at the position or positions required. This non-natural disulfide bond is in addition to or as an alternative to the natural disulfide bond(s) which may be present in the parental knob domain or antigen-binding portion thereof. The cysteine(s) in natural positions can be replaced by an amino acid such as serine which is incapable of forming a disulfide bridge. Introduction of engineered cysteines can be performed using any method known in the art. These methods include, but are not limited to, PCR extension overlap mutagenesis, site-directed mutagenesis or cassette mutagenesis (see, generally, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-Interscience, NY, 1993). Site-directed mutagenesis kits are commercially available, e.g. QuikChange® Site-Directed Mutagenesis kit (e.g. Stratagene, La Jolla, CA). Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be made by total gene synthesis by annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.

In one aspect, it may be useful to decrease or remove the cysteine residues and/or disulfide bonds in a knob domain, antigen-binding portion thereof, or overall antigen-binding region, e.g. to lower the risk of immunogenicity, i.e. of side reactions occurring during or after the administration to a patient. In such aspect, one or all of the cysteine(s) in natural positions can be replaced by an amino acid such as serine which is incapable on forming a disulfide bridge. It will be appreciated that alternative bridging moieties may be used to stabilise and/or form a cyclised knob domain, or antigenbinding portion thereof, in the absence of cysteine residues. In one embodiment, a variant knob domain, or antigen-binding portion thereof, has been engineered to remove the cysteine residues and which comprises at least one bridging moiety as defined herein. In one embodiment, the knob domain, or antigen-binding portion thereof, is a variant which has been engineered to contain only one, or only two, or only three, or only four, cysteine residues, and/or to contain only one or only two disulphide bonds and which optionally further comprises at least one bridging moiety as defined herein.

Additional modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of tyrosinyl, seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (Creighton, T. E., Proteins: Structure and Molecular Properties, W.H. Freeman and Co., San Francisco, 1983, pp. 79-86).

The knob domain, or antigen-binding portion thereof, may be cyclised. Cyclisation may be advantageous to confer more resistance to proteolysis, resulting notably in an improved stability. Therefore, in one embodiment, the knob domain or antigen-binding portion thereof further comprises a bridging moiety between two amino acids.

Cyclised knob domains, and antigen-binding portions thereof, include those that have as part of their structure one or more cyclic features such as a loop, bridging moiety, and/or an internal linkage. As used herein, the term "bridging moiety" refers to one or more components of a bridge formed between two adjacent or non-adjacent amino acids in knob domain, or antigen-binding portion thereof. Bridging moieties may be of any size or composition.

In one embodiment, a bridging moiety may be between the amino acid residue in N-terminal position and the amino acid residue in C-terminal position such as to create a head-to-tail cyclisation. In one embodiment, a bridging moiety may be between amino acids which are not in terminal position.

In one embodiment, the knob domain, or antigen-binding portion thereof, comprises only one bridging moiety between two amino acids. In another embodiment, the knob domain, or antigen-binding portion thereof, comprises more than one bridging moiety between two amino acids, e.g. two, or three, or five bridging moieties, each one being between two amino acids.

In one embodiment, the bridging moiety comprises a disulphide bond. In one embodiment, the disulphide bond is formed between two naturally occurring cysteine residues. In another embodiment, the disulphide bond is formed between cysteine residues, with at least one cysteine residue being engineered, as described above.

In one embodiment, the knob domain or antigen-binding portion thereof is fully bovine. In such embodiment, each and every residue is derived from a bovine germline sequence. In some embodiments, each and every residue is derived from a bovine germline sequence which can have undergone affinity maturation for an antigen.

In one embodiment, the knob domain or antigen-binding portion thereof employed in the invention is chimeric.

The term "chimeric" refers to a knob domain or antigen-binding portion thereof comprising at least two portions, one being derived from a particular source or species, such as bovine, while the other portion is derived from a different source or species, such as human. In one embodiment, the antibody fragment is human/bovine chimeric. In one embodiment, the antibody fragment comprises at least one residue derived from a human sequence.

An antigen-binding protein of the present invention comprises at least one knob domain or antigen-binding portion thereof that binds to an antigen of interest and preferably binds specifically to an antigen of interest. “Specifically,” as employed herein is intended to refer to a knob domain, or antigen-binding portion thereof, that only recognises the antigen to which it is specific or that has significantly higher binding affinity to the antigen to which is specific compared to affinity to antigens to which it is non-specific, for example 5, 6, 7, 8, 9, 10 times higher binding affinity. Preferably, a knob domain, or antigen-binding portion thereof, has a specific binding affinity (as measured by its dissociation constant K_D) for its cognate antigen of 10'⁵ M or less, 10'⁶ M or less, 10'⁷ M or less, 10'⁸ M or less, 10'⁹ M or less, IO ¹⁰ M or less, or 10¹¹ M or less. In one embodiment, it has a specific binding affinity (as measured by its dissociation constant K_D) for its cognate antigen between 1. 10'⁷ M and 1. 10'⁸ M, or between 1. 10'⁸ M and 1. 10'⁹ M, or between 1. 10'⁹ M and 1. IO ¹⁰ M. In one embodiment, the antigen-binding protein as a whole may have such affinity.

In one particularly preferred embodiment, a knob domain, or antigen-binding portion thereof fused at its N-terminus, C-terminus, or at both termini, to a helical peptide has a higher specific binding affinity (as measured by its dissociation constant K_D) for its cognate antigen) than in comparison to the knob domain or antigen-portion thereof on their own. In one embodiment, the comparison will be performed between an antigen-binding protein of the present invention comprising a single knob domain, or antigen-binding portion thereof, versus the knob domain or antigen-binding portion thereof on their own without other sequences. In another embodiment, the knob domain, or antigen-binding portion thereof, fused to a helical peptide or peptide as set out therein has at least the same affinity as the knob domain or antigen-binding portion thereof on its own.

Affinity can be measured by known techniques such as surface plasmon resonance techniques including Biacore™. Affinity may be measured at room temperature, 25°C or 37°C. Affinity may be measured at physiological pH, i.e. at about pH 7.4. In one embodiment, the affinity values as described above are measured using Biacore, notably Biacore 8K, at pH 7.4. In one embodiment, the affinity of a knob domain (or antigen-binding portion thereof), the knob domain (or antigen-binding portion thereof) fused to a helical peptide or peptides, or the overall antigen-binding protein may be measured using the Biacore method set out in the Examples herein. Biacore may also be used to determine the ability of an overall antigen-binding protein to bind more than one antigen at the same time, whether that be two different antigens, two molecules of the same antigen, or two different epitopes of the same antigen. In one preferred embodiment, such measurement may be performed using a method set out in the Examples.

It will be appreciated that the affinity of antigen-binding proteins provided by the present invention may be altered using any suitable method known in the art, such a mutagenesis, for instance to increase affinity.

