Field of the invention

The present invention relates to fusion proteins comprising an antibody fragment having a biologic activity and half-life extending polypeptide(s), and to uses of such fusion proteins.

Background

Antibodies constitute a major class of biological therapeutics on the market today, where their ability to bind to other proteins, and either block or activate them, comes into their full right.

However, some inherent properties of antibodies can limit their use as therapeutics in certain applications. The most prominent ones are their size, their bivalent binding, and their interplay with the rest of the immune system. Particularly in instances where a pure blocking function is desired, it may be beneficial to harness the binding properties of antibodies and reformat the antigen interacting parts.

There are five major classes of antibodies, or immunoglobulins (Ig): IgG, IgA, IgE, IgD and IgM. Primarily the IgG class is used as biotherapeutics and is as such further divided into 4 subclasses; namely lgG1 - 4. IgG antibodies have two chains, the heavy and the light chain. The heavy chain (HC) is made up by four domains of the Ig fold: the variable heavy (VH), constant heavy 1 (CH1 ), hinge region, constant heavy 2 (CH2) and constant heavy 3 (CH3) domains. The light chain (LC) are made up by the variable light (VL) and the constant light (CL) domain. A heterodimer is formed between the HC and the LC facilitated by a disulfide bond between the CH1 and CL domains. The heterodimer in turn forms a dimer with intermolecular disulfide bonds being formed between the hinge regions in the HCs.

In order to isolate the binding properties of antibodies and to avoid the above mentioned limitations, various antibody fragments have been designed to minimize size, get univalent binding, and to remove interactions with other parts of the immune system as well as FcRn. Amongst these, the fragment antigen-binding (Fab) entity is the largest one. The Fab constitutes one antigen binding arm of the antibody and incorporates the domains VH-CH1 and VL-CL which are joined by a disulfide bond between the CH1 and CL domains. Second in size and complexity is the single-chain variable fragment (ScFv) in which the VH and VL are joined by a linker sequence and wherein either the VH or VL can act as N-terminal domain. The ScFv entity

incorporates all the antigen binding regions of a full antibody. The smallest entity is the domain antibody (dAb), which is either an isolated VH or VL domain, and which thus incorporates half of the antigen binding region from an antibody. In addition, there are natural domain antibodies that are present in certain species such as Camelids (VHH) and Sharks (VNAR).

When the CH2 and CH3 domain which constitute the Fc region is removed, the size of the resulting antibody fragment is reduced as compared to the size of a full-length antibody. By removing the Fc region, the antibody fragment’s capability to interact with FcRn is removed. Antibody fragments therefore typically display a short half-life in vivo as they are readily cleared from circulation by filtration by the kidneys.

As biologies most often are administrated by either intravenous (i.v., iv) or subcutaneous (s.c., sc) injection, the time span between each dose is of great importance. Meanwhile, these routes of administration, in particular intravenous injection, typically require the assistance of healthcare

professionals and may also be uncomfortable, even painful, to the patient. Thus, more frequent dosing increases patient discomfort and inconvenience and demands healthcare resources. This is in great contrast to dosing of a small molecule drug, which can often be administrated by less invasive routes, such as orally, intranasally or topically, as often as required, with much less effort and inconvenience.

One of the earliest attempts to address the problem of rapid clearance of biologies or biopharmaceuticals from circulation was to chemically attach a polyethylene glycol (PEG) polymer chain to a protein or peptide to increase the hydrodynamic radius of the drug. This gives the drug an increased apparent size in solution, such that it reaches a size that is not readily cleared by the kidneys. This technology, termed PEGylation, has shown to be successful, and is currently used in approved pharmaceutical products.

However, the step of chemical attachment adds another process step to the manufacturing, resulting in an increased cost of the manufactured drug.

Furthermore, attachment of a PEG moiety can occur at various sites of a protein or peptide, resulting in a product of greatly increased inhomogeneity in which the location of the PEG chain varies among individual molecules. The nature of the PEG polymer itself also adds a degree of inhomogeneity as the polymer is not monodisperse, but rather a collection of PEG polymers of similar, but not equal, length.

Contrary to the original belief that it was non-immunogenic and even capable of reducing immunogenicity also towards molecules to which it was linked, PEG has later been found to be immunogenic. In one example this led to a significantly increased clearance of the drug to which it was linked (PEG- uricase; Ganson NJ et al., 2005).

Half-life extending technologies based on randomly non-repetitive protein sequences that can be used as fusion partners to prolong the biological half- life of therapeutic proteins and peptides have been developed by i.a. Amunix Inc and XL-Protein GmbH (Podust et al. 2016 J Control Release, Schlapschy et al. 2013 Protein Eng Des Sel ).

However, despite the advancements described above, there remains a need in the art for new means of prolonging the half-life of antibody fragments.

Summary of the invention

It is an object of the present invention to at least partly reduce or avoid the problems of the prior art, and to provide new means of extending the biological half-life of antibody fragments.

These and other objects, which will be apparent to a skilled person from the present disclosure, are achieved by the different aspects of the invention as defined in the appended claims and as generally disclosed herein.

In one aspect, the invention relates to a fusion protein comprising

i) an antibody fragment having a biologic activity, said antibody fragment being selected from the group consisting of a Fab, a ScFv, a dAb, a VHH and a VNAR; and ii) one or more half-life extending polypeptide moieties, each moiety comprising 2 to 136 units, wherein each unit is independently selected from the group consisting of all amino acid sequences according to SEQ ID NO:1 :

X1 -X2-X3-X4-X5-X6-D-X8-X9-X10-X11 (SEQ ID NO:1 ) i

X

X

with the proviso that said protein in total comprises 10 to 136 units.

The 2-136 units of amino acid sequences according to SEQ ID NO:1 may be the same or different within the definition of SEQ ID NO:1 set out above. Stated differently, said one or more half-life extending polypeptide moieties comprise from 2 to 136 units, wherein each unit is an amino acid sequence independently selected from the group consisting of the individual sequences falling within the definition of SEQ ID NO:1. Preferably, each unit may be an amino acid sequence independently selected from the group consisting of SEQ ID NOs:2-11.

The present inventors surprisingly found that a polypeptide moiety as defined above, which is based on or derived from the C-terminal domain of human bile salt-stimulated lipase (BSSL), can provide an excellent in vivo half-life extending moiety when fused to an antibody fragment having a biologic activity. The half-life extending polypeptide moiety has a generally unfolded conformation under physiological conditions and provides a fusion protein with a large hydrodynamic radius, and thus avoids, or at least reduces the rate of, renal clearance of the antibody fragment. Thus, the fusion protein including one or more half-life extending polypeptide moieties may have a biological half-life which is extended as compared to the biological half-life of the antibody fragment alone.

As used herein, the expressions“fused” and“fusion” refer to the artificial joining of two or more portions of chemical entities of the same kind, such as peptides, polypeptides, proteins, or nucleic acid sequences. A fusion protein as referred to herein typically comprises at least two polypeptide portions, which are of different origin; for instance, one or more half-life extending polypeptide moieties, which may be derived from BSSL, and an antibody fragment. The fusion protein of the present invention is typically a non- naturally occurring entity. The fusion protein of the invention may also be referred to as a chimeric protein.“Chimeric protein” is understood to mean a hybrid protein encoded by a nucleotide sequence consisting of two or more complete or partial genes that originally coded for distinct proteins, which may be of the same or different species. The fusion protein, or chimeric protein, of the invention is produced by recombinant DNA technology.

The expression“biological half-life” refers to the time it takes for the

concentration of the substance in question in blood, serum or plasma to decrease to half of the initial concentration. The biological half-life may be determined according to conventional methods known to persons of skill in the art. For instance, the biological half-life can be determined based on the concentration in serum, plasma or whole blood. The term biological half-life is to be understood as referring to the half-life of a substance in at least one species, such as human.

In this context,“biological activity” refers to any activity of an antibody fragment that may lead to a therapeutic effect in vivo, and may be exemplified as a binding activity. Non-limiting examples include enzymatic activity, agonist activity, and antagonist activity.

As used herein, the term“antibody fragment” refers to a Fab, ScFv, dAb, VHH or VNAR that exerts a desired biological activity in vivo. The fusion protein may have a biological half-life which is extended by a factor of at least 2 relative to the biological half-life of the antibody fragment alone. As a result of the increased biological half-life, the effect of the antibody fragment might be prolonged.

From a dosing perspective, using one or more half-life extending polypeptide moieties as disclosed herein allows for less frequent administration, which is beneficial for the patient as well as from an economic perspective. For instance, instead of administration twice a week of a drug, the same or a similar biological or therapeutic effect may be attained by only one

administration per week. Such a difference means a great improvement for patients, especially those who are required to come to a hospital or clinic to receive treatment, and/or where administration is physically uncomfortable or even painful. Additionally, by fewer doses and/or a longer time period between doses, adverse reactions caused by the mode of administration may be avoided; for instance, for subcutaneous injection, injection site reactions such as pain, eczema and rashes can be reduced or avoided, and for intravenous administration, infusions reactions involving e.g. fever or nausea can be reduced or avoided.

Another benefit of the half-life extending polypeptides used in the present invention resides in the increased hydrophilicity of the fusion protein due to the high number of hydrophilic residues in the half-life extending polypeptide. The increased hydrophilicity may improve bioavailability of the fusion protein (relative to the bioavailability of the antibody fragment as such) and increase systemic concentration, potentially allowing smaller and/or less frequent doses. As used herein,“bioavailability” refers to the dose fraction of a substance that reaches systemic circulation following administration via a different route than intravenous administration.

Another practical implication of the increased hydrophilicity is that

subcutaneous administration may be an option instead of intravenous administration. Where possible, subcutaneous administration is often preferred over intravenous infusion as subcutaneous injections in general are faster, less uncomfortable and require less medical training to perform compared to intravenous administration. Additionally, the increased hydrophilicity of the fusion protein according to the invention may also be an advantage during the purification of a crude expression product. It has been found that fusion proteins according to embodiments of the invention eluted earlier than the antibody fragment as such using hydrophobic interaction chromatography (HIC) using gradient elution. This is considered a potentially very useful effect that could be the solution to problems relating to undesirable host cells proteins eluting simultaneously with the antibody fragment. Hence, it may be possible to reduce the number of chromatography unit operations required to obtain a fusion protein of high purity.

Another advantage of using one or more half-life extending polypeptide moieties as described herein is that it may allow for more accurate prediction of the biological half-life of the resulting fusion protein, based on its size in terms of hydrodynamic radius (or apparent size) in solution. The increased biological half-life of the fusion protein may be exclusively or at least mainly reliant on the size increase. In fact, the half-life extending polypeptide moiety as used in embodiments of the present invention may be devoid of binding to the major recycling receptor, the neonatal Fc receptor, and may thus avoid the complex interplay between protein size and recycling through receptor interaction, which otherwise makes prediction and fine-tuning of biological half-life very uncertain.

