Hexameric protein

Abstract

The present invention provides hexameric proteins, methods of making them and uses thereof. The present invention relates to proteins that are hexamerized via a tailpiece peptide. The present invention provides hexameric antibodies that may be used as non-hormonal contraceptives, in the treatment of a pathogenic infection or in the treatment of cancer.

Description

[0001] HEXAMERIC PROTEIN

[0002] FIELD OF THE INVENTION

[0003] The present invention provides hexameric proteins, methods of making them and uses thereof. The present invention relates to proteins that are hexamerized via a tailpiece peptide. The present invention relates to hexameric antibodies, wherein the hexamerization is mediated by a tailpiece peptide, particularly hexameric antibodies that may be used as non-hormonal contraceptives.

[0004] BACKGROUND TO THE INVENTION

[0005] Antibodies are glycoproteins belonging to the immunoglobulin superfamily of proteins. There are five primary classes of immunoglobulin, including IgG, IgM, IgA, I g E and IgD. Antibodies are typically made of basic structural units comprising two heavy and light chains. The IgG immunoglobulin molecule, for example, comprises four polypeptide chains, two of which are identical heavy (H) chains and two of which are identical light (L) chains. Each heavy chain comprises an N-terminal variable domain (VH) and three constant domains (CH1 , CH2 and CH3), with a hinge region linking CH1 and CH2. Each light chain comprises an N-terminal variable domain (VL) and a constant domain (CL), which together associate with the VH and CH1 domain of the heavy chain.

[0006] An antibody may recognise an antigen via the fragment antigen-binding (Fab) variable region, which comprises the VL, CL, VH and CH1 domains. The heavy chain variable region (VH) refers to the fragment of the heavy chain of an antibody that comprises the three complementarity determining regions (CDRs) which are interposed between the framework regions, the latter of which form a scaffold which supports the CDRs. Similarly, the light chain variable region (VL) comprises three CDRs and their surrounding framework regions.

[0007] The antibody may activate the immune system through the fragment crystallisable region (Fc region), which is the tail region of the antibody comprising the CH2 and CH3 domains. Many antibodies depend on the Fc region for function, largely through interactions with complement components (e.g. C1q) and Fc gamma receptors (FcyRs). To date, there has been little clinical application of such multimeric antibodies in effective therapeutics.

[0008] In particular, there remains a need for improved antibody therapies, such as through the production of hexameric proteins and antibodies for use as anti-microbial, anti-cancer and contraceptive agents. For instance, there remains a global demand for non-hormonal contraceptives in order to overcome the real and / or perceived side effects of traditional hormonal contraception. Currently available non- hormonal contraceptive methods have several disadvantages. For example, the copper intrauterine device (IUD) may result in heavy menstrual bleeding and dysmenorrhea. Spermicidal agents, such as nonoxynol-9 [N9]-based non-hormonal contraceptives, may cause mucosal inflammation, disruption to the vaginal epithelial barrier function, alter the vaginal microbiome and increase acquisition of HIV-1.

[0009] Two lines of in vivo evidence suggest that human anti-sperm antibodies may be effective.

[0010] First, an agglutinating anti-sperm antibody was shown to be effective as a contraceptive in rabbits (Castle et al., 1997). And second, anti-sperm antibodies are observed in women suffering from immune infertility (Ustay, 1967; Bronson 1999). For example, the HC4 antibody was cloned from circulating B-cells of an infertile woman with high titer anti-sperm antibodies in her blood and genital tract, and without other explanation for her infertility (Isojima S, 1989). Recently, in a phase 1 , first-in-woman, open-label clinical trial HC4 was demonstrated to be safe and efficacious as a contraceptive when applied before intercourse (Thruman et al., 2023).

[0011] There therefore remains a need for the production of hexameric proteins, wherein by generating a hexameric protein, particularly advantageous properties may be imparted. For example, there remains a need for a non-hormonal contraceptive that demonstrates both safe and efficacious contraceptive properties.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is based on the inventors’ development of hexameric proteins which display particularly advantageous and surprising properties.

[0014] For example, in one embodiment, the present invention relates to a hexameric antibody that demonstrates improved agglutination and antibody functional responses.

[0015] The present inventors surprisingly demonstrate that the hexameric antibody of the present invention, wherein the hexameric antibody recognises CD52g on the surface of sperm cells, shows improved agglutination, complement activation and / or immobilization of said sperm cell when compared to a monomeric reference standard.

[0016] In one aspect, the present invention provides a hexameric protein, wherein the hexameric protein is composed of six units and wherein each unit comprises: (a) an N-terminal immunoglobulin, or a fragment thereof;

[0017] (b) a linker domain; and

[0018] (c) a C-terminal tailpiece derived from IgM.

[0019] In one aspect, the present invention provides a hexameric protein as disclosed herein, wherein the N-terminal immunoglobulin, or a fragment thereof, comprises an immunoglobulin Fc region.

[0020] In one aspect, the present invention provides a hexameric protein as disclosed herein, wherein the hexameric protein is a hexameric antibody.

[0021] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal immunoglobulin or a fragment thereof is independently an N-terminal IgG or IgM, or an antigen-binding fragment thereof, which is capable of binding one or more ligand molecules.

[0022] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 29 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0023] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 30 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0024] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 31 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0025] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 32 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0026] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 33 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0027] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 34 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0028] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 35 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0029] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein each unit comprises an N-terminal IgG, wherein the heavy chain has a sequence with at least 70% sequence identity to SEQ ID NO: 36 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0030] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 37 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0031] In one aspect, the present invention provides a hexameric antibody as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 38 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0032] In one aspect, the present invention provides a hexameric protein or antibody as disclosed herein, wherein the linker domain has an amino acid sequence according to SEQ ID NO: 40 to SEQ ID NO: 49.

[0033] In one aspect, the present invention provides a hexameric protein or antibody as disclosed herein, wherein the tailpiece has an amino acid sequence according to any one of SEQ ID NO: 1 to SEQ ID NO: 28. In one aspect, the present invention provides a hexameric protein or antibody as disclosed herein, wherein the tailpiece is fused to the C-terminus of an immunoglobulin Fc region via a linker amino acid sequence of any one of SEQ ID NO: 40 to SEQ ID NO: 49.

[0034] In one aspect, the present invention provides a hexameric protein or antibody as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 50 to SEQ ID NO: 68

[0035] In one aspect, the present invention provides a hexameric protein or antibody as disclosed herein wherein the IgG or IgM domain is capable of binding one or more ligand molecules.

[0036] In one aspect, the present invention provides a hexameric protein or antibody as disclosed herein wherein the IgG or IgM domain is capable of binding to CD52g and human spermatozoa.

[0037] In one aspect, the present invention provides a hexameric protein or antibody or pharmaceutical composition as disclosed herein for use in therapy.

[0038] In one aspect, the present invention provides a hexameric protein or antibody or pharmaceutical composition as disclosed herein for use as a contraceptive agent.

[0039] In one aspect, the present invention provides a hexameric protein or antibody or pharmaceutical composition as disclosed herein for use in the treatment or prevention of bacterial, viral or fungal infection.

[0040] In one aspect, the present invention provides a hexameric protein or antibody or pharmaceutical composition as disclosed herein for use in the treatment or prevention of cancer.

[0041] DESCRIPTION OF DRAWINGS

[0042] Figure 1 : Representative hexameric antibody KB15A hlgG1.

[0043] Figure 2: Representative protein gel of hexameric KB15A.14 hlgG1 ptp_m and KB15A.16 hlgG1 ptp_m with / without DTT reducing reagent. Nicotiana Benthamiana produced KB15A.14 hlgG1 ptp_m and KB15A.16 hlgG1 ptp_m were purified using protein A chromatography followed by Capto Core 400. Figure 3: Representative SEC-HPLC of hexameric KB15A.14 and 16 hlgG1 ptp_m. Nicotiana Benthamiana produced KB15A.14 and 16 hlgG1 ptp_m was purified using protein A chromatography followed by Capto Core 400.

[0044] Figure 4: Representative SEC-HPLC of hexameric KB15A antibodies which were purified from Nicotiana Benthamiana using protein A chromatography.

[0045] Figure 5: Hexameric KB15A antibodies effectively agglutinate human sperm. KB15A.14 hlgG1 utp_m (KB15A.14 hlgG1) and KB15A.16 hlgG1 utp_m (KB15A.16 hlgG1) show strong agglutination activity than KB15A.14 lgG1 and KB15A.16 lgG1. Agglutination kinetics assay using ~37x106 / mL washed sperm and various concentrations of KB15A.

[0046] Figure 6: Hexameric KB15A antibodies show strong complement-dependent sperm immobilization. HCA = KB15A antibody; C = complement; HiC = heat inactivated complement.