Linkers

In one embodiment, one or more linker may be used to join together different regions of an antigen-binding protein of the present invention. Hence, in one embodiment, an antigen-binding protein may comprise one or more linker, particularly one or more peptide linker, for example one or more of the specific peptide linkers set out herein. Linkers may be employed to fuse a knob domain, or antigenbinding portion thereof to a helical peptide. A knob domain, or antigen-binding portion thereof, optionally fused at its N-terminus, C-terminus, or both termini, to a helical peptide may be thought of as a unit and linkers may also be used to joined together two such units in embodiments where an antigen-binding protein comprises a plurality of knob domains, or antigen-binding portions thereof. In one embodiment, the shorter linkers described herein are preferred for fusing a knob domain, or an antigen-binding portion thereof, to a helical peptide, whereas the longer linkers are preferred for joining together units of a knob domain, or antigen-binding portion thereof, and fused helical peptides in antigen-binding proteins with a plurality of knob domains, or antigen-binding portions thereof.

In one embodiment, a knob domain, or antigen-binding portion thereof, may be fused to a helical peptide via a linker. In one preferred embodiment, an antigen-binding protein may comprise in N-terminal to C-terminal order: a first helical peptide; a first linker; a knob domain or antigen-binding portion thereof; a second linker; and a second helical peptide, where each is fused to the next in the linear amino acid sequence of the polypeptide. In an alternative embodiment, the antigen-binding protein may comprise in N-terminal to C-terminal order: a first helical peptide; a knob domain or antigen-binding portion thereof; and a second helical peptide, where each is fused to the next in the linear amino acid sequence of the polypeptide.

A preferred linker for employing in the present invention is a peptide linker comprised of amino acids, though also non-peptide linkers may also be employed. A range of suitable linkers will be known to the person of skill in the art. In one embodiment, the linker is a short linker that limits flexibility. Such short linkers are particularly preferred for fusing a knob domain, or antigen-binding portion thereof to a helical peptide or peptides. In one embodiment, a linker consists of two to three amino acids. In one preferred embodiment, a linker consists of two amino acids. Such short linkers are particularly preferred for fusing a knob domain, or antigen-binding portion thereof, to a helical peptide. In one embodiment, the linker has a sequence up to 5 amino acids in length and is comprised of Glycine and Serine residues. In one embodiment, the linker has a sequence selected from the list of GS, GG, SG, GGS, GSG, GGG. In one embodiment, the linker has the sequence GSG. One especially preferred linker for fusing a knob domain, or antigen-binding portion thereof, to a helical peptide is a GS linker. In one preferred embodiment, a knob domain, or antigen-binding portion thereof, is fused at its N- terminus to a helical peptide via a GS linker. In one preferred embodiment, a knob domain, or antigenbinding portion thereof, is fused at its C terminus to a helical peptide via a GS linker. In one particularly preferred embodiment, a knob domain, or antigen-binding portion thereof, is fused at its N-terminus to a helical peptide via a GS linker and also at its C-terminus via a GS linker.

In one embodiment, longer linkers may be employed. Longer linkers are particularly preferred for joining together units of knob domains, or antigen-binding portions thereof, fused to helical peptides, in antigen-binding proteins with more than one knob domain, or antigen-binding portion thereof. Using a flexible linker helps the different knob domains, or antigen-binding portions thereof in binding antigen, particularly as the same time. In one embodiment, a linker is selected from a sequence comprised in the list consisting of SEQ ID NO: 6 to SEQ ID NO: 74 which represent longer linkers preferred for joining together different knob domain, or antigen-binding portion thereof, units. Table 1. Flexible linker sequences

(S) is optional in sequences 8 and 12 to 16.

Table 2. Hinge linker sequences

Examples of rigid linkers include the peptide sequences GAPAPAAPAPA (SEQ ID NO:59),

PPPP (SEQ ID NO: 60) and PPP.

In one embodiment a peptide linker is an albumin binding peptide.

Examples of albumin binding peptides are provided in W02007/106120 and include: Table 3. Albumin binding peptides

As well as peptide linkers other formers of linkers may also be used in fusing a knob domain, or antigen-binding portion thereof, at its N-terminus, C-terminus, or both termini to a helical peptide. Such non-peptide linkers may also be used to join together different units of knob domain, or antigenbinding portion thereof, with fused helical peptide(s) in the generation of antigen-binding proteins with more than one knob domain, or antigen-binding portion thereof. An example of one suitable linker is an ADH (Adipic Acid dihydrazide) linker.

Specific knob domains

Illustrative specific knob domain sequences are described in Table 5 of Example 1. Further illustrative specific knob domain sequences are set out in Table 4 below, with the knob domains being specific for human complement component C5. The specific knob domain sequences set out in Tables 4 and 5, and antigen binding portions thereof, may be employed in antigen-binding proteins, as may variants of either. However, the present invention is not limited to a specific knob domain, or antigen-binding portion thereof, sequence and the approach described herein may be applied to any knob domain or antigen-binding portion thereof. Table 7

Variants and portions

As well as the specific antigen-binding proteins (or antigen-binding portions thereof), knob domains, helical peptides, and linkers set out herein variants and portions of them may be employed, provided that they retain the ability to perform the necessary function.

Hence, in one embodiment rather than one of the specific sequences set out herein, a variant with a particular level of sequence identity or similarity may be employed. In one embodiment, the level of amino acid sequence identity or similarity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one preferred embodiment, the sequence may have such a level of sequence identity or similarity and also retain a desired function. For example, a knob domain or antigen-binding portion thereof may retain such a level of sequence identity or similarity to the specific sequence set out herein and retain the ability to bind the same antigen. A helical peptide may have such a level of sequence identity or similarity to the specific sequence and retain a function such as improving production compared to that of the knob domain, or antigen-binding portion thereof, on its own. In another embodiment, the overall antigen-binding protein may have such a level of sequence identity or similarity to the specific one set out herein.

“Identity", as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity", as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids which can often be substituted for one another include but are not limited to:

- phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains);

- lysine, arginine and histidine (amino acids having basic side chains);

- aspartate and glutamate (amino acids having acidic side chains);

- asparagine and glutamine (amino acids having amide side chains); and

- cysteine and methionine (amino acids having sulphur-containing side chains).

Degrees of identity and similarity can be readily calculated by methods well known, for example the BLAST™ software available from NCBI.

It may be in another embodiment, that a variant has a maximum number of sequence changes compared to the specific sequence. For instance, it may be that a variant has up to 10 amino acid sequence changes. In another embodiment, it may have from 1 to 10 sequence changes. In another embodiment, it may have from 1 to 7 amino acid sequence changes. In another embodiment, it may have from 1 to 5 amino acid sequence changes. In one preferred embodiment, it may have one, two, or three amino acid sequence changes compared to the specific sequence. In one preferred embodiment it may have only one or two amino acid sequence changes. In another preferred embodiment, it may have only a single amino acid sequence change. In one embodiment, sequence changes may be selected from insertions, deletions and substitutions. In a preferred embodiment, the sequence change(s) may be conservative amino acid sequence changes.

Antigens of interest

Antigens of interest recognised by antigen-binding proteins of the present invention may be, for instance, any medically relevant protein such as those proteins upregulated during disease or infection, for example receptors and/or their corresponding ligands. Particular examples of antigens include cell surface receptors such as T cell or B cell signalling receptors, co-stimulatory molecules, checkpoint inhibitors, natural killer cell receptors, Immunoglobulin receptors, TNFR family receptors, B7 family receptors, adhesion molecules, integrins, cytokine/chemokine receptors, GPCRs, growth factor receptors, kinase receptors, tissue-specific antigens, cancer antigens, pathogen recognition receptors, complement receptors, hormone receptors or soluble molecules such as cytokines, chemokines, leukotrienes, growth factors, hormones or enzymes or ion channels, epitopes, fragments and post translationally modified forms thereof.