In some embodiments, said one or more half-life extending polypeptide moieties may form a contiguous sequence of 10-136, such as 10-100, such as 10-70, such as 10-68, units of one or more sequence(s) as defined in SEQ ID NO: 1. Stated differently, the fusion protein may in some embodiments comprise one half-life extending polypeptide moiety wherein said moiety comprises 10-136, such as 10-100, such as 10-70, such as 10-68, units of one or more sequence(s) as defined in SEQ ID NO:1.

In embodiments, the fusion protein may comprise more than one half-life extending polypeptide moiety, with the proviso that said protein in total comprises 10 to 136 units. In such embodiments, each polypeptide moiety may comprise at least 2 units as defined above. Such multiple half-life extending polypeptides may be of the same length (having the same number of units), or may be of different lengths. For instance, the fusion protein may comprise two half-life extending polypeptide moieties, each moiety comprising 2 to 134 units as defined above.

In one embodiment, each of said one or more half-life extending polypeptide moieties comprises 5 to 70 units, such as 7 to 68 units, such as 7 to 51 units, such as 17 to 34 units, such as 12 to 34 units, such as 7 to 29 units.

In one embodiment, each of said one or more half-life extending polypeptide moieties individually comprises at least 7 units, such as at least 8 units, such as at least 9 units, such as at least 10 units, such as at least 11 units, such as at least 12 units, such as at least 13 units, such as at least 14 units, such as at least 15 units, such as at least 16 units, such as at least 17 units, such as at least 18 units, such as at least 19 units, such as at least 20 units, such as at least 21 units, such as at least 22 units, such as at least 23 units, such as at least 24 units, such as at least 25 units, such as at least 26 units, such as at least 27 units, such as at least 28 units, such as at least 29 units, such as at least 34 units. In particular, one of said one or more half-life extending polypeptide moieties may comprise 7 units, such as 8 units, such as 9 units, such as 10 units, such as 11 units, such as 12 units, such as 13 units, such as 14 units, such as 15 units, such as 16 units, such as 17 units, such as 18 units, such as 19 units, such as 20 units, such as 21 units, such as 22 units, such as 23 units, such as 24 units, such as 25 units, such as 26 units, such as 27 units, such as 28 units, such as 29 units, such as 34 units.

Alternatively, or additionally, at least one of the one or more half-life extending polypeptide moieties may constitute an insertion into, or replacement of a part of, the amino acid sequence of the antibody fragment. In the case of multiple half-life extending polypeptides, at least one of said half-life extending polypeptides moieties may optionally be positioned as an insertion into, or replacement of a part of, the amino acid sequence of the antibody fragment. An insertion or replacement may be made in a surface exposed loop of the tertiary structure of the antibody fragment, such that the half-life extending polypeptide moiety that constitutes an insertion into, or replacement of a part of, the amino acid sequence of the antibody fragment is exposed on the surface of the fusion protein. For instance, an insertion may be made between an VH and VL e.g. in a ScFv. This may partly or fully replace a linker used for joining an VFI and VL domain in a ScFv. In embodiments, at least one of said one or more half-life extending polypeptide moieties may be positioned at the amino terminal (N-terminal) or at the carboxy terminal (C-terminal) of said antibody fragment. In the case where more than one half-life extending polypeptide moiety is present, at least one of said half-life extending polypeptides moieties may be positioned N-terminally or C-terminally of said antibody fragment. In embodiments where at least one of said half-life extending polypeptides moieties is positioned at an N-terminal of said antibody fragment, said antibody fragment may be a ScFv, a dAb, a VHH or a VNAR.

In some embodiments, said antibody fragment is a ScFv and at least one of said one or more half-life extending polypeptide moieties may be positioned at the amino terminal (N-terminal) or at the carboxy terminal (C-terminal) of said ScFv. Such fusion proteins may for instance comprise in total from 10 to 100 units, such as 17 to 68 units, of one or more sequence(s) as defined above.

In some embodiments, said antibody fragment is a dAb and at least one of said one or more half-life extending polypeptide moieties may be positioned at the amino terminal (N-terminal) or at the carboxy terminal (C-terminal) of said dAb. Such fusion proteins may for instance comprise in total from 10 to 100 units, such as 17 to 68 units, of one or more sequence(s) as defined above.

In some embodiments, said antibody fragment is a VHH and at least one of said one or more half-life extending polypeptide moieties may be positioned at the amino terminal (N-terminal) or at the carboxy terminal (C-terminal) of said VHH. Such fusion proteins may for instance comprise in total from 10 to 100 units, such as 17 to 68 units, of one or more sequence(s) as defined above.

In some embodiments, said antibody fragment is a VNAR and at least one of said one or more half-life extending polypeptide moieties may be positioned at the amino terminal (N-terminal) or at the carboxy terminal (C-terminal) of said VNAR. Such fusion proteins may for instance comprise in total from 10 to 100 units, such as 17 to 68 units, of one or more sequence(s) as defined above.

In embodiments, at least one of said one or more half-life extending moieties may be positioned at a C-terminal of said antibody fragment. In embodiments where at least one of said half-life extending polypeptides moieties is positioned at a C-terminal of said antibody fragment, said antibody fragment may be a Fab, a ScFv, a dAb, a VHH or a VNAR. In particular, at least one of said one or more half-life extending moieties may be positioned at a C- terminal of a light or heavy chain of said antibody fragment. In the case where said fusion protein for example comprises two or more half-life extending moieties, one half-life extending moiety may be positioned at a C-terminal of a heavy chain, and one half-life extending moiety may be positioned at a light chain of said antibody fragment. In such embodiments, the antibody fragment may for example be a Fab or a ScFv.

In some embodiments, said antibody fragment is a Fab and at least one of said one or more half-life extending moieties may be positioned at a C- terminal of said Fab. Such fusion proteins may for instance comprise in total from 10 to 100 units, such as 17 to 68 units, of one or more sequence(s) as defined above.

When the antibody fragment is a Fab, the Fab may comprise a heavy chain which consists of a variable heavy domain and a constant heavy chain domain 1. Alternatively, the heavy chain of such a Fab may in some instances comprise up to 5 amino acid residues from the hinge region of an antibody.

In some embodiments, said antibody fragment is a ScFv and at least one of said one or more half-life extending moieties may be positioned at a C- terminal of said ScFv. Such fusion proteins may for instance comprise in total from 10 to 100 units, such as 17 to 68 units, of one or more sequence(s) as defined above.

The antibody fragment of the fusion protein, whose half-life it is desirable to prolong by fusion with one or more half-life extending polypeptide moieties, may be selected from a group of antibody fragments having IgG, IgA, IgE, IgM or IgD origin. In other embodiments, the antibody fragment may be of IgG origin.

In embodiments, said antibody fragment has a binding activity. For example, said binding activity may be a binding activity to a particular in vivo target. Non-limiting examples of such in vivo targets are FIX/FIXa, CD40L, CD28, IL2 receptor alpha chain (CD25), vascular endothelial growth factor (VEGF), CD16, and P-selectin. In such embodiments, said antibody fragment or plurality of antibody fragments thus may bind to FIX/FIXa, CD40L, CD28, IL2 receptor alpha chain (CD25), VEGF, CD16, or P-selectin, such as any one of FIX/FIXa, CD40L, VEGF, CD16 and P-selectin, such as any one of FIX/FIXa and CD40L.

The fusion protein may comprise at least one antibody fragment. In

embodiments, the fusion protein may comprise a plurality of antibody fragments, such as two antibody fragments, three antibody fragments, or four antibody fragments. It is to be understood that the plurality of antibody fragments may have the same or different origin, structure, and biological activity. For example, the fusion protein may comprise two antibody fragments which have different biological activities, such as different binding activities. The fusion protein may for instance thus have bispecific or trispecific activity. Said bispecific or trispecific fusion protein may for example comprise multiple antibody fragments, each antibody fragment individually having a binding activity to a target selected from FIX/FIXa, CD40L, CD28,

IL2 receptor alpha chain (CD25), VEGF, CD16, and P-selectin. For example, a bispecific fusion protein as disclosed herein comprising a plurality of antibody fragments may comprise one antibody fragment binding to CD40L and one antibody fragment binding to CD28. Other examples include fusion proteins comprising one antibody fragment binding to CD28 and one antibody fragment binding to CD25, and one antibody fragment binding to CD40L and one antibody fragment binding to CD25.

In embodiments wherein the antibody fragment or plurality of antibody fragments bind(s) to FIX/FIXa, the fusion protein may further comprise a cleavage site enabling in vivo cleavage of said one or more half-life extending polypeptide moieties, said cleavage site optionally being selected from an activated protein C (APC) cleavage site from factor VIII and a protease cleavage site, wherein the protease is factor Xla, factor Vila or factor IXa. Such a cleavage site may enable cleavage of the half-life extending polypeptide moiety or moieties from the fusion protein, such as to disable its interaction with an antigen and/or to provide a fusion protein without half-life extending polypeptide moieties.

Furthermore, in some embodiments of FIX/FIXa binding fusion proteins, the protein may comprise a peptide which binds to the platelet membrane, said peptide optionally being a phosphatidylserine binding peptide.

In embodiments of the invention, at least one of the residues X3 and X4 of SEQ ID NO:1 may be P. In some embodiments, at least one of X4 and X5 of SEQ ID NO:1 may be T. In some embodiments, at least one of X10 and X11 of SEQ ID NO:1 may be A or P. In some embodiments, X1 is P and X2 is V.

In some embodiments, at least one of said one or more half-life extending polypeptide moieties may have an amino acid unit having a sequence as set out in SEQ ID NO:2 in its N-terminal end, as is typically the case of naturally occurring sequences of human origin. For instance, the half-life extending polypeptide moiety may comprise at least 4 contiguous units in the following order: [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5], optionally preceded by SEQ ID NO: 2.

In embodiments of the invention, said one or more half-life extending polypeptide moieties may comprise at least one sequence selected from the group of amino acid sequences consisting of SEQ ID NOs:12-34 and SEQ ID NO: 108-126. For example, each of said one or more half-life extending polypeptide moieties may be selected from the group of sequences consisting of SEQ ID NO: 12-34 and SEQ ID NO: 108-126. Alternatively, the half-life extending polypeptide moiety may comprise multiple copies, e.g. 2, or 3, optionally contiguous, copies of a sequence selected from the group consisting of SEQ ID NO: 12-34 and SEQ ID NO: 108-126.