[0047] Figure 7: Representative SEC-HPLC of hexameric C4BP, hexameric SAL4 hlgG1 utp_m, hexameric ZAC3 hl gG 1 utp_m, hexameric LA2 hl gG 1 utp_m, hexameric HSV8 h IgG 1 utp_m, hexameric F2-5 h IgG 1 utp_m. Nicotiana Benthamiana produced hexamers were purified using protein A chromatography followed by Capto Core 400.

[0048] Figure 8: SEC-MALS representative figure of KB15A.16.3 polish purified. KB15A.16.3 is 93% pure hexamer with 6% HMW and 1% LMW antibody with estimated molecular weights of 900 kDa, 1800 kDa and 350 kDa, respectively.

[0049] Figure 9: Hexamer KB15A.16.3 (i.e. hexameric KB15A.16 hlgG1), stored at 4 °C or -80 °C, is 10X more potent than the monomer version (reference standard).

[0050] Figure 10A: Demonstrates the complement-dependent sperm immobilization of hexameric KB15A.16 hlgG1 (i.e. KB15A.16.3) using two different sperm donors versus the reference standard KB15A.16.2 (KB15A.16.2 refers to a monomer).

[0051] Figure 10B: Demonstrates the complement-dependent sperm immobilization of hexameric KB15A.16 hlgG1 (KB15A.16.3) and KB15A.16 hlgG4 using three different sperm donors versus the reference standard KB15A.16.2.

[0052] DETAILED DESCRIPTION Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0053] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0054] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes", "containing", or "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0055] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0056] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. All publications mentioned in the specification are herein incorporated by reference.

[0057] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0058] Sequence Identity

[0059] “Percent (%) amino acid sequence identity” with respect to the peptide sequences herein is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in the sequences recited herein after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be carried out by the skilled person using techniques well known in the art, for example using publicly available software such as EMBOSS Needle, BLAST, BLAST2 or Align software. The percentage sequence identities used herein in the context of the present invention may be determined using these programs with their default settings. More generally, the skilled worker can readily determine appropriate parameters for determining alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0060] In some embodiments, any substitutions compared to the sequences recited herein are conservative substitutions.

[0061] Structure of the hexameric protein

[0062] The present invention relates to a hexameric protein wherein the hexameric protein is composed of six units. A typical structure of such a hexameric protein, wherein in this embodiment the hexameric protein is a hexameric antibody, can be visualised in Figure 1.

[0063] In one embodiment, the present invention relates to a hexameric protein, wherein the hexameric protein is composed of six units and wherein each unit comprises:

[0064] (a) an N-terminal immunoglobulin, or a fragment thereof;

[0065] (b) a linker domain; and

[0066] (c) a C-terminal tailpiece derived from IgM.

[0067] The hexameric protein of the present invention comprises six units wherein each unit is comprises an N-terminal immunoglobulin which is connected to a C-terminal tailpiece via a linker domain.

[0068] In one embodiment, the hexameric protein is comprised of six units, wherein each unit is the same.

[0069] In one embodiment, the hexameric protein is comprised of six units, wherein each unit is different.

[0070] In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, five units are identical and one is different.

[0071] In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, four units are identical to each other and the remaining two units are identical to each other but different from the first four units.

[0072] In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, three units are identical to each other and the remaining three units are identical to each other but different from the first three units. In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, four units are identical to each other, and the other two units are independently different from the first four units such that they are also different from one another.

[0073] In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, three units are identical to each other, a separate two units are identical to each other and the remaining one unit is different from both the first three units and the other two units.

[0074] In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, two units are identical to each other, a separate two units are identical to each other but different from the first two units and the remaining two units are identical to each other but different from the first two units and the second two units.

[0075] In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, three units are identical to each other and the remaining three units are individually different such that they are different from the first three units and different from each other.

[0076] In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, two units are identical to each other, a separate two units are identical to each other but different from the first two units, and the remaining two units are individually different such that they are different from each other and also different from the first two units and the second two units.

[0077] In one embodiment, the hexameric protein is comprised of six units, wherein of the six units, two units are identical to each other and the remaining four units are individually different such that they are different from each other and also different from the first two units.

[0078] In one embodiment, the hexameric protein is comprised of six units, wherein in each unit there is the same N-terminal immunoglobulin.

[0079] In one embodiment, the hexameric protein is comprised of six units, wherein in each unit there is a different N-terminal immunoglobulin.

[0080] In one embodiment, the hexameric protein is comprised of six units, wherein the hexameric protein comprises any combination of six N-terminal immunoglobulins. In one embodiment, the hexameric protein is comprised of six units, wherein in each unit there is the same linker domain.

[0081] In one embodiment, the hexameric protein is comprised of six units, wherein in each unit there is a different linker domain.

[0082] In one embodiment, the hexameric protein is comprised of six units, wherein the hexameric protein comprises any combination of six linker domains.

[0083] N-terminal immunoglobulin

[0084] The present invention relates to a hexameric protein, wherein the hexameric protein is composed of six units. In each unit, there is an N-terminal immunoglobulin. The N-terminal immunoglobulin may comprise an antibody, an antigen-binding fragment of an antibody, or an Fc region.

[0085] In some embodiments, the N-terminal immunoglobulin is responsible for specific binding or targeting of the hexameric protein or antibody to a specific target, antigen, epitope, ligand or any other target protein, peptide, compound or molecule to which the N-terminal immunoglobulin has a specific binding site. The N-terminal immunoglobulin is therefore able to ensure that the hexameric protein or antibody has specific and targeted action.

[0086] In one embodiment, the N-terminal immunoglobulin is an antibody, or an antigen-binding fragment thereof.

[0087] In one embodiment, the N-terminal immunoglobulin comprises an antibody heavy chain and an antibody light chain.

[0088] In one embodiment, the N-terminal immunoglobulin comprises an antibody heavy chain variable region and an antibody light chain variable region.

[0089] In one embodiment, the N-terminal immunoglobulin is an antibody Fc region, or an Fc portion thereof.

[0090] In one embodiment, the N-terminal immunoglobulin comprises or consists of a heavy chain according to any one of SEQ ID NO: 29 to SEQ ID NO: 38, as indicated in the table below (Table 1). Table 1 (underlined sequence corresponds to the Fc region):

[0091] In one embodiment, the N-terminal immunoglobulin comprises or consists of a light chain according to SEQ ID NO: 39:

[0092] SSELTQDPVVSVALGQTVRITCQGDSLRTYHASWYQQKPRQAPVLVIYDENNRPSGIPDRFSGST SGNTASLTITGAQAEDEADYYCNSRDSSGNRLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQAN KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS (SEQ ID NO: 39).

[0093] In one embodiment, the N-terminal immunoglobulin comprises or consists of a heavy chain according to any one of SEQ ID NO: 29 to SEQ ID NO: 38 and a light chain according to SEQ ID NO: 39.

[0094] In one embodiment, the N-terminal immunoglobulin comprises or consists of the following sequence (SEQ ID NO 69):

[0095] NCGPPPTLSFAAPMDITLTETRFKTGTTLKYTCLPGYVRSHSTQTLTCNSDGEWVYNTFCIYKRC RHPGELRNGQVEIKTDLSFGSQIEFSCSEGFFLIGSTTSRCEVQDRGVGWSHPLPQCEIGGSGDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA

[0096] KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGN VFSCSVM H EALH N HYTQKSL In one embodiment, the N-terminal immunoglobulin comprises or consists of a heavy chain according to any one of SEQ ID NO: 70 to SEQ ID NO: 74, as indicated in the table below (Table 1A).

[0097] Table 1A

[0098] In one embodiment, the N-terminal immunoglobulin comprises or consists of a light chain according to any one of SEQ ID NO: 75 to SEQ ID NO: 79, as indicated in the table below (Table 1 B).

[0099] Table 1 B

[0100] Linker domain

[0101] The present invention relates to a hexameric protein wherein the hexameric protein is composed of six units. Within each unit, an N-terminal immunoglobulin, or fragment thereof, and a C-terminal tailpiece are connected via a linker domain.

[0102] In one embodiment, the N-terminal immunoglobulin, or fragment thereof, and the C-terminal tailpiece are connected via covalent attachment to a linker domain.

[0103] In one embodiment, the linker domain is derived from an immunoglobulin.

[0104] In one embodiment, the linker domain is derived from IgM, lgA1 , lgA2, or lgG1 , lgG2, lgG3 & lgG4.

[0105] In one embodiment, the linker domain is derived from any one of an lgG1-CH3, lgG2-CH3, lgG3- CH3, lgG4-CH3 domain.