In one embodiment, the antigen of interest is a mammalian antigen. In one embodiment, the antigen is a human antigen.

In one embodiment, the antigen of interest is a cancer antigen. In another embodiment, the antigen is an autoimmune antigen.

In one embodiment, the antigen of interest is a bacterial antigen.

In one embodiment, the antigen of interest is a viral antigen. In one embodiment, the antigen of interest is a fungal antigen.

In one embodiment, the antigen of interest is not a bovine leukaemia virus antigen. In one embodiment, the antigen of interest is not that bound by the BVL12 antibody.

Effectors

In some embodiments, an antigen-binding protein of the present invention also comprises an effector. In one embodiment, the effector forms part of the same polypeptide as that comprising a knob domain, or antigen-binding portion thereof, and fused helical peptide(s). In one embodiment, the effector may be cleavable from the rest of the antigen-binding protein. In one embodiment, the effector region may be at the N terminus of the overall polypeptide. In another embodiment, the effector region may be at the C terminus of the overall polypeptide. In an alternative embodiment, rather than be part of the same polypeptide sequence an effector may be a conjugate and so be conjugated to them.

The term “effector molecule” as used herein includes, for example, biologically active proteins, for example enzymes, polypeptides, peptides, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.

Particular radioisotopes of interest are alpha emitting radioisotopes, in particular short-lived alpha-emitting isotopes such as Astatine isotopes. In one embodiment, 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.

Other effector molecules may include detectable substances useful for example in diagnosis. Examples of 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. In another embodiment 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. Examples of 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. In one embodiment, the effector molecule is palmitic acid. Palmitic acid has the advantageous property to bind albumin and improve interaction with cells. In one embodiment, the effector molecule is an activated form of palmitic acid such as palmitoyl.

Where 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.

Specific optional substituents which may be present on the above-mentioned synthetic polymers include one or more hydroxy, methyl or methoxy groups.

Specific examples of 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.

In one embodiment antigen-binding proteins for use in the present invention are attached to poly(ethyleneglycol) (PEG) moieties. In one particular example, 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. Suitably PEG molecules are covalently linked through a thiol group of at least one cysteine residue located in the antigen-binding protein. In one embodiment, 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.

As used herein, a "conjugate" refers to any molecule or moiety appended to another molecule. In the present invention, 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, sulfones, triazoles, tropanes, and vinca alkaloids. In one embodiment, the conjugation process involves palmitoylation. Palmitoylation can be employed to improve the pharmacokinetics of an antigen-binding protein of the invention.

In one embodiment, 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. In one embodiment, 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. In one embodiment, the antigen-binding protein is inserted into albumin, optionally via one or more, for example two, linker(s). For example, 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. In one embodiment, the linker or at least one of the linkers is SGGGS (SEQ ID NO: 7). Bispecific and multi-specific antigen binding proteins

In one preferred embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, an antigen-binding protein of the present invention may be biparatopic, that is it binds two different epitopes of the same antigen. In one embodiment, an antigen-binding protein of the present invention may be multi-paratopic, that is it binds multiple different epitopes of the same antigen. In one embodiment, 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. In another embodiment, it is bivalent for a given antigen, that is it has two binding sites specific for a given antigen. In another embodiment, an antigen-binding protein will be multi-valent, that is have multiple valencies for a given antigen.

In one aspect, 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. For brevity, 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. In one embodiment, 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.

In one embodiment, 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. In one embodiment, 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.

In one aspect, 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. In one embodiment, each of the three binds to a different epitope of the same antigen. In another embodiment, each bind to the same epitope of the same antigen. In another embodiment, each bind to a different antigen, i.e. bind to three different and distinct antigens. In one embodiment, 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.

In one embodiment, the antigen-binding proteins of the present invention may be monospecific, bispecific or multi-valent. In one particularly preferred embodiment, they are bispecific.

The “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. In one embodiment, 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. In one embodiment, 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. In one embodiment, the antigenbinding protein is a trivalent bispecific antigen-binding protein.

“Biparatopic” as used herein 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” as employed herein 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).

In one embodiment, 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.

Illustrative examples of preferred formats for antigen-binding proteins with more than one knob domain, or antigen-binding portion thereof, are shown in Figure 7 of the present application. For example, 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. In one preferred embodiment, a GSG linker is employed to joined together the units. In another preferred embodiment, the two units are joined together via a G4P linker. In a further preferred embodiment, the two units are joined together by a G4P linker. In one embodiment, a longer linker is employed, for example the 127 amino acid sequence linker used in Figure 7D is employed.

In one embodiment, as well as 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.

In one embodiment, a linker may be used to join together units of knob domain (or antigenbinding portions thereol) and fused helical peptide(s).

In one aspect, the polypeptide comprising at least two knob domains, or antigen-binding portions thereof, and fused helical peptide(s) units is cyclised. In some embodiments, the polypeptide comprises at least one bridging moiety between two amino acids.

When the polypeptide is cyclic and does not have end-amino acids, it may be referred to as a macrocycle.

The definitions of bridging moiety described above in connection with cyclised antibody fragments also apply to the cyclised polypeptides of the present disclosure. In particular, in one embodiment, the bridging moiety may be a disulphide bond. In one preferred embodiment, 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.

In one preferred embodiment, an antigen-binding protein of the present invention shows thermal stability. For example, in one preferred embodiment 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.

In another embodiment, the antigen-binding protein provided does not have an antigen-binding site specific for bovine leukaemia virus. In one embodiment, an antigen-binding protein of the invention does not comprise a Fab region. In one embodiment, 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. In one preferred embodiment, 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. In one preferred embodiment, an antigen-binding protein of the present invention does not comprise an antibody constant region.

In one preferred embodiment, 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. In one embodiment, 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.

Methods of production

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.

It will be appreciated that in the context of a 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. Alternatively, 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. Hence, 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.

Also provided is 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.

In one aspect, there is provided a process for producing an antigen-binding protein of the present invention, said process comprising expressing such a protein from a host cell of the present invention. Preferably, the method further comprises recovering the protein. The method may comprise cleavage and/or purification steps. In one embodiment, the method may further comprise formulating the antigen-binding protein into a pharmaceutical composition.

Knob domain generation

The 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.

Illustrative methods are described in more detail below. Step a) immunising a bovine with an immunogenic composition

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.

In one embodiment, 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. In one embodiment, 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.

By “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.

In one embodiment, 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. In one embodiment, 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.

In one embodiment, the immunogenic composition is administered by sub-cutaneous injection, for example into the shoulder.

In one embodiment, the antigen of interest is the component C5 of the Complement. In another embodiment, the antigen is IL-2 or a portion thereof. Peptides representing particular epitopes may be employed.