In embodiments of the invention, said one or more half-life extending polypeptide moiety may comprise 2-136 units of one or more amino acid sequence(s) independently selected from the group consisting of SEQ ID NOs:2-11. These sequences represent human variants of SEQ ID NO: 1. In embodiments of the invention, each of said one or more half-life extending polypeptide moieties may comprise at least 4, at least 5, at least 6, at least 7, at least 8, at least 10 units, at least 11 units, at least 12 units, such as at least 13 units, such as at least 14 units, such as at least 15 units, such as at least

16 units, such as at least 17 units, such as at least 18 units, such as at least

19 units, such as at least 20 units, such as at least 21 units, such as at least

22 units, at least 23 units, at least 24 units, at least 25 units, at least 26 units, at least 27 units, at least 28 units, at least 29 units or at least 34 units of one or more amino acid sequence(s) according to SEQ ID NO: 1. Furthermore, in embodiments of the invention, each of said one or more half-life extending polypeptide moieties may comprise up to 8, up to 10, up to 18, up to 34, up to 51 , up to 68 or up to 70 units of one or more amino acid sequence(s) according to SEQ ID NO: 1. Thus for example, each of said one or more half- life extending polypeptide moieties may comprise from 5 to 70, such as 7 to 68, such as 7 to 51 , such as 17 to 68, such as 17 to 34, units of one or more amino acid sequence(s) according to SEQ ID NO: 1. In another embodiment, each of said one or more half-life extending polypeptide moieties may comprise from 5 to 70, such as from 7 to 68, such as 7 to 51 , such as 17 to 68, such as 17 to 34, units independently selected from the group consisting of SEQ ID NO: 2-11.

In some embodiments, the half-life extending polypeptide, or, in the case where the fusion protein comprises more than one half-life extending polypeptide moieties, at least one of the half-life extending polypeptides, comprises at least two different amino acid sequences according to SEQ ID NO: 1.

In embodiments of the invention, the half-life extending polypeptide may be fused to an antibody fragment which alone has an apparent size in solution of at least 12 kDa, such as from about 12 kDa to about 50 kDa. In particular for small antibody fragments, the present half-life extending polypeptide moiety may be of great benefit, as it may increase the size enough to avoid renal clearance. As a whole, the fusion protein may typically have an apparent size in solution of at least 60 kDa as determined by size exclusion

chromatography. In embodiments, the apparent size in solution of the fusion protein is larger than the apparent size in solution of the antibody fragment alone, by a factor of at least 2, such as by a factor of at least 3, such as a factor of at least 4, such as a factor of at least 5, and up to a factor of 300. In terms of hydrodynamic radius, the fusion protein as a whole may exhibit a hydrodynamic radius of at least 3.8 nm. In embodiments, the hydrodynamic radius of the fusion protein may be at least 1.4 times as large, for instance 1.6 times as large, and for instance twice as large, as the hydrodynamic radius of the antibody fragment alone.

The apparent size increase provided by the half-life extending polypeptide may be at least partly explained by the unstructured or unfolded conformation of the half-life extending polypeptide. For instance, the half-life extending polypeptide may lack secondary structure elements such as a-helices and b- sheets, and thus the half-life extending polypeptide may be characterized as not contributing to the a-helix and/or b-sheet content of the fusion protein.

In embodiments of the invention, an amino acid sequence according to SEQ ID NO: 1 may be of human origin. Thus, each unit of the one or more half-life extending polypeptide moieties may be of human origin. For example, the half-life extending polypeptide moiety or moieties may correspond to one or more naturally occurring human amino acid sequences. The use of a sequence of human origin may be advantageous as it is expected to contribute to a lower immunogenicity in human subjects. Nevertheless, sequences comprising or corresponding to naturally occurring repeating units of other species are also contemplated for use in a half-life extending polypeptide, alone or in combination with repeating units of human origin. Such other species particularly include non-human primates, e.g. gorilla, chimpanzee, orangutan, bonobo, and macaque.

In embodiments of the invention, each repeating unit according to SEQ ID NO: 1 has one, or at most one, potential O-glycosylation site. Moreover, when the half-life extending polypeptide moiety as disclosed herein has been produced in a mammalian expression system, each unit may comprise at most one O-glycosylation, and typically a majority, but not all, of said units comprises one O-glycosylation each. For instance, a certain number or share of said units may lack glycosylation. While some glycosylation may be beneficial as it may further contribute to the size increase, unspecific or an unknown glycosylation pattern may present practical problems during protein characterization. Flence, the limited and relatively well-defined glycosylation pattern of the half-life extending polypeptide moiety according to

embodiments of the present invention is advantageous in this respect. In some embodiments however, in particular where the fusion protein is produced in non-mammalian cells, said one or more half-life extending polypeptide moieties may completely lack glycosylation. In such

embodiments, one or more half-life extending polypeptide moieties of the fusion protein may each comprise 2 to 136 units of one or more amino acid sequence(s) independently selected from the group consisting of SEQ ID NOs: 637-643, 644 and 648-712, such as from the group consisting of SEQ ID NOs: 637-643.

In some embodiments, said fusion protein comprises a polypeptide having an amino acid sequence selected from any one of sequences SEQ ID NO: 37- 42, 50-53, 59-78, 80-83, 86-89, 98-103, 105, 127-260, 262, 265-401 , 405- 520, 524-595, 622, 625, 630-631 , 633-636 and 646-647.

In another aspect, the invention provides a method of prolonging the biological half-life of an antibody fragment, or a method of producing a fusion protein according to the above-mentioned first aspect of the invention, comprising the steps of:

a) providing a polynucleotide, typically a DNA construct, encoding a fusion protein as described above, comprising the antibody fragment and one or more half-life extending polypeptide moieties;

b) introducing said polynucleotide into a cell;

c) maintaining said cell under conditions allowing expression of said fusion protein; and

d) isolating said fusion protein.

In some embodiments, said method is an in vitro method. In some

embodiments, the cell is a mammalian cell. Expression in mammalian expression systems may be beneficial as it may provide glycosylation of the fusion protein. In other embodiments, the cell may be a non-mammalian eukaryotic cell, such as a yeast cell, a plant cell or a non-mammalian animal cell. In yet other embodiments, the cell may be a prokaryotic cell, such as E. coli. In some embodiments, the fusion protein may be co-expressed with a a2,6- sialyltransferase (EC: 2.4.99.1 ; an alternative name is B-cell antigen CD75). Such methods may comprise the steps of

i) providing a polynucleotide, typically a DNA construct, encoding a a2,6-sialyltransferase or promoting expression of endogenous a2,6-sialyltransferase,

ii) introducing said polynucleotide into a cell, which may be the same cell that is used in step b) above for expression of a fusion protein according to embodiments of the invention, and

iii) maintaining said cell under conditions also allowing expression of said a2,6-sialyltransferase.

The polynucleotide may be the same construct that of step a) above encoding a fusion protein. Alternatively, it may be a different DNA construct. In embodiments using different DNA constructs encoding the fusion protein and encoding, or promoting expression of, the a2,6-sialyltransferase, respectively, the DNA constructs may be introduced into the same cell, simultaneously or at different points in time. Alternatively, different cells may be used, in which case the cells may be cultured together and thus maintained together under conditions allowing, simultaneously or sequentially, expression of the fusion protein and the a2,6-sialyltransferase.

In other aspects, the invention provides a polynucleotide encoding a fusion protein as described herein, an expression vector comprising such a polynucleotide, and a cell, which may be a mammalian cell or a non- mammalian cell, comprising such an expression vector.

In another aspect, the invention provides a pharmaceutical composition comprising the fusion protein as described herein and a pharmaceutically acceptable carrier. In embodiments, the pharmaceutical composition may be formulated for subcutaneous administration, and/or for intravenous

administration.

In yet another aspect, the invention provides a fusion protein for use as a medicament, and in particular for use as a medicament intended to be administered subcutaneously to a subject. Hence, in further aspects, invention provides a method of treatment of a condition, disorder or disease, comprising the step of administering to a patient suffering from said condition, disorder or disease a fusion protein comprising an antibody fragment useful for treatment of said condition, disorder or disease, fused to a half-life extending polypeptide moiety as described herein. The patient may be a mammal, such as a human. In such a method, administration may occur less frequently compared to a treatment regimen involving administration of the biologically active antibody fragment alone.

In further aspects, the invention relates to the use of a half-life-extending polypeptide as defined herein for increasing the biological half-life of an antibody fragment, as well as to the use of one or more half-life-extending polypeptide moieties as defined herein for increasing the bioavailability of an antibody fragment. The antibody fragment is typically selected from the group consisting of a Fab, a ScFv, a dAb, a VHH and a VNAR. In particular, the invention relates to use of one or more half-life-extending polypeptide moieties as defined herein, for increasing the bioavailability of an antibody fragment having a biologic activity, said use comprising in vitro expression of a fusion protein comprising said one or more half-life-extending polypeptide moieties and an antibody fragment, or a plurality of antibody fragments, wherein said antibody fragment(s) is/are selected from the group consisting of a Fab, a ScFv, a dAb, a VHH and a VNAR. As mentioned above, a distinct benefit of the half-life extending polypeptide moiety described herein is the increased hydrophilicity of the resulting fusion protein due to the high number of hydrophilic residues in the half-life extending polypeptide. The increased hydrophilicity may improve bioavailability and increase systemic concentration (e.g., serum concentration), potentially allowing smaller or less frequent doses.

Brief description of the drawings

Figure 1A-E are schematic representations of exemplary Fab containing fusion proteins according to embodiments of the invention. Fig 1 A represents a Fab with a heavy chain (FIC), containing a VFI and a CH 1 domain, linked with a light chain (LC), containing a VL and a CL domain, through a disulfide bond between the CH1 and CL domains. The disulfide bond is marked with a black line. Fig. 1 B represents a Fab with a half-life extending polypeptide moiety (striped) at the C-terminus of the HC. Fig. 1 C represents a Fab with a half-life extending polypeptide moiety at the C-terminus of both the HC and the LC. Fig. 1 D represents a Fab with a half-life extending polypeptide moiety at the C-terminus of the LC. Fig. 1 E represents a Fab with a half-life

extending polypeptide moiety at both the N- and C-terminus of both the FIC and the LC.

Figure 2A-D are schematic representations of exemplary fusion proteins, containing ScFvs, according to embodiments of the invention. The N-terminus of the ScFv based fusion proteins is marked with an”N-”. Fig. 2A represents a VFI-VL (left) and VL-VFI (right). Fig. 2B represents two ScFvs (VFI-VL, left; VL- VFI, right) with C-terminal fusions to half-life extending polypeptide moieties (striped). Fig. 2C represents two ScFvs (VFI-VL, left; VL-VFI, right) with N- terminal fusions to half-life extending polypeptide moieties. Fig. 1 D represents two ScFvs (VFI-VL, left; VL-VFI, right) with N- and C-terminal fusions to half- life extending polypeptide moieties, and with an insertion of a half-life extending polypeptide moiety into the linkers between VFI and VL domains.