[0106] In one embodiment, the linker domain is derived from any one of an lgA1-CH3 or lgA2-CH3 domain.

[0107] In one embodiment, the linker domain is derived from an lgM-CH4 domain.

[0108] In one embodiment the linker domain has an amino acid sequence according to SEQ ID NO: 40 to SEQ ID NO: 49, as shown in the table below (Table 2).

[0109] Table 2: In one embodiment, the linker domain of any one of SEQ ID NO: 40 to SEQ ID NO: 49 is attached to the C-terminus of any one of SEQ ID NO: 29 to SEQ ID NO: 38.

[0110] In one embodiment, the linker domain of any one of SEQ ID NO: 40 to SEQ ID NO: 49 is covalently attached to the C-terminus of any one of SEQ ID NO: 29 to SEQ ID NO: 38.

[0111] In one embodiment, the linker domain of any one of SEQ ID NO: 40 to SEQ ID NO: 49 is attached to the N-terminus of any one of SEQ ID NO: 1 to SEQ ID NO: 28.

[0112] In one embodiment, the linker domain of any one of SEQ ID NO: 40 to SEQ ID NO: 49 is covalently attached to the N-terminus of any one of SEQ ID NO: 1 to SEQ ID NO: 28.

[0113] In one embodiment, the linker domain of any one of SEQ ID NO: 40 to SEQ ID NO: 49 is covalently attached to:

[0114] (a) the C-terminus of any one of SEQ ID NO: 29 to SEQ ID NO: 38, and

[0115] (b) the N-terminus of any one of SEQ ID NO: 1 to SEQ ID NO: 28.

[0116] C-terminal tailpiece

[0117] The present invention relates to a hexameric protein wherein the hexameric protein is composed of six units. Within each unit, the N-terminal immunoglobulin, or fragment thereof, is connected to the C-terminal tailpiece via a linker domain, wherein the C-terminal tailpiece is responsible for interacting with other C-terminal tailpieces to mediate hexamerization.

[0118] The C-terminal tailpieces may interact with other tailpieces through a variety of mechanisms, including covalent and non-covalent interactions between the tailpieces.

[0119] In one embodiment, the C-terminal tailpieces form disulfide bridges between each other in order to form a hexameric tailpiece.

[0120] In one embodiment, the C-terminal tailpiece comprises or consists of about 5-40 amino acids, such as about 10-30 amino acids, such as about 11-29 amino acids, such as about 12-28 amino acids, such as about 13-27 amino acids, such as about 14-26 amino acids, such as about 15-25 amino acids, such as about 16-24 amino acids.

[0121] In one embodiment the C-terminal tailpiece comprises or consists of 14 amino acids. In one embodiment the C-terminal tailpiece comprises or consists of 15 amino acids.

[0122] In one embodiment the C-terminal tailpiece comprises or consists of 16 amino acids.

[0123] In one embodiment the C-terminal tailpiece comprises or consists of 17 amino acids.

[0124] In one embodiment, the C-terminal tailpiece comprises or consists of 18 amino acids.

[0125] In one embodiment the C-terminal tailpiece comprises or consists of 19 amino acids.

[0126] In one embodiment the C-terminal tailpiece comprises or consists of 20 amino acids.

[0127] In one embodiment the C-terminal tailpiece comprises or consists of 21 amino acids.

[0128] In one embodiment the C-terminal tailpiece comprises or consists of 22 amino acids.

[0129] In one embodiment the C-terminal tailpiece comprises or consists of 23 amino acids.

[0130] In one embodiment the C-terminal tailpiece comprises or consists of 24 amino acids.

[0131] In one embodiment, the C-terminal tailpiece comprises or consists of 18 amino acids, wherein the penultimate amino acid is a cysteine (C).

[0132] In one embodiment the penultimate amino acid of the C-terminal tail piece is a cysteine (C) which undergoes disulfide bridge formation via its side chain with another cysteine of a C-terminal tailpiece in order to form a hexameric tailpiece.

[0133] In one embodiment, the C-terminal tailpiece comprises or consists of 18 amino acids, wherein the penultimate amino acid is a cysteine (C) which undergoes disulfide bridge formation via its side chain with another cysteine of a C-terminal tailpiece in order to form a hexameric tailpiece.

[0134] In one embodiment the hexameric tailpiece may be derived from IgM.

[0135] A hexameric tailpiece derived from IgM may be referred to as a ptp.

[0136] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is derived from a mammalian, reptilian or avian IgM. In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is derived from a human, mouse, pig, horse, Abington island giant tortoise, tuatara or chicken IgM.

[0137] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece comprises or consists of an amino acid sequence according to any one of SEQ ID NO: 1 to SEQ ID NO: 28, as shown in the table below (Table 3).

[0138] Table 3:

[0139]

[0140] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is attached to the C-terminus of an immunoglobulin. In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is covalently attached to an immunoglobulin.

[0141] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is covalently attached to the C-terminus of an immunoglobulin.

[0142] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is attached to the C-terminus of a linker domain.

[0143] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is covalently attached to the C-terminus of a linker domain.

[0144] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is covalently attached to the C-terminus of a linker domain that is covalently attached to an immunoglobulin Fc region.

[0145] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is fused to the C-terminus of an immunoglobulin Fc region via a linker amino acid sequence.

[0146] In one embodiment, the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein the tailpiece is fused to the C-terminus of an immunoglobulin Fc region via a linker amino acid sequence of any one of SEQ ID NO: 40 to SEQ ID NO: 49.

[0147] In one embodiment, the present invention relates to a hexameric protein or antibody as disclosed herein, wherein the tailpiece is covalently attached to the C-terminus of an Fc region of any one of SEQ ID NO: 29 to SEQ ID NO: 38, wherein the tailpiece is attached via a linker amino acid sequence of any one of SEQ ID NO: 40 to SEQ ID NO: 49.

[0148] Hexameric Fc Proteins

[0149] In one embodiment, the present invention relates to a hexameric protein, wherein the hexameric protein is composed of six units and wherein each unit comprises:

[0150] (a) an N-terminal Fc region, or a fragment thereof;

[0151] (b) a linker domain; and

[0152] (c) a C-terminal tailpiece derived from IgM. In one embodiment, the N-terminal Fc region may be conjugated to a peptide, a protein, an extracellular domain of a receptor, a protein binding domain, a cytokine, an enzyme, a drug, a toxin or a combination thereof.

[0153] In one embodiment, the present invention relates to a hexameric fusion protein, wherein the hexameric fusion protein is composed of six units and wherein each unit comprises:

[0154] (a) an N-terminal peptide;

[0155] (b) an Fc region;

[0156] (c) a linker domain; and

[0157] (d) a C-terminal tailpiece derived from IgM, wherein the N-terminal peptide may be an extracellular domain of a receptor, a protein binding domain, a cytokine, an enzyme, a drug, a toxin or a combination thereof.

[0158] In one embodiment, the present invention relates to a hexameric protein, wherein the hexameric protein comprises the Fc region of any of SEQ ID NO: 29 to SEQ ID NO: 38.

[0159] In one embodiment the Fc region is covalently attached to any of SEQ ID NO: 1 to SEQ ID NO: 28.

[0160] In one embodiment, the present invention relates to a hexameric protein, wherein the hexameric protein comprises the Fc region of any of SEQ ID NO: 29 to SEQ ID NO: 38, wherein said Fc region is covalently attached to any of SEQ ID NO: 1 to SEQ ID NO: 28 via a linker domain.

[0161] In one embodiment, the present invention relates to a hexameric protein, wherein the hexameric protein comprises the Fc region of any of SEQ ID NO: 29 to SEQ ID NO: 38, wherein said Fc region is covalently attached to any of SEQ ID NO: 1 to SEQ ID NO: 28 via a linker domain according to any of SEQ ID NO: 40 to SEQ ID NO: 49.

[0162] In one embodiment, the present invention relates to a hexameric fusion protein, wherein:

[0163] (a) the hexameric fusion protein comprises the Fc region of any of SEQ ID NO: 29 to SEQ ID NO: 38, wherein said Fc region is covalently attached to any of SEQ ID NO: 1 to SEQ ID NO: 28 via a linker domain according to any of SEQ ID NO: 40 to SEQ ID NO: 49; and

[0164] (b) a peptide, a protein, an extracellular domain of a receptor, a protein binding domain, a cytokine, an enzyme, a drug, a toxin or a combination thereof is covalently attached to the N-terminus of the Fc region of any of SEQ ID NO: 29 to SEQ ID NO: 38. Hexameric Antibodies

[0165] In one embodiment, the present invention relates to a hexameric antibody, wherein the hexameric antibody is composed of six units and wherein each unit comprises:

[0166] (a) an N-terminal antibody, or an antigen-binding fragment thereof;

[0167] (b) a linker domain; and

[0168] (c) a C-terminal tailpiece derived from IgM.