“Adjuvant” refers to an immune stimulator. Adjuvants are substances well known in the art. Traditional adjuvants, which act as immune stimulators or antigen delivery systems, or both, encompass, for example, Alum, polysaccharides, liposomes, nanoparticles based on biodegradable polymers, lipopolysaccharides. For example, 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

Methods for isolating total RNA from PBMC or secondary lymphoid organ are well known in the art.

Step c) amplifying the cDNA of the ultralong CDR-H3

It will be appreciated that step c) generally comprises a first step of obtaining cDNA from the total RNA obtained at step b), using RT-PCR. Advantageously, at step c) 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.

In one embodiment, the method for amplifying the cDNA of CDR-H3 comprises:

1) a primary PCR using primers flanking CDR-H3, annealing to the conserved framework 3 and framework 4 of the VH, to amplify all CDR-H3 sequences, and

2) a second round of PCR using stalk primers to specifically amplify ultralong sequences from the primary PCR.

In one embodiment, the primers used at step 1) comprise or consist of SEQ ID NO: 154 and SEQ ID NO: 311. In one embodiment, 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.

Optional screening step

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.

Alternatively, the 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.

In one embodiment, the carrier is an Fc polypeptide. An “Fc polypeptide” as used herein is a polypeptide comprising a Fc fragment. In one embodiment, 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.

In one embodiment, 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.

Additional scFc sequences and variants useful in the context of the present disclosure have been described in W02008/012543. Although 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.

Additionally, 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.

In one embodiment, 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. A phage display library of knob domains, or antigenbinding portions thereof, optionally displayed within the full sequences of CDR-H3. In one embodiment, 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. In one embodiment, the knob domains or antigen-binding portions thereof, optionally displayed within the full sequences of CDR- H3, 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).

In one aspect, 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. Alternatively, 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. In one embodiment, 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.

In one aspect, 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.

In one aspect, there is provided a method for generating an immune phage display library of ultralong CDR-H3 sequences, said method 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. In one embodiment, the method for amplifying the cDNA of CDR-H3 comprises:

In one embodiment, the primers used at step 1) comprise or consist of SEQ ID NO: 154 and SEQ ID NO: 311). In one embodiment, 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.

In one aspect, the disclosure provides a method for producing a fusion protein of the invention, which binds to an antigen of interest, said method comprising: a) generating a phage display library of ultralong CDR-H3 or knob domains (or antigen-binding portions thereof) of ultralong CDR-H3; and, b) enriching the phage display library against the antigen of interest to produce an enriched population of phage which bind the antigen of interest; and, c) sequencing an ultralong CDR-H3 from the enriched population of phage obtained in step b); and, d) expressing the knob domain, or antigen-binding portion thereof, of the ultralong CDR-H3 or portion thereof derived from the ultralong CDR-H3 obtained in step c) as a fusion protein according to the invention.

Steps a) to d) are methods well known in the art. For example, enriching the phage display library against the antigen of interest at step b) may be performed by panning the library obtained at step a) against the antigen of interest.

At step c), the sequence of the ultralong CDR-H3 sequences may be amplified using PCR using appropriate primers, for example sequencing primers annealing to the phagemid vector. In one embodiment, the primers used comprise or consist of SEQ ID NO: 316 and/or SEQ ID NO:317.

Further screening may be performed where an identified knob domain (or antigen-binding portion thereof) is assessed in the context of different helical peptides, with the present invention also providing a library encoding a plurality of antigen-binding proteins where the proteins comprise the same knob domains, or antigen-binding portions thereof, but different helical peptides to help determine the optimal helical peptide. Different linkers may also be screened.

Advantageously, libraries of CDR-H3 sequences can be cloned and screened for binding to an antigen, or a panel of antigens, using in vitro display technologies.

Pharmaceutical compositions and medical uses

In one aspect, the invention provides a pharmaceutical composition comprising an antigenbinding protein of the present invention in combination with one or more of a pharmaceutically acceptable excipient. Although, pharmaceutical compositions comprising an antigen-binding protein of the present invention are preferred, in other embodiments a sequence encoding the protein may be employed wherever reference is made herein to a pharmaceutical composition. Hence, in another aspect, the invention provides a pharmaceutical composition comprising a polynucleotide or vector encoding an antigen binding protein of the present invention in combination with one or more of a pharmaceutically acceptable excipient.

The term “pharmaceutically acceptable excipient” as used herein refers to a pharmaceutically acceptable formulation carrier, solution or additive to enhance the desired characteristics of the compositions of the present disclosure. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. The formulation will generally be provided in a substantially sterile form employing sterile manufacture processes. This may include production and sterilization by filtration of the buffered solvent solution used for the formulation, aseptic suspension of the antigen-binding protein of the invention in the sterile buffered solvent solution, and dispensing of the formulation into sterile receptacles by methods familiar to those of ordinary skill in the art.

The pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, poly glycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

The antigen-binding proteins of the present invention can be delivered dispersed in a solvent, e.g., in the form of a solution or a suspension. It can be suspended in an appropriate physiological solution, e.g., physiological saline, a pharmacologically acceptable solvent or a buffered solution.

A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991).

The pharmaceutical compositions suitably comprise a therapeutically effective amount of the antigen-binding proteins of the present invention. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. For any antigen-binding protein, the therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician.

Compositions may be administered individually to a patient or may be administered in combination (e.g. simultaneously, sequentially or separately) with other agents, drugs or hormones. Agents as employed herein refers to an entity which when administered has a physiological affect. Drug as employed herein refers to a chemical entity which at a therapeutic dose has an appropriate physiological affect.

The dose at which the antigen-binding protein of the present invention is administered depends on the nature of the condition to be treated, and on whether the antigen-binding protein is being used prophylactically or to treat an existing condition.

The frequency of dose will depend on the half-life of the antigen-binding protein and the duration of its effect. If the antigen-binding protein has a short half-life (e.g. 2 to 10 hours) it may be necessary to give one or more doses per day. Alternatively, if the antigen-binding protein has a long half-life (e.g. 2 to 15 days) it may only be necessary to give a dosage once per day, once per week or even once every 1 or 2 months.

A pharmaceutical composition of the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention.

Suitable forms for administration include forms suitable for parenteral administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the antigen-binding protein or pharmaceutical composition comprising it may be in dry form, for reconstitution before use with an appropriate sterile liquid.

Direct delivery of the compositions will be generally accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a specific tissue of interest. Dosage treatment may be a single dose schedule or a multiple dose schedule.

In one embodiment the formulation is provided as a formulation for topical administrations including inhalation.

Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases (such as nebulisable solutions or suspensions). Inhalable powders according to the disclosure containing the active substance may consist solely of the abovementioned active substances or of a mixture of the above-mentioned active substances with physiologically acceptable excipient. The propellent gases which can be used to prepare the inhalable aerosols are known in the art. Suitable propellent gases are selected from among hydrocarbons such as n-propane, n-butane or isobutane and halohydrocarbons such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The abovementioned propellent gases may be used on their own or in mixtures thereof. The propellent-gas-containing inhalable aerosols may also contain other ingredients such as cosolvents, stabilisers, surface -active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art. The propellant-gas-containing inhalable aerosols according to the invention may contain up to 5 % by weight of active substance. Aerosols according to the invention contain, for example, 0.002 to 5 % by weight, 0.01 to 3 % by weight, 0.015 to 2 % by weight, 0.1 to 2 % by weight, 0.5 to 2 % by weight or 0.5 to 1 % by weight of active.