Figure 3A-E are schematic representations of exemplary fusion proteins, containing VFIFH, dAb or VNAR, according to embodiments of the invention. Fig. 3A represents a VFIFH, dAb or VNAR (white). Fig. 3B represents a VFIFH, dAb or VNAR (white) with a C-terminal fusion to a half-life extending polypeptide moiety (striped). Fig. 3C represents a VFIFH, dAb or VNAR (white) with a N-terminal fusion to a half-life extending polypeptide moiety. Fig. 3D represents a VFIFH, dAb or VNAR with N- and C-terminal fusions to half-life extending polypeptide moieties. Fig. 3E represents a VFIFH, dAb or VNAR with an N-terminal fusion to a half-life-extending polypeptide moiety and with an insertion of a half-life extending polypeptide moiety in a surface exposed loop.

Figure 4A-C are schematic representations of exemplary fusion proteins according to embodiments of the invention. Fig. 4A represents a Fab (FIC and LC linked through a disulfide bond) fused to a half-life extending polypeptide moiety (striped) at the C-terminus of the heavy chain (FIC: VFH-CFH 1 ). A ScFv is moreover fused to the C-terminus of the half-life extending polypeptide moiety. Fig. 4B represents a Fab fused to a half-life extending polypeptide moiety (striped) at the C-terminus of both the FIC and LC chains. The half-life extending polypeptide moiety fused to the FIC chain of the Fab furthermore has a dAB, VNAR or VFIFH (white) fused to its C-terminus, and the half-life extending polypeptide moiety fused to the LC chain of the Fab furthermore has a ScFv (VFI-VL) fused to its C-terminus. Fig. 4C represents a ScFv (VFI- VL) with a c-terminal fusion to a half-life extending polypeptide moiety

(striped), the half-life extending moiety furthermore having a dAB, VNAR or VHH (white) fused to its C-terminus.

Figure 5 provide schematic representations of exemplary fusion proteins according to embodiments of the invention. 5A: From the N-terminus (-N): a ScFv (VFI-VL) linked to a second ScFv protein with APC cleavage sites (marked by arrows). Fused to the C-terminus of the second ScFv is a phosphatidylserine binding peptide (dotted), followed by a thrombin cleavage site (black) and a half-life extending polypeptide moiety (striped). 5B: From the N-terminus (-N): a ScFv (VL-VFI) linked to a second ScFv protein with APC cleavage sites (marked by arrows). Fused to the C-terminus of the second ScFv is a phosphatidylserine binding peptide (dotted), followed by a thrombin cleavage site (black) and a half-life extending polypeptide moiety (striped). 5C: From the N-terminus (-N): a ScFv (VL-VFI) linked to a second ScFv protein with an APC cleavage site (marked by arrow) between the two ScFvs. Fused to the C-terminus of the second ScFv is a phosphatidylserine binding peptide (dotted), followed by a thrombin cleavage site (black) and a half-life extending polypeptide moiety (striped).

Figure 6 is a graph illustrating the relationship between size and number of repeating units in the half-life extending moiety for different fusion proteins according to embodiments of the invention; the Y axis represents apparent size in solution, the X-axis represents number of repeating units of half-life extending polypeptide moiety. 0 correspond to an antibody fragment without a half-life extending polypeptide moiety.

Detailed description

The human lactating mammary gland and pancreas produce a lipolytic enzyme, bile salt-stimulated lipase (BSSL), also referred to as bile salt- activated lipase (BAL) or carboxylic ester lipase (CEL). The protein is arranged in two domains, a large globular amino-terminal domain and a smaller but extended carboxy-terminal (C-terminal) domain (for a review, see e.g. Wang & Hartsuck (1993) Biochim. Biophys Acta 1166: 1-19). The present inventors surprisingly found that repetitive sequences based on or derived from the C-terminal domain of human BSSL can be successfully fused to biologically active proteins or peptides and confer increased biological half-life of the fusion partner, thereby extending its biological or therapeutic effect in vivo, as demonstrated in the Examples below.

The C-terminal domain of human BSSL consists of repeating units of, or similar to, the amino acid sequence“PVPPTGDSGAP” (SEQ ID NO: 5). Table 2 in Example 1 below lists the repeating units from human BSSL variants.

The most common form of the C-terminal domain contains 18 repeating units (UniProt entry P19835). However, there are variations in the human population, both with regard to the number of repeating units, and the amino acid sequence of the individual repeating units. Furthermore, each repeating unit has one site that may be O-glycosylated, increasing the hydrophilicity and size of the region (Stromqvist et al. Arch. Biochem. Biophys. 1997). The C- terminal end of the domain is however hydrophobic, and has been shown to bind into the active site of BSSL and cause auto-inhibition of the enzyme. The most frequent human sequence of this hydrophobic portion is“QMPAVIRF” (SEQ ID NO: 596) (Chen et al. Biochemistry 1998).

It has previously been speculated that the C-terminal domain may be responsible for the stability of BSSL in vivo, for example its resistance to denaturation by acid and aggregation under physiological conditions (Loomes et al., Eur. J. Biochem. 1999, 266, 105-111 ). In contrast, another study of the cholesterol esterase structure showed that the C-terminal domain, which is enriched with Pro, Asp, Glu, Ser and Thr residues, is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that the protein may have a short half-life in vivo due to the repetitive sequences in the C-terminal domain (Kissel et al., Biochimica et Biophysica Acta 1989, 1006).

In the present invention, the extended biological half-life of a fusion protein comprising a half-life extending polypeptide moiety as defined herein, based on or derived from the C-terminal domain of human BSSL, is believed to be due mainly to the increased hydrodynamic radius of the protein. However, it is also envisaged that other mechanisms may contribute to the increased biological half-life.

As used herein, the expressions“fused” and“fusion” refer to the joining of two or more portions of chemical entities of the same kind, such as peptides, polypeptides, proteins, or nucleic acid sequences. A fusion protein as referred to herein typically comprises at least two polypeptide portions, which may be of different origin; for instance, an antibody fragment, and one or more half- life extending polypeptide moiety, which may be derived from BSSL.

Generally, a fusion may contain the fused portions in any order and at any position; however, a fusion of genes is typically made in-frame (in-line), such that the open reading frames (ORFs) of the fused genes are maintained, as appreciated by persons of skill in the art.

In the context of the present invention, the amino acid sequences of the fusion partners of the fusion protein are referred to using the terms

“polypeptide” and“polypeptide moiety”. Notably, these terms are intended to include amino acid sequences as short as 18 amino acids, which effectively represents the smallest version of the half-life extending polypeptide moiety (2 units each of 9 amino acids). An amino acid sequence of up to about 50 amino acids may sometimes be referred to as“peptide”; however, for the sake of simplicity, in the present specification, the amino acid sequences of the fusion protein will be referred to as“polypeptide” or“polypeptide moiety” throughout.

The antibody fragment(s) constituting the fusion partner(s) of the half-life extending polypeptide moiety may be any antibody fragment, or combination of antibody fragments, that may be suitable for use in treatment or prevention of any condition or disorder, where the biological function requires a certain systemic concentration of the antibody fragment.

Typically, the antibody fragment may be or may be derived from a

biopharmaceutical, also referred to as a biologic. Examples of suitable antibody fragments include the antigen binding fragment of an antibody (Fab), a single chain variable fragment of an antibody (ScFv), a domain antibody (dAb), i.e. a VFI or VL domain of an antibody, a camelid antibody (VHH) and shark antibody (VNAR).

The antibody fragment as such may be a naturally occurring polypeptide, such as a naturally occurring VHH or VNAR, or it may be a non-naturally occurring polypeptide. Flowever, fused to one or more half-life extending polypeptide moieties, the resulting fusion protein will always be a non- naturally occurring entity. The fusion protein comprising a naturally or non- naturally occurring polypeptide may be recombinantly produced, e.g. as described in the examples below.

The antibody fragment as such may be a non-naturally occurring polypeptide, one example being a Fab. It is to be understood that the light chain of a Fab is identical with the light chain of a full-length antibody. The heavy chain is however truncated in a Fab compared with a full-length antibody. The heavy chain of a full-length antibody contains a variable heavy domain (VFI), a constant heavy chain domain 1 (CH 1 ), a hinge region, a constant heavy chain domain 2 (CFI2) and a constant heavy chain domain 3 (CFI3). The truncated heavy chain of a Fab only contains a VFI and a CH 1 and possibly the first amino acid residues of the hinge region. Thus, an antibody may be truncated either directly after the CH 1 domain or after the fifth amino acid residue of the hinge region, thus constituting a heavy chain of a Fab. A FAb thus

encompasses VH-CH 1 or VFI-CFI1 -(5aa from hinge), this difference is exemplified by SEQ ID NO: 106 compared to SEQ ID NO: 36, where the first sequence includes only the VH-CH1 , while the second sequence also includes the first five residues of the hinge region.

As will be explained below with reference to the Figures, the one or more half- life extending polypeptide moieties may in some embodiments be located at the C-terminal of the antibody fragment. In embodiments of the invention, the one or more half-life extending polypeptide moieties may be located at the N- terminal of the antibody fragment, or half-life extending polypeptide moieties may be located both at the N-terminal and C-terminal. In other embodiments, one or more half-life extending polypeptides may be inserted at a position within the antibody fragment, for example in a position located in a surface- exposed loop of the antibody fragment.

In some embodiments, a half-life extending polypeptide moiety may replace a specific sequence segment of the antibody fragment. For instance, when positioned as an insert, a half-life extending polypeptide moiety may replace a part of a surface-exposed loop on the antibody fragment, a part of the joining linker within a ScFv antibody fragment, or a part of a joining linker joining a plurality of antibody fragments and/or half-life extending polypeptide moieties. In yet other embodiments, an in-line inserted half-life extending polypeptide moiety may be combined with either an N-terminal moiety, a C-terminal moiety, or both N-terminal and C-terminal half-life extending polypeptide moieties. Notably, in embodiments of the invention comprising multiple half- life extending moieties, located at different positions, each such half-life extending moiety may be independently defined as described herein.

Otherwise stated, each such half-life extending moiety may comprise from 2 to 134 units of an amino acid sequence according to SEQ ID NO: 1.

Figure 1 schematically illustrates exemplary fusion proteins according to embodiments disclosed herein. Figure 1 illustrates the antibody fragment as being a Fab (Fig. 1 A) with the two chain chains joined by a disulfide bonds, and the half-life extending polypeptide moiety forming a tail at a C-terminal or N-terminal end of one or more chains of the antibody fragment. The one or more half-life extending polypeptide moieties may be located C-terminally of the heavy chain (Fig. 1 B), of the light chain (Fig. 1 D), or C-terminally of both the light and heavy chains (Fig. 1 C). Fig. 1 E illustrates a Fab having half-life extending polypeptide moieties located both N-terminally and C-terminally of both the heavy and light chains. Alternatively, a half-life extending polypeptide moiety may be inserted within the boundaries of the Fab. In such

embodiments, half-life extending polypeptide moieties may optionally be present at multiple sites, e.g. at one or more sites as shown in Fig. 1 , or more sites as desired, as long as the insertion does not disrupt the tertiary or folding structure of the Fab.