[0169] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal antibody, or antigen-binding fragment thereof, is independently an N-terminal IgG or IgM, or an antigen-binding fragment thereof, which is capable of binding one or more ligand molecules.

[0170] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM is independently selected from any one of an IgM, lgG1 , lgG2, lgG3 or lgG4.

[0171] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 29 to SEQ ID NO: 38.

[0172] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 70 to SEQ ID NO: 74.

[0173] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 39.

[0174] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 75 to SEQ ID NO: 79. In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 29 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100 sequence identity to SEQ ID NO: 39.

[0175] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0176] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 31 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0177] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 32 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0178] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 33 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0179] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 34 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0180] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 35 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0181] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein each unit comprises an N-terminal IgG, wherein the heavy chain has a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 36 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0182] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 37 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0183] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 38 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0184] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 69 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0185] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 70 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 75.

[0186] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 71 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0187] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 72 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 77.

[0188] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 73 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.

[0189] In one embodiment the present invention relates to a hexameric antibody, as disclosed herein, wherein within each unit the N-terminal IgG has a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 74 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 79. The term modified heavy chain is used herein to denote the full amino acid sequence that comprises or consists of the N-terminal immunoglobulin, the linker domain and the tailpiece.

[0190] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 50 to SEQ ID NO: 68, as shown in the table below (Table 4).

[0191] Table 4:

[0192] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 50 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0193] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0194] 51 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0195] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0196] 52 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0197] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0198] 53 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0199] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0200] 54 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0201] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0202] 55 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39. In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0203] 56 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0204] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0205] 57 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0206] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0207] 58 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0208] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0209] 59 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0210] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0211] 60 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39. In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0212] 61 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0213] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0214] 62 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0215] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0216] 63 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0217] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0218] 64 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0219] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0220] 65 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0221] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0222] 66 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0223] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0224] 67 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0225] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0226] 68 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0227] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0228] 80 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0229] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0230] 81 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 75.

[0231] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 82 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0232] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0233] 83 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 77.

[0234] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0235] 84 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.

[0236] In one embodiment the present invention relates to a hexameric protein or antibody, as disclosed herein, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:

[0237] 85 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 79.

[0238] Antibody fragments

[0239] Whilst the disclosure herein discusses IgG at greater length, the scope of embodiments is not limited to IgG, but includes other naturally occurring antibodies, such as IgM, IgA, IgE and IgD and their respective subtypes and allotypes. The scope of embodiments further includes antigen-binding fragments of said antibody, including but not limited to an antigen-binding fragment (Fab), a fragment antibody (F(ab’)2), single chain antibody (scFv) and a single-domain antibody (sdAb).

[0240] A fragment antibody (F(ab’)2), refers to a region on an antibody that remains following digestion of the Fc region which leaves intact portions of the hinge region.

[0241] A single chain antibody (scFv) refers to an antibody that has been engineered to consist of a light and heavy chain variable region that are connected by a peptide-linker sequence. The peptide linker sequence is typically in the length of 10-25 amino acids, rich in glycine for flexibility and rich in serine or threonine for solubility. The peptide linker may connect the N-terminus of the heavy chain variable region with the C-terminus of the light chain variable region.

[0242] A single domain antibody (sdAb), which is often referred to as a nanobody, refers to an antigenbinding fragment of an antibody that consists of a single monomeric variable antibody domain. Therefore, an sdAb may be a light chain variable region or a heavy chain variable region. Examples of single-domain antibodies include, but are not limited to, VHH fragments and VNAR fragments. VHH and VNAR fragments comprise the antigen-binding fragment of the heavy chain.

[0243] In one embodiment, the antigen-binding fragment of the antibodies disclosed herein may be any fragment of the antibodies disclosed herein.

[0244] In another embodiment the antigen-binding fragment of the antibodies disclosed herein may be a genetically-engineered product of one or more of the fragments of the antibody.

[0245] Whilst the disclosure herein discusses IgG at greater length, the scope of embodiments is not limited to IgG, but includes other naturally occurring antibodies, such as IgM, IgA, IgE and IgD and their respective subtypes.

[0246] In one embodiment the fragment is an antigen-binding fragment (Fab), a fragment antibody (F(ab’)2), a single-chain variable fragment (scFv) or a single domain antibody (sdAb), or a camelid antibody (VHH).

[0247] The antibodies, or antibody-binding fragments thereof, disclosed herein may be combined to produce an engineered antigen binding protein that displays specificity to two or more different antigens or epitopes. Further, the antibodies, or antigen-binding fragments thereof, disclosed herein may be combined with alternative antibodies to produce an engineered antigen binding protein that displays specificity to two or more different antigens or epitopes.

[0248] Hexameric protein-drug conjugates

[0249] In one embodiment, the hexameric protein or hexameric antibody of the present invention may be conjugated to a drug molecule.

[0250] Antibody-drug conjugates (ADCs), for example, are therapeutic molecules that may be specifically targeted to an antigen via the antibody, or an antigen-binding fragment thereof, enabling precise delivery of a drug to a specific location. For example, ADCs have been used as chemotherapeutic agents to target cytotoxic drugs to antigen-expressing tumour cells, whereupon they are internalized by said tumour cells, enabling the cytotoxic drug to take effect in specific tumour cells. This strategy has been further advanced through the conjugation of drugs to antibodies through cleavable linker molecules, enabling the ADC to be cleaved once internalised into a specific cell, releasing the cytotoxic drug such that it has greater activity.

[0251] In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein.

[0252] In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein and a coupling moiety.

[0253] In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein and a coupling moiety connected by a cleavable linker-molecule.

[0254] In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein and a coupling moiety connected by a non-cleavable linker-molecule.

[0255] In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein and a coupling moiety wherein the coupling moiety is a drug.

[0256] In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein and a coupling moiety wherein the coupling moiety is a toxin.

[0257] In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein and a coupling moiety wherein the coupling moiety is a cytokine. In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein a coupling moiety wherein the coupling moiety is an enzyme.

[0258] In one embodiment, the present invention provides a hexameric protein-drug conjugate or hexameric antibody-drug conjugate comprising the hexameric protein or hexameric antibody, disclosed herein and a coupling moiety wherein the coupling moiety is a drug, toxin, cytokine, enzyme or a combination thereof.

[0259] Hexameric Protein-Toxin Fusion Proteins

[0260] In one embodiment, the hexameric protein or hexameric antibody of the present invention may be conjugated to a toxin moiety.

[0261] An anti body- toxin fusion protein, for example, such as a recombinant immunotoxin, comprises an antibody, or antigen-binding fragment thereof, or immunoglobulin that is linked to a peptide toxin. Such an antibody-toxin fusion protein may be encoded by a recombinant DNA sequence that is subsequently expressed. The recombinant DNA sequence, also known as a fusion gene, may be expressed via an expression vector or plasmid that comprises the DNA segments which thus direct the synthesis of such a fusion protein. The antibody-toxin fusion protein, or recombinant immunotoxin, therefore comprises an amino acid sequence representing an antibody, antigenbinding fragment thereof, or immunoglobulin linked to peptide toxin.

[0262] In other words, a recombinant immunotoxin is a polypeptide in which a peptide toxin is genetically linked to an antibody or protein component, often by a contiguous polypeptide linker.

[0263] Much like antibody-drug conjugates, anti body- toxin fusion proteins enable targeted accumulation and activation of the toxin for specific effect, promoting efficiency while minimising harm to normal cells.

[0264] In one embodiment, the present invention provides a hexameric recombinant fusion protein wherein the hexameric fusion protein comprises an antibody, antigen-binding fragment thereof or Fc region thereof, as disclosed herein, that is linked to a peptide toxin.

[0265] In one embodiment, the present invention provides a hexameric recombinant fusion protein wherein the hexameric fusion protein comprises an antibody, or antigen-binding fragment thereof or Fc region thereof, as disclosed herein, wherein at least one of the heavy or light chain regions are linked to a peptide toxin.

[0266] Formulations

[0267] In another aspect the invention provides a pharmaceutical composition comprising a compound as disclosed herein, or pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition of the invention comprises a compound as disclosed herein, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or vehicle.

[0268] Any of the hexameric proteins or hexameric antibodies of the invention may be in the form of a pharmaceutically acceptable salt. All references to “a compound as disclosed herein”, “a hexameric protein as disclosed herein” or “a hexameric antibody as disclosed herein” herein should be considered to encompass any pharmaceutically acceptable salt thereof, regardless of whether “pharmaceutically acceptable salt” is explicitly recited.