Alternatively, topical administrations to the lung may also be by administration of a liquid solution or suspension formulation, for example employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).

In one embodiment the formulation is provided as discrete ampoules containing a unit dose for delivery by nebulisation.

In one embodiment the antigen-binding protein or pharmaceutical composition comprising it is supplied in lyophilised form, for reconstitutions or alternatively as a suspension formulation.

The antigen-binding protein of the present invention can be delivered dispersed in a solvent, e.g., in the form of a solution or a suspension. It can be suspended in an appropriate physiological solution, e.g., physiological saline, a pharmacologically acceptable solvent or a buffered solution. Buffered solutions known in the art may contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrate per 1 ml of water so as to achieve a pH of about 4.0 to 5.0. As mentioned supra a suspension can made, for example, from lyophilised antigen-binding protein.

Nebulisable formulation according to the present invention may be provided, for example, as single dose units (e.g., sealed plastic containers or vials) packed in foil envelopes. Each vial contains a unit dose in a volume, e.g., 2 ml, of solvent/solution buffer.

The antigen-binding proteins and pharmaceutical compositions of the present invention may be delivered via nebulisation. In one embodiment, they are in a form specific for delivery via nebulisation.

The present invention also provides a process for preparation of a pharmaceutical or diagnostic composition comprising adding and mixing the antigen-binding protein of the present invention together with one or more of a pharmaceutically acceptable excipient, diluent or carrier.

In one aspect, the invention provides an antigen-binding protein of the present invention, or a pharmaceutical composition comprising it for use in therapy.

Antigen-binding proteins of the invention may be used for the treatment of diseases or disorders including inflammatory diseases and disorders, immune diseases and disorders, complement-related diseases and disorders, autoimmune diseases, vascular indications, neurological diseases and disorders, kidney -related indication, ocular diseases. In some embodiments, an antigen-binding protein of the present invention, and pharmaceutical composition thereof according to the present invention may be useful in the treatment of diseases, disorders and/or conditions.

The present invention also provides for the use of antigen-binding protein of the present invention in the manufacture of a medicament to treat or prevent any of the conditions mentioned here. It further provides a method of treatment of any of the conditions referred to herein comprising administering an effective amount of the antigen-binding protein to a subject in need thereof to treat or prevent the condition. The present invention also provides a pharmaceutical composition as set out herein for use in treating or preventing any of the conditions referred to herein.

Please note that where terms such as “comprising” or “comprises” are used, also provided are embodiments “consisting essentially of’ what is set out and also that “consist” of what is set out. Please also note that where the singular is used, the plural is also provided or may be employed unless indicated otherwise.

All documents mentioned herein are incorporated by reference in their entirety, as well as specifically in relation to the topic being discussed where they are mentioned.

Examples

Example 1: Generation of fusion proteins comprising a knob domain and a helical peptide or peptides

Helical peptides were identified that could enhance the recombinant expression of knob domains, in particular in mammalian cells, where the expression yields of the knob domains were improved upon expression of the knob domains-helical peptide fusion proteins as compared to knob domains expressed on its own. Fusion proteins comprising knob domains of bovine ultralong CDR-H3 fused to at least one helical peptide were generated according to the methods described below. Figure 1 A represents the native conformation of a knob domain, when expressed as part of the bovine antibody infrastructure (notably comprising the stalk).

In the present Examples, knob domains comprising the sequences as listed in Table 5 below were used.

Table 5: Amino acid sequences of knob domains

In a first aspect, knob domains were fused to two helical peptides assembling into a coiled-coil structure. Knob domains were grafted onto stretches of anti-parallel coiled-coil dimers taken from Sin Nombre orthohantavirus nucleocapsid protein (SNV-N, residues 4-11 and 61-68, PDB 2IC6), and human autophagy-related protein Beclin-1 (BECN1, residues 103-121 and 68-86, UniProt K7ELY9) as represented in Figure IB for illustration purposes.

In a second aspect, knob domains were fused with one or two helical peptide(s) comprising N- or N- and C-terminal motifs from human haemoglobin beta subunit (HBB, residues 1-16, PDB 4N8T) and HSA (residues 1-13 or both 1-13 and 573-585, PDB 1BM0). A knob domain fused to a HBB peptide at its N-terminus is represented in Figure IB for illustration purposes.

The ability of the synthetic helical peptide(s) to increase expression yields of knob domain peptides was evaluated by screening a panel of constructs comprising an anti-IL2 binding knob domain (aIL2_l) and two unrelated anti-complement component 5 (C5) binding knob domains (referred to as K8 and K57) fused to helical peptides.

The knob domains expression constructs were N-terminally fused with a leader peptide derived from murine immunoglobulin heavy chain (MEWSWVFLFFLSVTTGVHS, UniProt AON1R4 MOUSE - SEQ ID NO: 81) and synthesised with a C-terminal 8*His purification tag. DNA sequences were codon-optimised for human expression, synthetised and cloned into pcDNA3.1(+) vector by GenScript. Sequences are presented in Table 6A below. Helical peptide sequences are provided in Table 6B below.

Table 6A: Amino acid sequences of the knob domains-helical peptide fusion proteins

Table 6B: Amino acid sequences of synthetic helical peptides

Expression screening was carried out as a series of small-scale transient transfections of Expi293F cells as described below, to permit cell supernatants to be enriched by IMAC and analysed by SDS-PAGE. Plasmids encoding the knob domains expressed alone were used as negative controls.

Expression screening was conducted by transiently transfecting Expi293F cells using ExpiFectamine 293 Transfection Kit (Gibco), as per manufacturer’s instructions. Briefly, the cells were seeded in 24-well culture blocks as individual 2 mL cultures at 3 * 10⁶ cells/mL and transfected with 2 pg of plasmids. 96 h post-transfection, cell media were harvested by centrifugation at 16,000 g for 5 min. Secreted His-tagged knob domain constructs were enriched in an automated 12-channel PhyNexus MEA 2 protein purification system using PhyTip columns packed with 10 pl of Ni-IMAC resin. First, the columns were equilibrated with 1 mL of PhyNexus Capture Buffer. 900 pL of media samples were then mixed with 100 pL of 0.5 M sodium phosphate, 1.5 M NaCl, 100 mM imidazole (pH 8.0) and loaded on the columns by slowly pipetting up and down. The columns were washed two times with 1 mL of 1:4 diluted PhyNexus Wash Buffer, and bound fractions were eluted with 30 pL of PhyNexus Elution Buffer. The eluates were assayed for knob domain fusion proteins by SDS-PAGE under reducing and non-reducing conditions.

The results are shown in Figure 2. The results show that fusing the knob domains with antiparallel coiled-coil dimers taken from SNV-N and BECN1 dramatically improved expression yields of all peptides. Similar results were seen after N-terminal fusion with the first 16 residues taken from mature HBB sequence, as well as grafting both N- and C-terminal helices from HSA onto respective ends of the knob domains.