Figure 2 schematically illustrates exemplary fusion proteins according to embodiments disclosed herein wherein the antibody fragment is a ScFv having a VFI and a VL chain joined by a linker (Fig. 1A), where the VFI chain is either at the ScFv’s N-terminal end (left) or at its C-terminal end (right). One example of a ScFv containing fusion protein is a ScFv having a half-life extending polypeptide moiety at its C-terminal end (Fig. 2B), either at the VL chain (left) or at the VH chain (right). Another example is a fusion protein containing a ScFv to which a half-life extending polypeptide is fused at its N- terminal end (Fig. 2C), either at the VH chain (left) or at the VL chain (right). Alternatively, half-life extending polypeptide moieties may be fused to both the N- and C-terminal end of the ScFv (Fig. 1 D). In addition, or alternatively, a half-life extending polypeptide moiety may be inserted into, or may replace, the linker between the VH and the VL of the scFc (Fig. 1 D).

Figure 3 schematically illustrates exemplary fusion proteins according to embodiments disclosed herein wherein the antibody fragment is a VHH, dAb or VNAR. A VHH, dAb or VNAR may have a C-terminal half-life extending polypeptide moiety (Fig. 3B), or a N-terminal half-life extending polypeptide moiety (Fig. 3C). Alternative fusion proteins according to the invention have half-life extending polypeptide moieties fused to both the N- and C-terminal of the VHH, dAb or VNAR (Fig. 3D). A further example of a fusion protein according to the invention is a VHH, dAb or VNAR with a N-terminal fusion to a half-life-extending polypeptide moiety and with an insertion of a half-life extending polypeptide moiety in a surface exposed loop (Fig. 3E).

Finally, the present invention is not limited to the use of a single antibody fragment as fusion partner; rather, as illustrated in Fig. 4, it is envisaged that in some embodiments the fusion protein may comprise multiple antibody fragments separated by linkers, and/or, as in the examples of Fig. 4, by a half-life extending polypeptide.

Figure 4 schematically illustrates exemplary fusion proteins according to embodiments disclosed herein. The fusion proteins may contain multiple antibody fragments individually selected from a Fab, a ScFv, a dAb, a VHH and a VNAR. Each of said antibody fragments may individually have the same or a different binding affinity, for example a binding affinity to one of FIX/FIXa, CD40L, CD28, IL2 receptor alpha chain (CD25), VEGF, CD16, and P-selectin. In one embodiment, the fusion protein comprises a Fab, a ScFv and a half-life extending polypeptide moiety. The half-life extending

polypeptide moiety can for example be located at the C-terminus of the heavy chain of the Fab (Fig. 4A), but may alternatively be located at the C-terminus of the light chain of the Fab (not shown). The ScFv can be fused to the C- terminus of the half-life extending polypeptide moiety. The ScFv may be fused to the half-life extending polypeptide moiety either at its VFI (Fig. 4A) or its VL chain (not shown).

Another exemplary fusion protein according to the invention comprises multiple antibody fragments and multiple half-life extending polypeptide moieties. In one embodiment, the fusion protein comprises a Fab, a ScFv, dAB, VNAR or VHH; and two half-life extending polypeptide moieties. One half-life extending polypeptide moiety can for example be located at the C- terminus of the heavy chain of the Fab and one half-life extending polypeptide moiety can for example be located at the C-terminus of the light chain of the Fab (Fig. 4B). The ScFv can be fused to the C-terminus of either of the two half-life extending polypeptide moieties, Fig. 4B illustrates the ScFv being fused to the half-life extending polypeptide moiety that is fused to the light chain of the Fab. The dAB, VNAR or VHH can be fused to the C-terminus of the half-life extending polypeptide moiety that is fused to the heavy chain of the Fab.

Another exemplary fusion protein according to the invention comprises multiple antibody fragments and multiple half-life extending polypeptide moieties. In one embodiment, the fusion protein comprises a ScFv and a dAB, VNAR or VHH wherein a half-life extending polypeptide moiety constitutes a linker between the two antibody fragments. The ScFv may be C- terminally fused to a half-life extending polypeptide moiety (Fig. 4C).

Alternatively, or additionally, one or more half-life extending polypeptide moiety or moieties may also be located at the N- or C-terminal of a fusion protein comprising multiple antibody fragments. In the case of multiple antibody fragments, these may be the same or different. Moreover, the antibody fragments may have the same or different biological activity, including optionally the same or different binding activity. For example, the fusion protein may comprise two different antibody fragments, optionally separated by a linker or spacer sequence and/or a half-life extending polypeptide moiety. Such a fusion protein may be referred to as having bispecific activity. Alternatively, the fusion protein may comprise three different antibody fragments. Such a fusion protein may be referred to as having trispecific activity.

One example of a fusion protein comprising multiple antibody fragments which have different binding activities is a fusion protein comprising an antibody fragment having binding activity for CD40L and an antibody fragment having binding activity for CD28. Another example of a bispecific fusion protein is a fusion protein comprising an antibody fragment having binding activity for CD40L and an antibody fragment having binding activity for the IL2 receptor alpha chain (CD25). Yet another example of a bispecific fusion protein is a fusion protein comprising two or more antibody fragments individually having binding activity for two or more of CD40L, CD28 and IL2 receptor alpha chain (CD25).

In other embodiments, the fusion protein may further comprise a biologically active polypeptide such as a polypeptide selected from the group consisting of growth factors, cytokines, enzymes and ligands. As an example, the half- life extending polypeptide moiety may be positioned as a linker between different biologically active polypeptides and/or antibody fragments.

According to the invention, the one or more half-life extending polypeptide moieties used for fusion with an antibody fragment comprises an amino acid sequence comprising 2 to 136 repeating units, each unit being independently selected from the group of amino acid sequences defined by SEQ ID NO: 1 :

X1 -X2-X3-X4-X5-X6-D-X8-X9-X10-X11 (SEQ ID NO: 1 ) in which, independently,

XI is P or absent;

X2 is V or absent;

The fusion protein may comprise one or more half-life extending polypeptide moieties, said protein in total comprising 10 to 136 units.

As used herein, a“unit” refers to an occurrence of an amino acid sequence of the general formula according to SEQ ID NO: 1 as defined above, including for instance any of the sequences according to SEQ ID NOs: 2-11. The one or more half-life extending polypeptide comprises from 2 to 136 such units, which may be the same or different, within the definition set out above. The units of the half-life extending polypeptide may also be referred to as “repeating units” although there is some variation in the amino acid sequence between individual units, and hence“repeating units” is not to be understood exclusively as the repetition of one and the same sequence. Stated

differently, the half-life extending polypeptide moiety comprises from 2 to 136 units, wherein each unit is an amino acid sequence independently selected from the group consisting of the individual sequences falling within the definition of SEQ ID NO: 1.

A half-life extending polypeptide moiety contained in a fusion protein as disclosed herein may comprise a contiguous sequence of at least 18 amino acid residues (corresponding to two units that are both 9-meric versions of SEQ ID NO: 1 ), and typically up to 1496 amino acid residues (corresponding to 136 units which are all 11 -mer versions of SEQ ID NO: 1 ). The repeating units may be contiguous with one another, although it is also possible that the repeating units are separated by short spacing sequences. For instance, two repeating units may be separated by up to 10 amino acid residues that do not correspond to SEQ ID NO: 1 ; for instance, the short spacing sequence may be a peptide linker of the formula (G ₄ S)2. In some embodiments, a spacing sequence may be up to 5 amino acid residues. In some embodiments one or more amino acid residue(s) may be positioned between two repeating units, e.g. to impart a desired functionality such as an N-glycosylation site, or to provide a site for another type of modification, for instance employing a single Cys residue. In some embodiments, a linker, such as one or more G ₄ S linkers, may be used as spacing sequences between adjacent repeating units. Hence, in view of this possibility, the contiguous sequence comprising up to 136 repeating units may be longer than 1496 amino acid residues.

The repeating units of the one or more half-life extending polypeptide moieties are defined by SEQ ID NO: 1 , which is based on the repeating units of human variants of the BSSL C-terminal domain, and which allows some variation of amino acid residues in positions X3, X4, X5, X6, X8, X9, X10 and X11. In contrast, the residues at positions X1 , X2 and X7 are fixed, although positions X1 and X2, may be absent. Typically, both X1 and X2 are absent, and in such embodiments, a repeating unit consists of 9 amino acids only. A half-life extending polypeptide moiety comprising 2 to 136 units (repeating units) typically comprises several variants of the amino acid sequence motif generally defined by SEQ ID NO: 1 , such as at least two different variants according to SEQ ID NO: 1. For instance, in embodiments of the invention where the half-life extending polypeptide moiety comprises at least 4 units, it may comprise at least one unit of each of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. In embodiments of the invention where the half-life extending polypeptide moiety comprises at least 2 units, these may be independently selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. Advantageously, the half-life extending polypeptide moiety may comprise SEQ ID NOs: 3-5 in this order, optionally preceded by SEQ ID NO: 2. A unit according to SEQ ID NO: 2 may especially be located at the N- terminal end of the half-life extending polypeptide moiety, representing the first unit of the half-life extending polypeptide moiety. While other specific variations of the repeating units (e.g. the units according to SEQ ID NOs: 3- 11 ) may appear repeatedly, SEQ ID NO: 2, if present, typically only appears once, as the first repeating unit of the half-life extending polypeptide moiety. The conformation of the half-life extending polypeptide moiety is generally unstructured. For instance, in embodiments of the invention, the one or more half-life extending polypeptide moieties do not contribute to the a-helix and/or b-sheet content of the fusion protein as determined by circular dichroism or FTIR (Fourier Transform Infrared Spectroscopy).

In embodiments of the invention, a repeating unit defined by SEQ ID NO: 1 is of human origin, and preferably all of the repeating units of the half-life extending polypeptide moiety/moieties correspond(s) to naturally occurring repeating units of a variant of the C-terminal domain of human BSSL. Such repeating units are represented by SEQ ID NOs: 2-11 (See also Table 2 in the Examples). In embodiments of the invention, all repeating units of the half-life extending polypeptide moiety are selected from the group consisting of SEQ ID NOs: 2-11 , e.g. SEQ ID NOs:3-11. That is, the half-life extending polypeptide moiety may comprise 2-136 units, each independently selected from the group consisting of SEQ ID NO: 2-11 , e.g. SEQ ID NOs: 3-11. The use of a sequence of human origin may be advantageous as it is expected to contribute to a lower immunogenicity in human subjects compared to half-life extending moieties with repeating units of non-human or partly human origin, whether polypeptide based or other as used in the prior art.