[0269] In one embodiment, a therapeutically effective amount of the hexameric protein or hexameric antibody as disclosed herein may be administered to a subject. As will be apparent to one skilled in the art, a “therapeutically effective amount” of a compound or pharmaceutical composition of the present invention will vary depending upon, amongst other factors, the age, weight and / or gender of the subject (patient) to be treated. Such factors and their relationship in determining a therapeutically effective amount are well known in the medical arts, therefore the determination of therapeutically effective dosage levels to achieve the desired therapeutic effect will be within the ambit of the skilled person.

[0270] Medical uses and methods of treatment

[0271] In a further aspect, the present invention provides a hexameric protein or hexameric antibody, as disclosed herein, or a pharmaceutically acceptable salt thereof, for use in a method of medical treatment or prophylaxis. In other words, the present invention provides a method of medical treatment or prophylaxis comprising administering the hexameric protein or hexameric antibody of the invention to a subject or patient.

[0272] The present inventors find that the hexameric protein or hexameric antibody, as disclosed herein, for use in therapy, prophylaxis or a method of medical treatment, displays improved and advantageous properties. For example, the hexameric protein or hexameric antibody is capable of inducing agglutination, complement activation and / or an immunogenic response.

[0273] In one embodiment, the hexameric protein or hexameric antibody, as disclosed herein, is capable of inducing agglutination.

[0274] Agglutination occurs due to antigen-antibody interaction, wherein the antibody binds multiple antigens to form a lattice like structure. Agglutination thus results in the aggregation of antigen expressing cells or particles, enabling enhanced entrapment and clearance of said antigen expressing cells or particles, such as by phagocytosis. In the context of motile cells, agglutination may result in the loss of motility of said cells.

[0275] In one embodiment, the hexameric protein or hexameric antibody, as disclosed herein, is capable of inducing complement activation.

[0276] Antibodies may activate the complement cascade through directly binding to complement components. Activation of the complement cascade may result in a number of consequences. The complement system may directly result in lysis of cells or pathogens, cells may be tagged by complement proteins and marked for phagocytosis or complement proteins may recruit leukocytes to the site of infection.

[0277] In one embodiment, the hexameric protein or hexameric antibody, as disclosed herein, is capable of inducing an immunogenic response.

[0278] Immunogenicity is defined as the ability of a molecule or a cell to generate an immune response. As indicated above, an antibody may do so through activation of the complement cascade and recruitment of leukocytes. An antibody may also mediate various immune responses through its Fc effector functions, which include antibody-dependent cellular cytotoxicity (ADCC) and antibodydependent cellular phagocytosis (ADCP).

[0279] Contraceptive agent

[0280] There remains a global demand for non-hormonal contraceptives in order to overcome the real and / or perceived side effects of traditional hormonal contraception. The present inventors surprisingly demonstrate that the hexameric protein or hexameric antibody of the present invention has advantageous properties when used as a contraceptive agent. In particular, the present inventors demonstrate improved agglutination and sperm immobilization. In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use as a contraceptive agent.

[0281] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use as a non-hormonal contraceptive agent.

[0282] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use with a hormonal contraceptive agent.

[0283] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use as a non-hormonal contraceptive agent, such as for use in or on vaginal film, a vaginal ring, vaginal gel, a cervical cap or diaphragm, a male condom or a female condom.

[0284] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use in a vaginal ring.

[0285] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use in a vaginal film.

[0286] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use in the prevention of sperm motility.

[0287] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use in the manufacture of a contraceptive agent.

[0288] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use in the manufacture of a non-hormonal contraceptive agent.

[0289] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use in the manufacture of a hormonal contraceptive agent.

[0290] In one embodiment, the present invention provides a hexameric antibody, as disclosed herein, for use in the manufacture of a vaginal ring.

[0291] In one embodiment, the present invention provides a method of contraception in a subject comprising administering to said subject the hexameric antibody, as disclosed herein. In one embodiment, the present invention provides a method of contraception in a subject comprising administering to said subject the hexameric antibody, as disclosed herein, as part of a vaginal ring.

[0292] In one embodiment, the present invention provides a hexameric protein or antibody or pharmaceutical composition as disclosed herein, wherein the hexameric protein or antibody binds to CD52g.

[0293] In one embodiment, the present invention provides a hexameric protein or antibody or pharmaceutical composition as disclosed herein, wherein the hexameric protein or hexameric antibody, or antigen-binding fragment thereof, binds to CD52g on the surface of human spermatozoa resulting in agglutination and / or immobilization of the spermatozoa.

[0294] Neutralization of pathogenic infection

[0295] In one aspect, the present invention provides a hexameric protein or hexameric antibody, as disclosed herein, for use in the treatment of a pathogenic infection.

[0296] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, for use in the treatment of a pathogenic infection, wherein the pathogenic infection is viral, bacterial or fungal.

[0297] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, for use in the treatment or prevention of a pathogenic infection, wherein the pathogenic infection is viral, and the virus that causes the infection is any one of human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), Epstein-Barr virus EBV, Coronavirus or herpes simplex virus (HSV).

[0298] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding glycoprotein F derived from RSV.

[0299] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding glycoprotein G derived from RSV.

[0300] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding glycoprotein F or G on the surface of the RSV virion. In one embodiment the hexameric antibody capable of binding glycoprotein F on the surface of the RSV virion has an N-terminal IgG comprising a a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 74 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 79.

[0301] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding glycoprotein F or G on the surface of the RSV virion and preventing infection by RSV.

[0302] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding gp41 derived from HIV.

[0303] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding gp120 derived from HIV.

[0304] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding gp41 or gp120 on the surface of the HIV virion and preventing infection by HIV.

[0305] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding gH / gL derived from EBV.

[0306] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding gp42 derived from EBV.

[0307] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding gH / gL or gp42 on the surface of the EBV virion and preventing infection by EBV.

[0308] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding spike proteins derived from Coronavirus.

[0309] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding spike protein on the surface of the Coronavirus virion and preventing infection by Coronavirus. In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding glycoprotein D derived from HSV.

[0310] In one embodiment the hexameric antibody capable of binding glycoprotein D derived from HSV has an N-terminal IgG comprising a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 73 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.

[0311] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding glycoprotein D on the surface of the HSV virion and preventing infection by HSV.

[0312] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, for use in the treatment of a pathogenic infection, wherein the pathogenic infection is bacterial, and the bacterium that causes the infection is Vibrio cholera, Salmonella sp., Borrelia burgdorferi, or Neisseria gonorrhoeae.

[0313] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding O5-antigen of STm lipopolysaccharide derived from Salmonella.

[0314] In one embodiment the hexameric antibody capable of binding O5-antigen of STm lipopolysaccharide derived from Salmonella has an N-terminal IgG comprising a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 70 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 75.

[0315] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding the core / lipid A region of 01 LPS derived from Vibrio cholera.

[0316] In one embodiment the hexameric antibody capable of binding the core / lipid A region of 01 LPS derived from Vibrio cholera has an N-terminal IgG comprising a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 71 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0317] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding the outer surface protein (OspA) derived from Borrelia burgdorferi.

[0318] In one embodiment the hexameric antibody capable of binding the outer surface protein (OspA) derived from Borrelia burgdorferi has an N-terminal IgG comprising a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 72 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 77.

[0319] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding the 2C7 epitope on gonococcal lipooligosaccharides and / or the major outer membrane porin protein (ProB) derived from Neisseria gonorrhoeae.

[0320] In one embodiment the hexameric antibody capable of binding the 2C7 epitope on gonococcal lipooligosaccharides and / or the major outer membrane porin protein (ProB) derived from Neisseria gonorrhoeae has an N-terminal IgG comprising a heavy chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 69 and a light chain with an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0321] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, for use in the treatment of a fungal infection

[0322] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding to a p-glucan.

[0323] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding to 1,3-p-glucan.

[0324] Oncology In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, wherein the hexameric protein or hexameric antibody is capable of recognising a tumour antigen.

[0325] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, wherein the hexameric protein or hexameric antibody is capable of inducing an immunogenic response.

[0326] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, for use in the treatment of cancer.

[0327] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, for use in the treatment of a cancer tumour.

[0328] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, for use in the treatment of a glioma.

[0329] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding to vimentin.

[0330] In one embodiment, the present invention provides a hexameric protein or antibody, as disclosed herein, that is capable of binding to vimentin on the surface of glioma cells.

[0331] Expression System

[0332] The present invention further relates to a method of production and engineering of hexameric proteins or antibodies, as disclosed herein.