Expression in large scale conditions

SNV-N, BECN1 and HBB constructs were further assessed by scaling up the transfections to enable the accurate quantification of expression yield and biophysical characterisation of knob domain fusions. Scale-up to 50 mL cultures was carried out in 250 mL Erlenmeyer flasks. To enable large-scale transfection, plasmid DNA was amplified using QIAGEN Plasmid Plus Maxi Kit. Culture media were harvested by centrifugation at 7,000 g for 1 h and passed through 0.22 pm filters. The knob domain constructs were purified by Ni-IMAC using an AKTA pure chromatography system (Cytiva) according to standard methods. Briefly, a 1 ml HisTrap excel column was equilibrated with PBS, 0.5 M NaCl prior to loading cell supernatants. The column was extensively washed with the same buffer followed by another wash with PBS, 0.5 M NaCl, 20 mM imidazole. Bound proteins were eluted with PBS, 0.5 M NaCl, 200 mM imidazole. The protein-containing fractions were pooled, quantified by measuring absorbance at 280 nm and stored frozen at -80 °C for subsequent analyses.

Purified constructs were tested to ascertain their identity and validate protein quality. SDS- PAGE analysis confirmed homogeneity of the samples, and no misfolded oligomers were detected by repeating SDS-PAGE under non-reducing conditions.

Analysis by LC-MS

LC-MS analysis was performed using a Waters ACQUITY UPLC System connected to a Waters Xevo G2 Q-ToF mass spectrometer operated by MassLynx™ Software. Ni-IMAC eluate samples (5 pL at 0.05-0.4 mg/mL) were injected on a BioResolveT RP mAb Polyphenyl, 450 A, 2.7 pm column held at 80 °C with a flow rate of 0.6 mL/min. The mobile phase buffers were water, 0.02% trifluoroacetic acid (TFA), 0.08% formic acid (Solvent A) and 95% acetonitrile, 5% water, 0.02% TFA, 0.08% formic acid (solvent B). A reverse phase gradient was run from 5% to 50% solvent B over 8.8 min with a 95% solvent B wash and re-equilibration at 5% solvent B. UV data were acquired at 260- 300 nm. For mass spectrometry, the system was configured as follows: ion mode, ESI positive; acquisition mode, resolution; mass range, 400-5,000 m/z; cone voltage, 30 V; capillary voltage, 3.2 kV; desolvation temperature, 350 °C; desolvation gas, 1,000 L/h; source temperature, 150 °C. Data analysis was performed using MassLynx™ and OpenLynx™ software.

Molecular weights determined by liquid chromatography-mass spectrometry (LC-MS) matched theoretical values calculated for monomeric proteins (Figure 3).

The mass spectra of HBB-fused knob domains exhibited peaks shifted by approximately +948 Da from their respective theoretical values, consistent with the presence of common O-linked tetrasaccharide -GalNAc(-NeuNAc)-Gal-NeuNAc occurring in recombinant proteins produced by mammalian kidney cell lines.

Analysis by SEC-HPLC

SEC-HPLC analysis was performed using Agilent 1100 Series HPLC and Waters ACQUITY UPLC systems. Ni-IMAC eluate samples (20 pL at 0.05-0.4 mg/mL) were injected on a Tosoh Bioscience TSKgel G3000SWXL column (7.8 x 300 mm) running at 1 mL/min in 0.2 M sodium phosphate buffer, pH 7. Chromatograms were obtained by monitoring absorbance at 280 nm and fluorescence intensity at Ex/Em = 280/345 nm. Molecular weights were determined using a set of five protein standards (670 kDa, 158 kDa, 44 kDa, 17 kDa, 1.35 kDa).

Analysis by SEC-HPLC confirmed the monomer purity indicating that all samples were free from high molecular weight aggregates as shown in Figure 4. Example 2: Assessment of binding and affinity of knob domains expressed as knob domains- helical peptide fusion proteins

Methods

The antigen-binding ability of the constructs was tested using ELISA. 96-well Nunc MaxiSorp plates were coated with 1-3 pg/mL solutions of either IL-2 or purified C5 in PBS. The plates were blocked with 10% Aquatic Block Reagent in PBS and incubated with the dilutions of purified knob domain constructs in PBS, 10% Aquatic Block Reagent, 0.05% Tween-20. Detection was performed using 1:2,000 rabbit anti-6-His-Tag primary antibodies and 1:5,000 HRP-conjugated goat anti-rabbit secondary antibodies. The washing steps comprised five wash cycles with PBS, 0.05% Tween-20. To reveal, the plates were incubated with 1-Step Ultra TMB-ELISA Substrate Solution (e.g.Thermo Scientific) and the optical density at 652 nm was measured using a BioTek Synergy Neo2 microplate reader.

More detailed data on antigen binding kinetics were obtained via multicycle SPR experiments, by comparing recombinant knob domains fusions with knob domains alone produced by solid-phase peptide synthesis.

The binding kinetics were measured using a Biacore 8K+ instrument. The antigens and knob domain constructs were immobilised on a Biacore CM5 chip via amine coupling; serial dilutions of their respective binding partners were prepared in HBS-EP+ buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). For each injection, a flow rate of 40 pL/min was used. Association was recorded for 300 s (K8 and K57) or 100 s (aIL2_l); dissociation was recorded for 6000 s (K8 and K57) or 1000 s (aIL2_l). The surface was regenerated by two sequential 30 s pulses of 2 M MgCL. To determine the binding kinetics, the data obtained after subtraction of reference measurements were fitted to 1:1 binding model using Biacore evaluation software.

Results

All K8 and K57 constructs were found to bind to C5 and all aIL2_l constructs displayed binding to IL-2 using ELISA (Figure 5).

The Biacore data showed that K8 and K57 exhibited tight binding to C5, with K_v values in low nanomolar range and relatively slow on- and off-rate kinetics (Table 7 below). Importantly, the results showed that fusing these knob domains with any of the helical peptides tested had little or no effect on the binding, suggesting that recombinant knob domains were able to fold properly and retained functionality. Table 7: Antigen-binding kinetics of knob domains produced by solid-phase peptide synthesis (aIL2_l, K8 and K57) and recombinant knob domain fusion constructs.

Example 3: Thermal stability of the knob domains-helical peptide fusion proteins

The ability of the knob domain fusion proteins to bind an antigen after a thermal stress was further analysed to assess the structural stability of the constructs. The samples were diluted to 5 pM in PBS and subjected to 30 min heating at 90°C. Cooled samples were briefly centrifuged (16,000 g, 5 min) to remove protein aggregates, and the supernatants were assessed by ELISA (method as described above). A control was used that represents the binding of knob domains to the antigen-free blocked surface. There was no significant impact on the binding activity, showing that all knob domain fusion proteins displayed high levels of thermal stability (Figure 6).