Furthermore, in embodiments of the invention, the one or more half-life extending polypeptide moieties comprise, or consist of, a sequence of repeating units that corresponds to a naturally occurring human amino acid sequence of repeating units. Examples of such natural human sequences of repeating units are presented in SEQ ID NO: 12-34 and SEQ ID NO: 108- 126. Typically, such sequences comprise, as the first five repeating units, in this order: [SEQ ID NO: 2] - [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5], or, alternatively, as the first four repeating units, in this order: [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5]

Thus, in embodiments of the invention, the one or more half-life extending polypeptide moieties comprise an amino acid sequence according to any one of in SEQ ID NO: 12-34 and SEQ ID NO: 108-126. In particular, fusion proteins comprising antibody fragment(s) binding to one of FIX/FIXa, CD40L, CD28, IL2 receptor alpha chain (CD25), VEGF, CD16, and P-selectin, may comprise an amino acid sequence according to any one of in SEQ ID NO: 12- 34 and SEQ ID NO: 108-126. In some embodiments the one or more half-life extending polypeptide moieties consists of a multiple of any one of SEQ ID NO: 12-34 and SEQ ID NO: 108-126. For instance, the half-life extending polypeptide moiety may consist of three contiguous multiples, or copies, of an amino acid sequence according to any one of SEQ ID NOs :12-34 and SEQ ID NO: 108-126; for instance SEQ ID NO: 20. SEQ ID NO: 20 comprises 17 units of an amino acid sequence according to SEQ ID NO: 1 , and thus a three-copy multiple of SEQ ID NO: 20 comprises at least 51 units. Flowever, it should be noted that the repeating units of the half-life extending polypeptide moiety can be independently selected from all units according to SEQ ID NO:

1 and the invention is thus not limited to certain sequences of units being repeated. Accordingly, for instance a 51 -unit half-life extending polypeptide moiety is not necessarily formed of three copies of a 17-unit sequence, but may be formed of any combination of units according to SEQ ID NO: 1 , and in particular of any combination of repeating units selected from SEQ ID NOs :2- 1 1 . It was found that each repeating unit as defined above carries one potential O-glycosylation site. That is, upon expression in a mammalian environment allowing glycosylation, each repeating unit may be glycosylated at at most one predetermined position, typically at a threonine (T, Thr) residue. For the repeating units of SEQ ID NOs: 2-11 , the potential sites of O-glycosylation are indicated in Table 2 (see Example 1 ). There may be an upper limit to the number of glycans, which is lower than the total number of units. That is, typically, less than all of the units of the half-life extending polypeptide moiety are glycosylated. For instance, out of a sequence of 17 units (such as SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 19) typically only 10 units are glycosylated. Flence, in embodiments, a majority of the units may be glycosylated, whereas a minority of the units may be non-glycosylated.

Furthermore, the degree of glycosylation (e.g. the ratio of glycosylated units to non-glycoslyated units, or the like) may be possible to adjust according to known measures, e.g. by appropriately selecting the expression system and/or controlling the cultivation or expression conditions of the producer cells. In some embodiments, in particular where the fusion protein is produced in mammalian cells, said one or more half-life extending

polypeptide moieties may completely lack glycosylation sites. In such embodiments, one or more half-life extending polypeptide moieties of the fusion protein may each comprise 2 to 136 units of one or more amino acid sequence(s) independently selected from the group consisting of SEQ ID NOs: 637-643, 644 and 648-712, such as from the group consisting of SEQ ID NOs: 637-643.

As mentioned above, the fusion protein comprising one or more half-life extending polypeptide moieties according to the invention benefits from an increased biological half-life compared to that of the antibody fragment alone. The increased biological half-life is mainly due to the increased size of the fusion protein vis-a-vis the antibody fragment alone. The size of the fusion protein according to the invention is large enough to decrease clearance from circulation by the kidneys (renal clearance).

The radius of the majority of the pores of the glomerular membrane are 4.5- 5 nm. The membrane is negatively charged and thus are proteins that are negatively charged less prone to be cleared by the kidneys. For instance, negatively charged molecules may be significantly protected from renal clearance already at a hydrodynamic radius of 2.5 nm, while neutral molecules need a size of 3.5 nm to get a similar protection of renal clearance (Haraldsson et al Physiological Reviews 88 (2) 451 -487). For an uncharged globular protein, the size limit for renal clearance (below which a protein is secreted) is a molecular weight of about 60 kDa.

The actual molecular weight of a protein, as determined for instance by Multi Angle Light Scattering (MALS), corresponds to the theoretical molecular weight based on the amino acid composition, and any glycans bound. In contrast, the apparent size (or apparent molecular weight) in solution of a protein can be determined by Size Exclusion Chromatography (SEC), e.g. as described in Example 3 and 8 below, and yields an apparent molecular weight, or apparent size, of a protein that corresponds to the actual molecular weight of a globular protein. For proteins and peptides that do not have a globular conformation, the actual molecular weight may differ from the apparent molecular weight, or apparent size, in solution.

Typically, a non-globular protein or polypeptide may exhibit an apparent size in solution that is larger than its actual molecular weight. In the case of the present half-life extending polypeptides moieties, which typically have an unstructured, unfolded conformation, the inventors found that each repeating unit represented approximately 9 kDa, as determined by SEC (Figure 6, described in more detail below), even though the actual molecular weight was only about 1 kDa. Hence, the apparent size in solution of the fusion protein can be increased by approximately 9 kDa for each unit contained in the fusion protein according to embodiments of the invention.

In total, the fusion protein may have an apparent size in solution, as determined by SEC, larger than the size of the antibody fragment alone by a factor of at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 50.

The size of the half-life extending polypeptide moiety and of the fusion protein, respectively, may also be defined by the hydrodynamic radius, also referred to as the Stokes radius, measured in nanometers (nm). Both the apparent size in solution and the hydrodynamic radius are determined by Size Exclusion Chromatography (SEC), e.g. as described in Example 3 and 8 below.

In accordance with what has been said above with regard to apparent size in solution, the hydrodynamic radius of the fusion protein is typically large enough to avoid renal clearance. For comparison, human serum albumin, which has a size above the limit of renal clearance, has a hydrodynamic radius of 3.8 nm. The fusion protein may have a hydrodynamic radius that is at least 1.4 times as large, or at least 1.6 times as large, as the hydrodynamic radius of the antibody fragment alone. For instance, the hydrodynamic radius of the fusion protein may represent an increase at least by a factor of 2, 3, 5,

10, 20 or 50 of the hydrodynamic radius of the antibody fragment alone.

In addition to the number of repeating units in the half-life extending polypeptide moiety/moieties, also the location of the polypeptide

moiety/moieties within the fusion protein may affect the size increase. For example, N-terminal or C-terminal location of a half-life extending polypeptide moiety is expected to provide a larger hydrodynamic radius compared to a half-life extending moiety located as an insert within the amino acid sequence of the antibody fragment (e.g. forming a surface loop).

Furthermore, the unfolded structure of the half-life extending moiety not only as such provides a large hydrodynamic radius, but it may contribute to the size increase because of the hydrophilic character of many of the amino acids of the repeating units, by binding of water molecules to the half-life extending polypeptide moiety, to further increase the hydrodynamic radius.

Finally, glycosylation of some of the repeating units may further contribute to a larger size, as demonstrated in Example 3 below. It was found that a half- life extending polypeptide moiety of 34 repeating units exhibited an apparent size of around further 20 kDa compared to the same sequence of repeating units without glycosylation.

Figure 6 illustrates the relationship between the number of repeating units and the apparent size in solution according to various embodiments of the invention. In these embodiments, described in the Examples hereinbelow, an antibody fragment was fused to half-life extending polypeptide moieties of various lengths (different number of units: 7, 17, 29, 34, 51 , and 68, respectively). The inventors have found that the correlation between the size in solution and number of repeating units is linear in the investigated area. It was also found that the size in solution of one unit corresponds to a globular protein with molecular weight of 9 kDa. Hence, the size increase achieved by addition of a given number of units can be predicted. For instance, a polypeptide moiety having 80 repeating units would have an apparent size in solution corresponding a globular protein of molecular weight of

approximately 720 kDa. These insights can be used for fine tuning the pharmacokinetic properties of a biologic, in particular half-life and mean residence time, by fusion with one or more half-life extending polypeptide moieties as described herein, wherein the one or more polypeptide moieties has a certain size, designed to provide a desired half-life in vivo. For each antibody fragment, the size of the half-life extending polypeptide in terms of the number of repeating units may be chosen with regard to the size and half- life of the antibody fragment as such, the route of administration, the dosing amount and the desired dosing interval; nevertheless, the linear relationship demonstrated between the size (Fig. 6) and the number of units allows for rational design of desired half-life extending polypeptides for a particular fusion protein of interest.

For market approved therapeutic products, accurate characterization is a necessary regulatory requirement, and for a glycosylated protein the exact position of any glycans must be known. The fact that each unit of the present half-life extending polypeptide moiety carries at most one O-glycosylation site may facilitate characterization of a fusion protein expressed in mammalian systems.

A suitable protease for characterization of the half-life extending polypeptides according to the invention is pepsin, which cleaves after the acidic residues: glutamic acid (Glu, E) and aspartic acid (Asp, D). However, as pepsin typically will not cleave proximal to a glycosylated residue due to steric interference of the glycan with the protease, the repeating units that carry an O-glycosylation will have different cleavage patterns compared to non- glycosylated units. Based on this knowledge and in view of the limited and relatively predictable glycosylation pattern, characterization of the present fusion proteins using established methods, such as chromatographic methods and mass spectrometry, is greatly simplified compared to half-life extended moieties that are potentially glycosylated to a massive or unknown extent, making industrial expression of the present fusion proteins in mammalian systems more practically feasible.

Another potential advantage of glycosylation of the half-life extending polypeptide moiety is that glycosylation may provide a means of increasing immune tolerance towards the fusion protein. Glycans of both the N-glycan and the O-glycan class (although only O-glycans are relevant in the context of the present invention) ending with a a2,6- linked terminal sialic acid, can bind to CD22 or to Siglec-10, which are two inhibitory receptors of the sialic acid binding immunoglobulin-like lectin (Siglec) family. These receptors act by damping the signal from the B-cell receptor (BCR), which may lead to development of B-cell tolerance towards the fusion protein. Glycans of human proteins possess both a2,6- and a2,3-linked terminal sialic acid. In order to increase the sialic acid content with a2,6- linked terminal sialic acid in fusion proteins expressed in cells of human origin, the fusion protein of interest may be co-expressed with a2,6-sialyltransferase. Fusion proteins produced in Chinese hamster ovary (CHO) cells only have a2, 3-linkage due to the absence of a2,6-sialyltransferase expression. In order to introduce a2,6- linked terminal sialic acid in the O-glycans of fusion proteins produced in CHO cells, the fusion protein of interest may be co-expressed with a2,6- sialyltransferase.