[0333] In some embodiments, a range of production hosts may be used for production of the antibody, or an antigen-binding fragment thereof, disclosed herein. Such hosts, which are utilized for the production of antibodies, or antigen-binding fragments thereof, may include, but are not limited to mammalian cells (eg. CHO cells), plant cells (eg. Nicotians benthamiana cells), eukaryotic cells (eg. yeast cells) and microbial cells.

[0334] In some embodiments within the scope of the present disclosure and as known in the art, one or more plasmid expression vectors may be assembled and transfected into living cells for transient expression by the host. If multiple expression vectors are used, they can be transfected into the cells either separately or jointly, and expressed by the host. In a preferred embodiment, an engineered Nicotiana benthamiana (Nb) plant strain is the production host into which one or more expression vectors containing nucleic acids encoding the antibody, or antigen-binding fragments thereof, of the present disclosure are transfected.

[0335] In one embodiment, the plasmid expression vector is transfected into Agrobacterium hosts.

[0336] In a preferred embodiment, the plasmid expression vector is transfected into Agrobacterium tumefaciens.

[0337] In a further embodiment, vector-carrying Agrobacterium are introduced into plant hosts by spray inoculation in the presence of mild abrasion. In a further embodiment, vector-carrying Agrobacterium are introduced into plant hosts by vacuum infiltration.

[0338] While not intending to limit the scope of the present embodiments, it is noted that plant based antibody production systems can avoid certain pitfalls of mammalian-based monoclonal antibody production. One such system, provided as a non-limiting example, utilizes an engineered Nicotiana benthamiana (Nb) plant strain expressing one or more expression vectors containing nucleic acid constructs for the formation of the desired Abs. In some embodiments within the scope of the present disclosure, and as known in the art, one or more plasmid expression vectors are assembled and then transfected into living cells for transient expression by the host. If multiple expression vectors are used, they can be transfected into the cells, for example as found in plant tissue of Nb, either separately or jointly, and expressed by the host. An exemplary transient expression process uses Agrobacterium strains (e.g., Agrobacterium tumefaciens), in which the Agrobacterium are grown and infiltrated into whole plants, infecting the plant and introducing the genes to the plant cells. After infiltration and transient expression of the genes of interest in the germinated plants, proper feeding and care of the infiltrated plants provides for desired plant growth and ultimately harvesting of the Ab-containing infiltrated plant samples. The desired Ab product then is extracted from the harvested parts of the plant, generally followed by purification and characterization of the extracted monoclonal antibodies.

[0339] As will be appreciated by persons of ordinary skill in the relevant art, a range of other production hosts can be suitable for use in protein and antibody production. Such hosts which are utilized for the production of proteins and antibodies in accordance with the present embodiments may include, but are not necessarily limited to, mammalian (e.g., CHO cells), plant (e.g., Nb plants or plant cells), yeast, and eukaryotic, and microbial cells. Through known steps available in the relevant literature, such hosts have been developed through genetic engineering, mutagenesis, or (in the case of plants) selective breeding aimed at enhancing mAb production or provide additional physicochemical characteristics in terms of increased or decreased molecular, metabolic, chemical, phenotypic, or other traits that affect protein formation by host.

[0340] Present embodiments provide for representative amino acid sequences of monoclonal antibodies derived from murine sources, which have been humanized to modify the amino acid sequences to increase their similarity to antibody sequence variants found in humans

[0341] Numbered Paragraphs

[0342] 1. A hexameric protein, wherein the hexameric protein is composed of six units and wherein each unit comprises:

[0343] (a) an N-terminal immunoglobulin, or a fragment thereof;

[0344] (b) a linker domain; and

[0345] (c) a C-terminal tailpiece derived from IgM.

[0346] 2. The hexameric protein of paragraph 1 , wherein the N-terminal immunoglobulin, or a fragment thereof, comprises an immunoglobulin Fc region.

[0347] 3. The hexameric protein of paragraph 1 or paragraph 2, wherein the hexameric protein is a hexameric antibody.

[0348] 4. The hexameric antibody of paragraph 3, wherein within each unit the N-terminal immunoglobulin or a fragment thereof is independently an N-terminal IgG or IgM, or an antigen-binding fragment thereof, which is capable of binding one or more ligand molecules.

[0349] 5. The hexameric antibody of paragraph 4, wherein within each unit the N-terminal IgG or IgM is independently selected from any one of an IgM, lgG1 , lgG2, lgG3 or lgG4.

[0350] 6. The hexameric antibody of paragraph 5, wherein within each unit the N-terminal IgG or IgM has an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 29 to SEQ ID NO: 38 and SEQ ID NO: 69 to SEQ ID NO: 74.

[0351] 7. The hexameric antibody of any one of paragraphs 3-6, wherein within each unit the N-terminal IgG or IgM has a light chain with an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 39 and SEQ ID NO: 75 to SEQ ID NO: 79. 8. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 29 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0352] 9. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 30 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0353] 10. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 31 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0354] 11. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 32 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0355] 12. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 33 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0356] 13. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 34 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0357] 14. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 35 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0358] 15. The hexameric antibody of any one of paragraphs 3-7, wherein each unit comprises an N- terminal IgG, wherein the heavy chain has a sequence with at least 70% sequence identity to SEQ ID NO: 36 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0359] 16. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 37 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0360] 17. The hexameric antibody of any one of paragraphs 3-7, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 38 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

[0361] 18. The hexameric antibody of any one of paragraphs 6 or 8 - 17 wherein the heavy chain has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the recited sequence.

[0362] 19. The hexameric antibody of any one of paragraphs 7 - 17 wherein the light chain has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the recited sequence.

[0363] 20. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the linker domain is derived from an immunoglobulin.

[0364] 21. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the linker domain is derived from IgM, lgA1 , lgA2, or IgG.

[0365] 22. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the linker domain has an amino acid sequence according to SEQ ID NO: 40 to SEQ ID NO: 49.

[0366] 23. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece is derived from a mammalian, reptilian or avian IgM.

[0367] 24. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece is derived from a human, mouse, pig, horse, Abington island giant tortoise, tuatara or chicken IgM. 25. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece has an amino acid sequence according to any one of SEQ ID NO: 1 to SEQ ID NO: 28.

[0368] 26. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece is fused to the C-terminus of an immunoglobulin.

[0369] 27. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece is fused to the C-terminus of an immunoglobulin Fc region.

[0370] 28. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece is fused to the C-terminus of an lgG1 , lgG2, lgG3, lgG4, IgGI (PtoS) or lgG3.17 Fc region.

[0371] 29. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece mediates hexamerization.

[0372] 30. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece mediates hexamerization through the formation of covalent or non-covalent interactions with other tailpieces.

[0373] 31 . The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece mediates hexamerization through the formation of disulfide bridges with other tailpieces.

[0374] 32. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece is fused to the C-terminus of an immunoglobulin Fc region via a linker amino acid sequence of any one of SEQ ID NO: 40 to SEQ ID NO: 49.

[0375] 33. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the tailpiece is fused to the C-terminus of an lgG1 , lgG2, lgG3, lgG4, IgGI (PtoS) or lgG3.17 Fc region via a linker amino acid sequence of any one of SEQ ID NO: 40 to SEQ ID NO: 49.

[0376] 34. The hexameric protein or antibody of any one of the preceding paragraphs, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 50 to SEQ ID NO: 68

[0377] 35. The hexameric protein or antibody of paragraph 34, wherein the modified heavy chain has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the recited sequence. 36. The hexameric protein or antibody of any one of the preceding paragraphs, wherein each unit comprises the same modified heavy chain.

[0378] 37. The hexameric protein or antibody of any one of the preceding paragraphs, wherein each unit comprises different modified heavy chains.

[0379] 38. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the hexameric antibody comprises any combination of six modified heavy chains, wherein the modified heavy chain has an amino acid sequence according to any one of SEQ ID NO: 50 to SEQ ID NO: 68.

[0380] 39. The hexameric protein or antibody of any one of paragraphs 3-38, wherein each unit comprises an N-terminal immunoglobulin or a fragment thereof, wherein the N-terminal immunoglobulin is an N-terminal IgG or IgM, or an antigen-binding fragment thereof, which is capable of binding one or more ligand molecules, wherein the IgG or IgM, or antigen-binding fragment thereof, comprises or consists of an antigen-binding fragment (Fab), a fragment antibody (F(ab’)2), single-chain variable fragment (scFv), a single-domain antibody (sdAb), variable domain of heavy-chain antibodies (VHH), variable new antigen receptor (VNARs), or designed ankyrin repeat protein (DARPin).