Example 4: recombinant expression of bispecific knob domains expressed as knob-helical peptide fusion proteins

As an example, we designed a panel of constructs based on the SNV-N- and BECN1 -fused knob domains (Figure 7), including: bispecific aIL2_l-K8 fusions joined via flexible linkers of varying length (GSG, GGGGP (SEQ ID NO: 6) and GGGGSGGGGS) (Fig.7A), bivalent aIL2_l-aIL2_l fusions joined via GGGGP linker (Fig.7B), bispecific/bivalent aIL2_l-aIL2_l-K8 fusions joined via GGGGP linkers (Fig.7C), and biparatopic K8-K57 fusions (Fig.7D) joined via 127 amino acid-long linker having the sequence:

GGS STASGSGSGGSGTAGS SGGAGS SGGSTTAGGSASGSGSTGSGTGGAS SGGASGASGEPKS SGGS S TASGSGSGGSGTAGSSGGAGS SGGSTTAGGSASGSGSTGSGTGGAS SGGASGASGGGGP (SEQ ID NO: 114)

Such an extensive linker was used to cover a 100 A distance between K8 and K57 epitopes located on the MG8 and MG5 domains of the C5 protein (PDB 3CU7) Macpherson el al. (2021) The allosteric modulation of complement C5 by knob domain peptides. Elife 10: e63586. All the designed constructs expressed at high levels in Expi293F cells and appeared as well- folded monomers on SDS-PAGE gel under non-reducing conditions (Figure 8A).

As control, we transfected the cells with the plasmids encoding aIL2_l-K8 and aIL2_l-aIL2_l knob domains alone, without the helical peptide(s). The resulting peptides were expressed at marginal levels (Figure 8B).

Molecular weights determined by LC-MS confirmed the identity and the monomeric state of the samples (Figure 9); absence of high molecular weight aggregates was further established by UPLC.

In addition, all constructs were able to engage their targets in an ELISA assay (Figure 10). Importantly, the knob domains retain their functionality and the binding kinetics of aIL2_l-GGGGP- K8 fusions determined by SPR were substantially similar to the parental knob domains as shown in Table 8 below.

Table 8. Antigen-binding kinetics of bispecific, bivalent knob domain fusions.

Having confirmed individual binding to IL2 or C5, aIL2_l-K8 and aIL2_l-aIL2_l-K8 were tested for simultaneous binding to IL2 and C5. For that, we employed a bridging SPR assay described in Gassner et al. (2015) Development and validation of a novel SPR-based assay principle for bispecific molecules. J Pharm Biomed Anal 102: 144-149.

Briefly, the surface of a Biacore CM5 chip was coated with amine-coupled C5 and the 200 nM knob domain fusions were injected twice for 300 s each. This was followed by a 300 s injection of 100 nM IL2. A single knob domain K8 was used as a control. While all samples showed initial association with C5, only aIL2_l-K8 and aIL2_l-aIL2_l-K8 were able to subsequently and simultaneously associate with IL2 while remaining bound to C5 (Figure 11, Fig.l lA SNV-N, Fig. 11B BECN1). The increase in signal upon IL2 binding by SNV-N-fused aIL2_l-aIL2_l-K8 was roughly double compared to SNV-N-fused aIL2_l-K8 (26 vs. 12 RU) indicating formation of quaternary complex.

Similarly, we showed that biparatopic K8-K57 fusions could simultaneously bind their epitope on C5, by comparing its binding kinetics to the kinetics of individual knob domains. Indeed, the dissociation of K8-K57 from C5 was about an order of magnitude slower than the most stable individual knob domain K8 or K57 suggesting that the K8-K57 construct is bound to C5 via both K8 and K57 (Figure 12). The observed association rates of biparatopic fusions were not compromised compared to K8 and K57.

Similarly, fusing two aIL2_l knob domains into bivalent constructs led to substantial slowing down of dissociation from IL2 -coated surface (Figure 13). This shows that the fusions were able to simultaneously engage two IL2 molecules on the solid phase leading to enhanced binding avidity. As expected, dissociation rates of the bivalent constructs were relatively similar to dissociation rate of individual aIL2_l when the antigen was flowed over the knob domain-coated chip surface (Table 4).

Example 5: Generation of additional knob-helical peptide fusion proteins and assessment of their binding activity

In this example, knob domain K8 was fused to coiled-coil forming peptides de novo-designed from sequences previously published (Monera et al. (1993) Comparison of antiparallel and parallel two-stranded alpha-helical coiled-coils. Design, synthesis, and characterization. J Biol Chem 268(26): 19218-19227; Oakley et al. (1998) A buried polar interaction can direct the relative orientation of helices in a coiled coil. Biochemistry 37(36): 12603-12610). The sequences designed are represented below:

Monera et al. (1993)

ECAE LKGE LAE LKGELGSVCPDGFNWGYGCAAGS SRFCTRHDWCCYDERADSHTYGFCTGNRVTGSLK

GELAELKGELEACK (SEQ ID NO: 115)

First helical peptide: ECAELKGELAELKGELGS SEQ ID NO: 116

Second helical peptide: GSLKGELAELKGELEACK SEQ ID NO: 117 Oakley et al. (1998)

LEKELDALEKELAQLGSVCPDGFNWGYGCAAGSSRFCTRHDWCCYDERADSHTYGFCTGNRVTGSLAQ LKEKLQRLKKKL SEQ ID NO: 118

First helical peptide: LEKELDALEKELAQLGS SEQ ID NO: 119

Second helical peptide: GSLAQLKEKLQRLKKKL SEQ ID NO: 120

Both fusion constructs were found to express well in Expi293F cells and produced bands consistent with the monomeric state of the protein on a non-reducing SDS-PAGE gel (Figure 14 A). Native folding of knob domains was further confirmed by an antigen-binding ELISA (Figure 14 B).

Example 6: assessment of the ability of shortened BECN1 based helical peptides to increase the expression of knob domain

Trimmed versions of K8-BECN 1 lacking one, two or three turns (4, 7 or 11 amino acids) of a- helix from either distal or proximal end of the helical peptide were produced according to the methods described in Example 1 in small scale production (constructs illustrated in Figure 15A; sequences represented in Table 5 below)

A canonical coiled coil is formed of heptad repeats in which positions 1 and 4 are reserved for hydrophobic residues that line the interface (Truebestein & Leonard (2016) Coiled-coils: The long and short of it. Bioessays 38(9): 903-916).

Within the sequence of shortened BECN 1 stalk, two such positions are taken by polar amino acids (glutamine, Q, and arginine, R) and another position is occupied by phenylalanine, F, whose side chain, despite its hydrophobic nature, is bulky and does not fit well to the interhelical space. We substituted these residues with aliphatic amino acids.

Table 9: Amino acid sequences of a K8 knob domain fused with shortened BECN1 stalks

BECN1 helical peptide size was reduced from 19 to 12 amino acids (each of the helices) and its molecular weight was reduced from 5.0 to 3.3 kDa. Expression levels of trimmed versions were benchmarked against the full-length K8-BECN 1. Removal of one or two turns from the distal end of the stalk had no impact on the ability to increase the knob domain expression level (Figure 15 B).

All samples producing the monomer band on a gel were recognising C5 in an ELISA assay (Figure 15C).

A trimmed version of K8-BECN 1 lacking two turns of a-helix from distal end and one turn of a-helix from proximal end was also considered. This further reduces the size of the BECN1 helical peptide to 8 amino acids (each of the helices) and its molecular weight - to 2.3 kDa. The shortened helical peptide was still able to rescue expression of K8 and other knob domains (K57, aIL2_l, aNotchl l, aNotchl_2, aNotchl_3). All samples produced monomer bands on non-reducing gel consistent with their native folding (Figure 15 D).