In addition to the potentially improved immune tolerance, an increased sialic acid content may also have a beneficial effect on the half-life of the fusion protein, as the sialic acid may serve to shield any potential epitopes for other glycan receptors present among the O-glycans, thereby reducing or abolishing binding of the fusion protein to endocytic receptors.

The specific embodiments of one or more half-life extending polypeptide moieties as disclosed above, as well as their associated advantages, are specifically intended to apply to the embodiments of different fusion proteins comprising defined antibody fragments as disclosed below.

The biological activity of said antibody fragment may be a binding activity, such as a CD40L binding activity. A fusion protein comprising an antibody fragment having binding activity to CD40L is also referred to as a CD40L binding fusion protein. Such a fusion protein thus may comprise a CD40L binding antibody fragment, and one or more half-life extending polypeptide moieties as defined herein.

The CD40L (CD154) - CD40 co-stimulatory signal axis is important for regulation of the immune system. CD40L are primarily expressed on activated T-cells but also present on other cell-types. Blockade of the system should be beneficial in autoimmune disorders, as well as in other indications where a decrease in activation of the immune system might be beneficial. An antibody with the generic name Ruplizumab has for instance previously been developed for Systemic Lupus Erythematosus (SLE). An example of an antibody fragment useful for blocking CD40 system is an antibody fragment based on Ruplizumab.

In some embodiments, the CD40L binding antibody fragment is a Fab. In such embodiments, said at least one of said one or more half-life extending moieties may be positioned at a C-terminal of said CD40L binding Fab. In particular, at least one of said one or more half-life extending moieties may be positioned at a C-terminal of a light or heavy chain of said Fab, or one half-life extending moiety may be positioned at a C-terminal of a light chain and one half-extending moiety may be positioned at a heavy chain of said Fab. It is to be understood that said one or more half-life extending polypeptide moieties may be fused directly to a C-terminal of a heavy chain and/or light chain with or without an intermediate linker. Examples of linkers are disclosed elsewhere herein. As disclosed above, the heavy chain of said Fab may in some instance include a few amino acid residues from the hinge region. In other instances, the heavy chain may lack such amino acid residues from a hinge region.

CD40L binding fusion proteins, and in particular CD40L binding Fabs, comprises in total 10 to 136 units, forming one or more half-life extending polypeptide moieties, such as in total at least 17, 29, 34, 36, 51 , or at least 68 units. Fusion proteins comprising a Fab as disclosed herein, and in particular a CD40L binding Fab, may for example comprise one half-life extending polypeptide moiety located at the C-terminal of a heavy chain or a light chain of said Fab. Said C-terminally located half-life extending polypeptide moiety may comprise 17 or at least 17 units, 34 or at least 34 units, 51 or at least 51 units, 68 or at least 68 units. Alternatively, one half-life extending polypeptide moiety may be located at a C-terminal of the heavy chain and one half-life extending polypeptide moiety may be located at a C-terminal of the light chain. Each of said half-life extending moieties may individually comprise at least 7 units, such as at least 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20,

21 , 22, 23, 24, 25, 26, 27, 28, 29 or 34 units.

Examples of different CD40L binding fusion proteins are listed in Table 3 of Example 2 and in Table 16 of Example 17. Thus, specific examples of Fab- based fusion proteins includes, but are not limited to, fusion proteins comprising a Fab and one or more half-life extending polypeptide moieties, said moieties comprising a defined number of units (#) and having a defined location as follows (# units HC/ # units LC): 16/18, 18/16, 15/19, 19/15, 14/20, 20/14, 13/21 , 21/13, 12/17, 17/12, 13/17, 17/13, 17/17, -/17, 171-, 29/7, 7/29, 17/34, 34/17, 34/-, -/34, 34/34, 51/-, -/51 , -/68 and 68/- units, where

indicates no units. Although specifically exemplified for fusion proteins comprising a CD40L binding Fab, it is to be understood that these examples of # units and location of half-life extending polypeptide moieties may be useful also for other Fab based fusion proteins as disclosed herein.

Said CD40L binding Fab, or plurality of CD40L binding Fabs, may comprise at least one heavy chain having an amino acid sequence selected from SEQ ID NO: 35 and SEQ ID NO: 106, and optionally at least one light chain having an amino acid sequence as defined in SEQ ID NO: 36.

In one embodiment, said CD40L binding fusion protein comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 37, 39, 41-42, 50-52, 99, 101-102, 105-106, 127-171 , 216-259, 594-595 and 646, and at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 38, 40, 53, 100, 103, 172-215 and 647. In Example 4 it is verified that the binding affinity to CD40 of such CD40L binding fusion proteins is not affected by fusion to one or more half-life extending polypeptide moieties as disclosed herein. For example, a CDL40 binding fusion protein may comprise a combination of amino acid sequences as set out in Table 3, such as a combination of polypeptides selected from the group consisting of: a combination of a polypeptide comprising SEQ ID NO:37 and a polypeptide comprising SEQ ID NO:38; a combination of a polypeptide comprising SEQ ID NO:41 and a polypeptide comprising SEQ ID NO:36; a combination of a polypeptide comprising SEQ ID NO:39 and a polypeptide comprising SEQ ID NO:36; a combination of a polypeptide comprising SEQ ID NO:35 and a polypeptide comprising SEQ ID NO:40; a combination of a polypeptide comprising SEQ ID NO:37 and a polypeptide comprising SEQ ID NO:36; a combination of a polypeptide comprising SEQ ID NO:35 and a polypeptide comprising SEQ ID NO:38; a combination of a polypeptide comprising SEQ ID NO:39 and a polypeptide comprising SEQ ID NO:38; a combination of a polypeptide comprising SEQ ID NO:37 and a polypeptide comprising SEQ ID NO:40; a combination of a polypeptide comprising SEQ ID NO:52 and a polypeptide comprising SEQ ID NO:53; a combination of a polypeptide comprising SEQ ID NO:102 and a polypeptide comprising SEQ ID NO:103; a combination of a polypeptide comprising SEQ ID NO:105 and a polypeptide comprising SEQ ID NO:46; a combination of a polypeptide comprising SEQ ID NO:99 and a polypeptide comprising SEQ ID NO:100; a combination of a polypeptide comprising SEQ ID NO:101 and a polypeptide comprising SEQ ID NO:100; a combination of a polypeptide comprising SEQ ID NO:149 and a polypeptide comprising SEQ ID NO:38; and a combination of a polypeptide comprising SEQ ID NO:166 and a polypeptide comprising SEQ ID NO:38.

In this context, a“combination of polypeptides” refers to a compound comprising at least two separate polypeptide chains forming a multimeric compound, in particular a dimeric compound, e.g. by action of surface complementarity and/or one or more disulfide bond(s). In the first-mentioned example above, a polypeptide comprising SEQ ID NO:37 thus forms a dimer with a polypeptide comprising SEQ ID NO:38.

Alternatively, the CD40L binding antibody fragment may be a scFv. In one embodiment, such a CD40L binding scFv comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 47- 48.

The biological activity of said antibody fragment may be a binding activity, such as a FIX/FIXa binding activity. A fusion protein comprising an antibody fragment having binding activity to FIX/FIXa is also referred to as a FIX/FIXa binding fusion protein. For hemophilia type A patients, the cause to the lack of coagulation is too low levels of FVIII. Upon coagulation activation, factor VIII (FVIII) forms a complex with activated factor IX (FIXa) thus forming the tenase complex which in turns activates factor X (FX). One example of a biologic under development for use in hemophilia A is Emicizumab. Emicizumab is a bispecific antibody

mimicking the FVIII function, by having one antigen binding arm binding to FIXa and the other binding to FX, thereby exhibiting procoagulant activity. An example of an antibody fragment useful as a FVIII mimetics, i.e. for binding to FIX/FIXa, is an antibody fragment based on Emicizumab. In particular, such an antibody fragment may comprise or be based on the FIXa antigen binding fragment or a variable portion thereof, e.g. a Fab or a ScFv, of a FVIII mimicking antibody such as Emicizumab, thus creating an antibody fragment with procoagulant activity.

In order to further increase the FVIII mimetic properties of a FIX/FIXa binding fusion protein, the fusion protein may further comprise a FX binding moiety, such as a peptide from FVIII with affinity to FX. In one embodiment, the fusion protein comprises an antibody fragment having a binding affinity for FIX/FIXa. In addition, the fusion protein may comprise one or more cleavage sites to enable separation of the binding portion of the antibody fragment from the half-life extending polypeptide in vivo. An antibody fragment binding to FIX/FIXa and functioning as a FVIII mimetic is not under the regulation of the coagulation system. It is therefore envisioned that incorporation of the regulatory elements that are present in native FVIII in the fusion protein according to the invention may further ensure safety for use of such a fusion protein. For example, one of the activated protein C (APC) cleavage sites from FVIII may be introduced; [PQLR^MKN, SEQ ID NO: 90] or [VDQR^GNQ, SEQ ID NO: 91] into a FIX/FIXa binding fusion protein (cleavage position is indicated by an arrow).

In other embodiments, a cleavage site for another protease in the coagulation cascade such as thrombin, Factor Xla, FVIIa or FIXa (e.g. SEQ ID NO: 95-97) may be introduced into a FIX/FIXa binding polypeptide. A fusion protein comprising an antibody fragment that binds to FIX/FIXa may furthermore comprise one or more insertions or mutations that enables localization to platelet membranes. One way of enabling localization of the fusion protein to the membrane of platelets is to introduce a peptide with binding propensity to phosphatidylserine (PS), which is enriched on platelet membranes. Two examples are sequences from protein kinase c gamma (PKCg; SEQ ID NO: 92) and phosphatidylserine decarboxylase (PSD; yeast SEQ ID NO: 93, human SEQ ID NO: 94). Other examples are SEQ ID NOs: 713 and 714 (Kapty et al 2012, Journal of Biomolecular Screening, 17(10), 1293-1301 ) and protein kinase c beta (PKCb; SEQ ID NO: 715). For native FVIII, localization to platelet membranes normally occur upon activation of FVIII by cleavage of FVIII by thrombin. In fusion proteins comprising a PS binding peptide, such a peptide can preferably be inserted C-terminally of one or more antibody fragment(s). In addition, the PS binding peptide may be followed directly by a thrombin sensitive peptide (SEQ ID NO: 95). A half-life extending polypeptide moiety may be positioned following the thrombin sensitive peptide.

By introduction of a cleavage site, a phosphatidylserine binding peptide, and a thrombin sensitive peptide, the half-life extending polypeptide moiety will be cleaved off upon initiation of coagulation. This will in turn expose the PS binding peptide, which may enable binding to platelets.