[0381] 40. The hexameric protein or antibody of any one of the preceding paragraphs wherein the IgG or IgM domain is capable of binding one or more ligand molecules, wherein the ligand molecules is selected from any one of spermatocyte CD52g, RSV glycoprotein F, RSV glycoprotein G, HIV gp41 , HIV gp120, EBV gH / gL, EBV gp42, coronavirus spike protein, Salmonella O5-antigen of STm lipopolysaccharide, Vibrio cholerae core / lipid A region of 01 LPS, Borrelia burgdorferi outer surface protein (OspA), Neisseria gonorrhoeae 2C7 epitope on gonococcal lipooligosaccharides and ProB, fungal 1 ,3-p-glucan, and HSV glycoprotein D.

[0382] 41. The hexameric protein or antibody of any one of the preceding paragraphs wherein the hexameric protein or antibody induces agglutination.

[0383] 42. The hexameric protein or antibody of any one of the preceding paragraphs wherein the hexameric protein or antibody induces complement activation.

[0384] 43. The hexameric protein or antibody of any one of the preceding paragraphs wherein the hexameric protein or antibody induces an immunogenic response. 44. The hexameric protein or antibody of any one of the preceding paragraphs, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 80 to SEQ ID NO: 85.

[0385] 45. A pharmaceutical composition comprising the hexameric protein or antibody according to any one of the preceding paragraphs, and a pharmaceutical excipient, adjuvant or carrier.

[0386] 46. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs, wherein the antibody binds to human spermatozoa.

[0387] 47. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs, wherein the antibody binds to CD52g.

[0388] 48. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs, wherein the antibody, or antigen-binding fragment thereof, binds to CD52g on the surface of human spermatozoa resulting in agglutination and / or immobilization of the spermatozoa.

[0389] 49. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the hexameric protein or antibody is conjugated to an active agent.

[0390] 50. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the hexameric protein or antibody is conjugated to a pharmaceutically active agent.

[0391] 51. The hexameric protein or antibody of any one of the preceding paragraphs, wherein the hexameric protein or antibody is conjugated to a peptide, a protein, an extracellular domain of a receptor, a protein binding domain, a cytokine, an enzyme, a drug, a toxin or a combination thereof.

[0392] 52. The hexameric protein or antibody of paragraph 51 , wherein the hexameric protein or antibody is conjugated to a peptide, a protein, an extracellular domain of a receptor, a protein binding domain, a cytokine, an enzyme, a drug, a toxin or a combination thereof, at the N-terminal immunoglobulin or fragment thereof.

[0393] 53. The hexameric protein or antibody of paragraph 52, wherein the N-terminal immunoglobulin or fragment thereof comprises an Fc region.

[0394] 54. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs for use in the manufacture of a medicament. 55. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs for use in therapy.

[0395] 56. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs for use in prophylactic therapy.

[0396] 57. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs for use in the manufacture of a contraceptive.

[0397] 58. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs for use in the manufacture of a non-hormonal contraceptive.

[0398] 59. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs for use as a contraceptive agent.

[0399] 60. The hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs for use as a non-hormonal contraceptive agent.

[0400] 61. A method of contraception comprising administering the hexameric protein or antibody or pharmaceutical composition of any one of the preceding paragraphs.

[0401] 62. The hexameric protein or antibody or pharmaceutical composition of any of paragraphs 1-45 for use in the treatment of a pathogenic infection.

[0402] 63. The hexameric protein or antibody or pharmaceutical composition of paragraph 62 for use in the treatment of a pathogenic infection, wherein the pathogenic infection is viral, bacterial or fungal.

[0403] 64. The hexameric protein or antibody or pharmaceutical composition of paragraph 63 for use in the treatment of a pathogenic infection, wherein the pathogenic infection is viral, and the virus that causes the infection is any one of HIV, RSV, EBV or Coronavirus.

[0404] 65. The hexameric protein or antibody or pharmaceutical composition of paragraph 64 for use in the treatment of a pathogenic infection, wherein the pathogenic infection is bacterial, and the bacterium that causes the infection is Vibrio cholerae, Salmonella sp., Borrelia burgdorferi, or Neisseria gonorrhoeae. 66. The hexameric protein or antibody or pharmaceutical composition of any of paragraphs 1-45, wherein the antibody is capable of recognising a tumour antigen.

[0405] 67. The hexameric protein or antibody or pharmaceutical composition of paragraph 66, wherein the antibody is capable of inducing an immunogenic response.

[0406] 68. The hexameric protein or antibody or pharmaceutical composition of any of paragraphs 1-45 for use in the treatment of cancer.

[0407] 69. The hexameric protein or antibody or pharmaceutical composition of paragraph 68 for use in the treatment of a cancer tumour.

[0408] 70. A method of treating or preventing a disease comprising administering to a subject the hexameric protein or antibody or pharmaceutical composition of any of paragraphs 1-45.

[0409] 71. A method of treating or preventing a pathogenic infection comprising administering to a subject the hexameric protein or antibody or pharmaceutical composition of any of paragraphs 1-45.

[0410] 72. A method of treating or preventing cancer comprising administering to a subject the hexameric protein or antibody or pharmaceutical composition of any of paragraphs 1-45.

[0411] EXAMPLES

[0412] Materials & Methods

[0413] Molecular Biology

[0414] KB15A IgGs were fused at their C-terminal lysine residues to the 18 amino-acid ptp directly, or with a linker derived from IgM (replacing 6 amino acid residues from IgGs). DNA fragments were ordered from Thermo Fisher with GeneArt codon optimization (Nicotiana Benthamiana). KB15A heavy and light chains were cloned into Magnicon-based vectors plCH38099 and plCH38100, respectively. Heavy chain and light chain containing vectors were transformed into agrobacterium cells ICF320.

[0415] Expression

[0416] 7KO Nicotiana Benthamiana was infiltrated with KB15A heavy chain and light chain at OD6oo of 0.008 each. At day post infiltration (DPI) 7, infiltrated leaf material was harvested and homogenized in the presence of 100 mM Sodium Phosphate, 1 mM EDTA, 40mM ascorbic acid, pH 7.3. The homogenized extract was centrifuged (10 min, 4°C , 10,000xg), and the supernatant filtered using miracloth.

[0417] Purification

[0418] KB15A hexamer was purified from green juice by protein A (ProA) chromatography with elution step using pH 3.0 buffer. Eluate was immediately neutralised to -pH 7.0 using 1M Tris pH 9.0. Capto core 400 chromatography was used to separate hexamer and monomer forms. Hexamer fractions were collected, concentrated, and formulated (20 mM Phosphate, 150 mM sodium chloride, 10mM histidine, pH 7.0) for further characterization.

[0419] Characterization

[0420] ProA and Capto core 400 purified hexamer was analysed with SEC-HPLC using PBS, pH 7.2, column TOSOH Bioscience TSKgel SuperSW3000, as well as non-reducing and reducing SDS- PAGE analysis.

[0421] Example 1 : Hexamer Assembly

[0422] Role of the linker in hexamer Assembly

[0423] The ptp (18 amino acid residues) can be fused to the IgG directly, or with an additional 6 amino acid residue linker from IgM (replacing 6 amino acid residues from IgG). The extent of hexamer was found to favour the conserved linker derived from IgM.

[0424] Role of the ptp in hexamer Assembly

[0425] IgM ptp (18 amino acid residues) and upstream linker (6 amino acid residues) were conserved very well from known different species. There are only two amino acid residue difference between human and mouse IgM ptp with identical linker. Pig, horse, abington island giant tortoise, tuatara IgM ptp are close to human and mouse. Chicken IgM ptp has slightly more differences from human and mouse, including the upstream linker. All of the species ptp mentioned above were fused into KB15A IgG and expressed from Nicotiana Benthamiana. Most constructs can produce similar KB15A hexamer yield and hexameric purity from ProA purification.

[0426] Role of the hinge in hexamer Assembly

[0427] IgG has four sub isotypes as lgG1 , lgG2, lgG3, and lgG4 with different hinge sequences. Wild-type lgG4 hinge has a relatively weaker disulfide connection due to the amino acid sequence “CPSC”. For commercial lgG4 antibody production, the hinge sequence “CPSC” is typically mutated into “CPPC” from lgG1. Since lgG1 hexamer has extra disulfide connection from the ptp, it was hypothesised that the lgG1 hexamer with hinge mutation “CPPC” into “CPSC” are stable. KB15A lgG1 hexamer with “CPSC” mutation indeed can form as stable a hexamer as wild-type lgG1 hexamer.