Example 7: generation of cyclic knob-helical peptide fusion proteins

A set of K8-SNV-N fusions with a pair of cysteines at the distal end of the coiled-coil dimer was generated, produced and purified according to the methods described in Example 1. Cyclisation may occur naturally during the recombinant production process. Constructs are illustrated in Figure 16A and sequences are presented in Table 6 below). Table 10: Amino acid sequences of knob domain K8 fused with the modified SNV-N stalks with an extra pair of cysteines.

In a small-scale test, all four cyclic peptides were found to express well (Figure 16 B).

Purified peptides migrated at a similar rate as the original K8-SNV-N and were capable of antigen binding (Figure 16 C), suggesting that the cyclised peptides retain their native folding.

Claims

1. 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 termini to a helical peptide, preferably an a-helical peptide.

2. The antigen-binding protein of claim 1, wherein (a) the helical peptide or helical peptides and (b) the knob domain or antigen-binding portion thereof do not occur together in the same natural protein and optionally (a) and (b) are heterologous to each other.

3. The antigen-binding protein of claim 1 or 2, wherein the knob domain, or antigen-binding portion thereof, fused at its N-terminus, C-terminus, or at both termini to a helical peptide is not present within an antibody or antigen-binding antibody fragment.

4. The antigen-binding protein of any one of the preceding claims, wherein the helical peptide or helical peptides are at least 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.

5. The antigen-binding protein of any one of the preceding claims, wherein the knob domain or antigen-binding portion thereof comprises at least two, or at least 3, or at least 4 or at least 5 or at least 6 disulphide bonds.

6. The antigen-binding protein of any one of the preceding claims, wherein the knob domain or antigen-binding portion thereof is a knob domain or antigen-binding portion thereof of an ultralong CDR H3.

7. The antigen-binding protein of claim 6, wherein the knob domain or antigen-binding portion thereof is a knob domain or antigen-binding portion thereof of a bovine ultralong CDR H3.

8. The antigen-binding protein of any one of the preceding claims, wherein the knob domain or antigen-binding portion thereof is fused, optionally via a linker, at both its N-terminus and C- terminus to a helical peptide, the two helical peptides forming a coiled-coil structure.

9. The antigen-binding protein of claim 8, wherein the two helical peptides are derived from a Sin Nombre orthohantavirus nucleocapsid protein (SNV-N) or a human Beclin-1 protein.

10. The antigen-binding protein of claim 9, wherein the two helical peptides are derived from a Sin Nombre orthohantavirus nucleocapsid protein (SNV-N) and comprise, or consist of, a pair of helical peptides selected from a pair of helical peptides with amino acid sequences selected from:

(i) SEQ ID Nos: 109 and 110;

(ii) SEQ ID Nos: 143 and 144;

(iii) SEQ ID Nos: 146 and 147;

(iv) SEQ ID Nos: 149 and 150;

(v) SEQ ID Nos: 152 and 153; and

(vi) a variant pair of helical peptides of any one of (i) to (v), wherein the pair of helical peptides is still able to form a coiled-coil structure and wherein the knob domain or portion thereof retains the ability to bind antigen.

11. The antigen-binding protein of claim 9, wherein the two helical peptides are derived from a human Beclin-1 protein and comprise, or consist of, a pair of helical peptides selected from a pair of helical peptides with amino acid sequences selected from:

(i) SEQ ID Nos: 111 and 112;

(ii) SEQ ID Nos: 122 and 123;

(iii) SEQ ID Nos: 125 and 126;

(iv) SEQ ID Nos 128 and 129;

(v) SEQ ID Nos: 131 and 132;

(vi) SEQ ID Nos: 140 and 141;

(vii) SEQ ID Nos: 319 and 320; and

(viii) a variant pair of helices of any one of (i) to (v), wherein the pair of helices are still able to form a coiled-coil structure and wherein the knob domain or potion thereof retains the ability to bind antigen.

12. The antigen-binding protein of claim 8, wherein the two helical peptides comprise, or consist of, a pair of helices selected from a pair of helical peptides with amino acid sequences selected from:

(i) SEQ ID Nos: 116 and 117;

(ii) SEQ ID Nos: 119 and 120; and

(iii) a variant pair of helical peptides of any one of (i) to (v), wherein the pair of helical peptides are still able to form a coiled-coil structure and wherein the knob domain or portion retains the ability to bind antigen.

13. The antigen-binding protein of any one of claims 1 to 7, wherein the knob domain or antigenbinding portion thereof is fused, optionally via a linker, to helical peptide either at its N- terminus or C-terminus, but not both termini.

14. The antigen-binding protein of claim 13, wherein the helical peptide is derived from a haemoglobin protein.

15. The antigen-binding protein of claim 14, wherein the helical peptide is derived from haemoglobin protein B and comprises, or consists of, the helical peptide of SEQ ID NO: 113 or a helical variant thereof.

16. The antigen-binding protein of any one of the preceding claims, wherein the knob domain or antigen-binding portion thereof comprises a (Zi) Xi C X₂ motif at its N-terminal extremity, 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;

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 is cysteine; and,

X₂ is an amino acid selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine.

17. The antigen-binding protein of any one of the preceding claims, wherein the knob domain or antigen-binding portion thereof is fused to a helical peptide at its N-terminus, C-terminus, or both termini via a peptide bond.

18. The antigen-binding protein of claim 17, wherein the knob domain or antigen-binding portion thereof is fused at its N-terminus, C-terminus, or both termini a helical peptide via an amino acid linker sequence.

19. The antigen-binding protein of any one of the preceding claims further comprising a second knob domain or a portion thereof capable of binding antigen, wherein the second 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.

20. The antigen binding protein of claim 19, wherein the antigen-binding protein is biparatopic with the first knob domain or antigen-binding portion thereof and the second knob domain or antigen-binding portion thereof each binding a different epitope of the same antigen.

21. The antigen-binding protein of claim 19, wherein the antigen-binding protein is bispecific with the first knob domain or antigen-binding portion thereof and the second knob domain or antigen-binding portion thereof each binding a different epitope of a different antigen.

22. The antigen-binding protein of claim 19 further comprising a third knob domain or a portion thereof capable of binding antigen, wherein the second 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:

23. The antigen-binding protein of claim 22 which is trispecific with the three knob domains or antigen-binding portions thereof each binding a different epitope on a different antigen.

24. The antigen-binding protein of any one of claims 19 to 22, wherein a linker is used to join together two different knob domains and fused helical peptide or peptides.

25. The antigen-binding protein of any one of the preceding claims which further comprises one or more effector molecule.

26. A polynucleotide encoding an antigen-binding protein according to any one of the preceding claims.

27. A vector comprising a polynucleotide according to claim 26.

28. A host cell comprising a polynucleotide according to claim 26 or a vector according to claim 27.

29. A method for producing an antigen-binding protein according to any one of claims 1 to 25 comprising expressing the antigen-binding protein from a host cell according to claim 28.

30. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 25 and a pharmaceutically acceptable excipient, diluent or carrier.

5 31. An antigen-binding protein according to any one of claims 1 to 25 or a pharmaceutical composition according to claim 30 for use in therapy.