[0428] IgG isotype in hexamer assembly

[0429] IgG isotypes have different Fc effector functions, which might translate into their hexamer isoform. Using this same strategy, we have generated KB15A clone 14 and 16 hexamer isotype lgG1 , lgG2, lgG3 and lgG4. lgG3 allotype17 was used in the protein expression for convenient ProA purification as wildtype lgG3 cannot bind ProA. Wildtype lgG4 was used for the hexamer production, and the hexamer is stable, even with the wild-type hinge “CPSC” sequence.

[0430] Example 2: Sperm agglutination and immobilization

[0431] Collection and processing of semen samples

[0432] Collection and processing of human semen samples followed the protocol from the following reference (Baldeon-Vaca et al. (2021), The Lancet, vol. 69, 103478). Sperm concentrations were adjusted to 25-40 million / mL for the agglutination kinetics assay and complement-dependent sperm immobilization test.

[0433] Agglutination kinetics assay

[0434] The effect of KB15A hexamer on sperm agglutination was determined using the sperm agglutination kinetics assay as described from previous publication (Baldeon-Vaca et al. (2021), The Lancet, vol. 69, 103478). All samples were run at 37 °C for the entirety of the assay. KB15A control monomer and hexamer samples have four-fold dilutions starting at 25pg / mL.

[0435] Complement-dependent sperm Immobilization test

[0436] The effect of KB15A hexamer on complement-dependent sperm immobilization was determined using the sperm immobilization test as described from previous publication (Baldeon-Vaca et al. (2021), The Lancet, vol. 69, 103478). All samples were incubated at 37 °C and analysed at room temperature. Briefly, washed sperm was sensitized with human serum in the presence of complement (C) or heat-inactivated complement (HiC), used as a negative control. KB15A control monomer has four-fold dilutions starting at 12.5pg / mL and hexamer sample has ten-fold dilutions starting at 1.56pg / mL.

[0437] Example 3: SEC-MALS of hexameric KB15A.16.3

[0438] Size-exclusion high performance liquid chromatography (SEC-HPLC) was performed with a Wyatt Technology WTC-050S5, SEC Analytical Column, 5pm 500A 7.8 x 300 mm. Test samples or reference standards are prepared to 2-10 mg / mL concentration (for a 20- 100 pg injection) and eluted with an isocratic gradient of mobile phase buffer (0.1 M sodium phosphate, 0.15 M NaCI, pH 7.2) with a flow rate of 0.5 mL / min over a run time of 30 minutes. Column effluent is monitored using a DAWN Multi-angle light scattering detector (MALS).

[0439] Figure 8 shows a representative SEC-MALS of hexameric KB15A.16 (also known as KB15A.16.3).

[0440] Example 4: Sperm Agglutination assay of hexameric KB15A.16.3

[0441] This assay was carried out according to Baldeon-Vaca et al. (2021), The Lancet, vol. 69, 103478, and assessed the ability of the antibody Fab region to agglutinate sperm. Five antibody dilutions (starting at 25 pg / ml,) and a media control were added to preparations of fresh washed motile human sperm, and time to 100% sperm agglutination was recorded. Main endpoint: the antibody dilution required to agglutinate 100% of sperm within 60 seconds. The experiment was run at 37 °C.

[0442] The results are shown in Figure 9, which demonstrates that the highly pure hexamer version KB15A.16.3 (KB15A.16 hlgG1) (stored at 4 °C or -80 °C)is 10X more potent than the monomer version (reference standard).

[0443] Example 5: Complement-dependent sperm Immobilization test of hexameric KB15A.16.3

[0444] This assay, carried out according to Baldeon-Vaca et al. (2021), The Lancet, vol. 69, 103478, assessed the ability of the antibody Fab region to bind to sperm and the antibody Fc region to activate complement to immobilize and kill sperm. Antibody dilutions starting at 10 pg / mL, a media control, and fresh serum complement were added to preparations of fresh washed human sperm, and the number of motile sperm were recorded at 15min, 30 min and 1 hour time points. Main endpoint: the antibody dilution required to kill 100% of sperm within 1 hr.

[0445] Results are shown in Figure 10A, which demonstrates the complement-dependent sperm immobilization of hexameric KB15A.16 hlgG1 (i.e. KB15A.16.3) using two different sperm donors versus the reference standard KB15A.16.2 (KB15A.16.2 refers to a monomer). In both donors the hexamer lgG1 outperformed the monomer lgG1. In both donors, KB15A.16 hlgG1 (KB15A.16.3) immobilized >95% of sperm at a concentration of 0.01 pg / ml, whereas the monomer standard showed comparable activity at a concentration of 10 pg ml. In the presence of heat-inactivated complement (HiC), antibodies did not immobilize sperm.

[0446] Further results are shown in Figure 10B, which demonstrates the complement-dependent sperm immobilization of hexameric KB15A.16 hlgG1 (KB15A.16.3) and KB15A.16 hlgG4 using three different sperm donors versus the reference standard KB15A.16.2. Both hlgG1 and hlgG4 outperformed the monomer standard with all donors, but there were noticeable differences between runs for hlgG1 and hlgG4 at lower concentrations. In all three donors, KB15A.16 hlgG1 (KB15A.16.3) immobilized >95% of sperm at a concentration of 0.01 pg / ml. In one donor KB15A.16 hlgG4 immobilized >95% of sperm at a concentration of 0.01 pg / ml, however in other two donors KB15A.16 hlgG4 immobilized >95% of sperm at a concentration of 0.1 pg / ml. The monomer standard showed comparable activity at a concentration of 10 pg ml.

Claims

CLAIMS1. A hexameric protein, wherein the hexameric protein is composed of six units and wherein each unit comprises:(a) an N-terminal immunoglobulin, or a fragment thereof;(b) a linker domain; and(c) a C-terminal tailpiece derived from IgM.

2. The hexameric protein of claim 1 , wherein the N-terminal immunoglobulin, or a fragment thereof, comprises an immunoglobulin Fc region.

3. The hexameric protein of claim 1 or claim 2, wherein the hexameric protein is a hexameric antibody.

4. The hexameric antibody of claim 3, wherein within each unit the N-terminal immunoglobulin or a fragment thereof is independently an N-terminal IgG or IgM, or an antigen-binding fragment thereof, which is capable of binding one or more ligand molecules, optionally wherein within each unit the N- terminal IgG or IgM is independently selected from any one of an IgM, I gG 1 , I gG2, lgG3 or lgG4.

5. The hexameric antibody of claim 4, wherein within each unit the N-terminal IgG or IgM has an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 29 to SEQ ID NO: 38 and SEQ ID NO: 69 to SEQ ID NO: 74.

6. The hexameric antibody of any one of claims 3-5, wherein within each unit the N-terminal IgG or IgM has a light chain with an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 39 and SEQ ID NO: 75 to SEQ ID NO: 79.

7. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 29 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

8. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 30 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

9. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 31 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

10. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 32 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

11. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 33 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

12. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 34 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

13. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 35 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

14. The hexameric antibody of any one of claims 3-6, wherein each unit comprises an N-terminal IgG, wherein the heavy chain has a sequence with at least 70% sequence identity to SEQ ID NO: 36 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

15. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 37 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

16. The hexameric antibody of any one of claims 3-6, wherein within each unit the N-terminal IgG or IgM has a heavy chain with an amino acid sequence with at least 70% sequence identity to SEQID NO: 38 and a light chain with an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 39.

17. The hexameric protein or antibody of any one of the preceding claims, wherein the tailpiece has an amino acid sequence according to any one of SEQ ID NO: 1 to SEQ ID NO: 28.

18. The hexameric protein or antibody of any one of the preceding claims, wherein the tailpiece is fused to the C-terminus of an immunoglobulin Fc region via a linker amino acid sequence of any one of SEQ ID NO: 40 to SEQ ID NO: 49.

19. The hexameric protein or antibody of any one of the preceding claims, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 50 to SEQ ID NO: 68.

20. The hexameric protein or antibody of any one of the preceding claims, wherein each unit comprises or consists of a modified heavy chain, wherein the modified heavy chain has an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NO: 80 to SEQ ID NO: 85.

21. The hexameric protein or antibody of any one of the preceding claims, wherein the antibody binds to human spermatozoa.

22. The hexameric protein or antibody of any one of the preceding claims, wherein the antibody binds to CD52g.

23. The hexameric protein or antibody of any one of the preceding claims, wherein the antibody, or antigen-binding fragment thereof, binds to CD52g on the surface of human spermatozoa resulting in agglutination and / or immobilization of the spermatozoa.

24. The hexameric protein or antibody of any one of the preceding claims for use as a non-hormonal contraceptive agent.

25. The hexameric protein or antibody of any of claims 1-20 for use in the treatment of a pathogenic infection.

26. The hexameric protein or antibody of any of claims 1-20 for use in the treatment of cancer.

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