Schematic representations of exemplary FIX/FIXa binding fusion proteins, comprising cleaveage site(s), a PS binding peptide and a thrombin sensitive peptide, are depicted in Figure 5. Cloning and production of examplary FIX/FIXa binding fusion proteins is described in Example 7.

For the provision of fusion proteins as disclosed herein, FIX/FIXa binding antibody fragment(s) are fused to one or more half-life extending polypeptide moieties, thus providing a fusion protein comprising in total 10 to 136 units, said units forming one or more half-life extending polypeptide moieties. In similarity with e.g. Fab-based CD40L binding fusion proteins, Fab-, dAb or scFv-based FIX/FIXa binding fusion proteins may comprise in total at least 17, 29, 34, 36, 51 , or at least 68 units. Fusion proteins may comprise one half-life extending polypeptide moiety, which may be located at a N- or C- terminal of the antibody fragment, said one half-life extending polypeptide moiety comprising e.g. 17 or at least 17 units, 34 or at least 34 units, 51 or at least 51 units, 68 or at least 68 units. When the antibody fragment is a Fab, said half-life extending polypeptide moiety may be located at a C-terminal of a heavy chain or a light chain. Alternatively, one half-life extending polypeptide moiety may be located at a C-terminal of the heavy chain and one half-life extending polypeptide moiety may be located at a C-terminal of the light chain. Each of said half-life extending moieties may individually comprise at least 7 units, such as at least 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20,

21 , 22, 23, 24, 25, 26, 27, 28, 29 or 34 units.

Examples of different FIX/FIXa binding fusion proteins are listed in Table 8 of Example 7. Specific examples of Fab-based fusion proteins includes, but are not limited to, fusion proteins comprising a Fab and one or more half-life extending polypeptide moieties, said moieties comprising a defined number of units (#) and having a defined location (# units HC/ # units LC): 16/18, 18/16, 15/19, 19/15, 14/20, 20/14, 13/21 , 21/13, 12/17, 17/12, 13/17, 17/13, 17/17, - /17, 171-, 29/7, 7/29, 17/34, 34/17, 34/-, -/34, 34/34, 51/-, -/51 , -/68 and 68/- units, where indicates no (zero) units.

In other embodiments, FIX/FIXa binding fusion proteins comprises a FIX/FIXa binding antibody fragment being a scFv. Specific examples of scFv-based fusion proteins comprising one half-life extending polypeptide moiety, includes, but are not limited to, fusion proteins comprising 17, 34, 51 and 68 units.

In one embodiment, said antibody fragment, or plurality of antibody

fragments, binding to FIX/FIXa comprises at least one heavy chain having an amino acid sequence selected from SEQ ID NO: 55, and optionally at least one light chain having an amino acid sequence as defined in SEQ ID NO: 54.

Additionally, or alternatively, said antibody fragment, or plurality of antibody fragments, binding to FIX/FIXa comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 56- 57, 618-621 , 623-624, 626-629, 632 and 716-723, such as SEQ ID NOs: 56- 57, 618-620, 627-629 and 720-722. In one embodiment, said fusion protein binds to FIX/FIXa and comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 59-66, 75-78, 98, 622, 625, 630, 631 and 633-636. Alternatively, said fusion protein comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 55 and 67-70 and at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 54 and 71 -74.

The biological activity of said antibody fragment may be a binding activity, such as a CD28 binding activity. A fusion protein comprising an antibody fragment having binding activity to CD28 is also referred to as a CD28 binding fusion protein.

Selective blockade of CD28, without direct inhibition of potentially tolerogenic signals through CTLA-4, is a relatively effective way to modulate pathogenic T cell responses. It is thus envisioned that various T-cell mediated

pathogenicities may benefit from CD28 blockade. One example of a biologic targeting CD28 is a single domain antibody with the generic name Lulizumab, previously developed for SLE and Sicca Syndrome. An example of an antibody fragment useful for blocking CD28 is an antibody fragment based on the single domain antibody Lulizumab.

In one embodiment, said antibody fragment, or plurality of antibody

fragments, is a domain antibody binding to CD28. In one example, such antibody fragment(s) comprises at polypeptide having an amino acid sequence as defined in SEQ ID NO: 79. CD28 binding fusion proteins may comprise such CD28 binding domain antibody and one or more half-life extending polypeptide moieties as disclosed herein. For example, a fusion protein binding to CD28 may comprise one or more half-life extending polypeptide moieties comprising 17, 34, 51 or 68 units. Such fusion proteins may comprise a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 80-83. Reference is made to Example 11 describing production and characterization of such example fusion proteins.

The biological activity of said antibody fragment may be a binding activity, such as an IL-2 receptor alpha chain (CD25) binding activity. A fusion protein comprising an antibody fragment having binding activity to one of the IL-2 receptor chains is also referred to as an IL-2 receptor binding fusion protein. The IL-2 system is considered central in the initiation and activation of immune responses. Blockade of this system leads to inhibition of the immune system. One example of an antibody blocking this system is an antibody with the generic name Basiliximab, which is approved as a drug for graft vs host reactions after organ transplantations. This antibody has binding affinity towards one of the chains of the IL-2 receptor. Other disorders where IL-2 blockade might prove useful are T-cell leukemia and a variety of autoimmune conditions. An example of an antibody fragment useful for blocking the IL-2 receptor is an antibody fragment based on Basiliximab, for example a Fab, dAb or ScFv based on Basiliximab.

In one embodiment, said antibody fragment, or plurality of antibody

fragments, is a Fab, dAb or scFv binding to CD25. In one example, such antibody fragment(s) is/are a Fab comprising at least one heavy chain having an amino acid sequence selected from SEQ ID NO: 84, and optionally at least one light chain having an amino acid sequence as defined in SEQ ID NO: 85. CD25 binding fusion proteins may comprise such CD25 binding Fab(s) and one or more half-life extending polypeptide moieties as disclosed herein. For example, a fusion protein binding to CD25 may comprise one or more half-life extending polypeptide moieties comprising 17, 34, 51 or 68 units. One half-life extending polypeptide moiety may be fused to the heavy chain of such a CD25 binding Fab, and/or one half-life extending polypeptide moiety may be fused to the light chain of said CD25 binding Fab. Such fusion proteins may comprise a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 84, 86, and 88-89, and at least one

polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 85 and 87. Reference is made to Example 12 describing production and characterization of such example fusion proteins.

In one embodiment, the biological activity of said antibody fragment is a binding activity, such as a VEGF binding activity. Binding to VEGF may provide blockade of VEGF-signaling which may inhibit growth of blood vessels. A fusion protein comprising an antibody fragment having binding activity to VEGF is also referred to as a VEGF binding fusion protein. One example of a biologic targeting VEGF is an antibody with the generic name Ranibizumab. An example of an antibody fragment useful for binding to VEGF is thus an antibody fragment, such as a dAb, Fab or scFv, based on

Ranibizumab. Non-limiting examples of a VEGF binding fusion protein include a fusion protein comprising a combination of polypeptides selected from the group consisting of: a combination of a polypeptide comprising SEQ ID NO:406 and a polypeptide comprising SEQ ID NO:403, and a combination of a polypeptide comprising SEQ ID NO:405 and a polypeptide comprising SEQ ID NO:403.

In one embodiment, said antibody fragment, or plurality of antibody

fragments, is a Fab binding to VEGF. In one example, such antibody fragment(s) binding to VEGF comprises at least one heavy chain having an amino acid sequence selected from SEQ ID NO: 402 and SEQ ID NO: 404, and optionally at least one light chain having an amino acid sequence as defined in SEQ ID NO: 403. It is to be understood that VEGF binding fusion proteins may comprise such VEGF binding Fab(s) and one or more half-life extending polypeptide moieties as disclosed herein. As an example, VEGF binding fusion proteins may comprise in total 10 to 136 units, forming one or more half-life extending polypeptide moieties, such as in total at least 17, 29, 34, 36, 51 , or at least 68 units. For example, a fusion protein binding to VEGF may comprise one or more half-life extending polypeptide moieties, each of said half-life extending moieties individually comprising at least 7 units, such as at least 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 34 units.

Said half-life extending polypeptide moiety may be located at a C-terminal of a heavy chain or a light chain of said VEGF binding Fab. Alternatively, one half-life extending polypeptide moiety may be located at a C-terminal of the heavy chain and one half-life extending polypeptide moiety may be located at a C-terminal of the light chain. Specific examples of Fab-based VEGF binding fusion proteins includes, but are not limited to, fusion proteins comprising a VEGF binding Fab and one or more half-life extending polypeptide moieties, said moieties comprising a defined number of units (#) and having a defined location (# units HC/ # units LC): 16/18, 18/16, 15/19, 19/15, 14/20, 20/14, 13/21 , 21/13, 12/17, 17/12, 13/17, 17/13, 17/17, -/17, 171-, 29/7, 7/29, 17/34, 34/17, 34/-, -/34, 34/34, 51/-, -/51 , -/68 and 68/- units, where indicates no units. Non-limiting examples of Fab-based VEGF binding fusion proteins are fusion proteins comprising at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 402, 404-474, and at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 403, 475-520. Reference is made to Example 13 describing production and characterization of such example fusion proteins.

In one embodiment, the biological activity of said antibody fragment is a binding activity, such as a CD16 binding activity. Binding to CD16 may provide CD16 blockade. Blocking of CD16 can prevent autoimmune blood cytopenia by blocking phagocytosis and antibody-dependent cellular cytotoxicity. A fusion protein comprising an antibody fragment having binding activity to CD16 is also referred to as a CD16 binding fusion protein. One example of a biologic targeting CD16 is a monoclonal antibody known as GMA-161. An example of an antibody fragment useful for binding to CD16 is thus an antibody fragment, such as a dAb, Fab or scFv, based on GMA-161.

In one embodiment, said antibody fragment, or plurality of antibody

fragments, is a Fab binding to CD16. In one example, such antibody fragment(s) binding to CD16 comprises at least one heavy chain having an amino acid sequence selected from SEQ ID NO: 263 and SEQ ID NO: 264, and optionally at least one light chain having an amino acid sequence as defined in SEQ ID NO: 261. It is to be understood that CD16 binding fusion proteins may comprise such CD16 binding Fab(s) and one or more half-life extending polypeptide moieties as disclosed herein. As an example, CD16 binding fusion proteins may comprise in total 10 to 136 units, forming one or more half-life extending polypeptide moieties, such as in total at least 17, 29, 34, 36, 51 , or at least 68 units. For example, a fusion protein binding to CD16 may comprise one or more half-life extending polypeptide moieties, each of said half-life extending moieties individually comprising at least 7 units, such as at least 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 34 units comprising 17, 34, 51 or 68 units.

Said half-life extending polypeptide moiety may be located at a C-terminal of a heavy chain or a light chain of said CD16 binding Fab. Alternatively, one half-life extending polypeptide moiety may be located at a C-terminal of the heavy chain and one half-life extending polypeptide moiety may be located at a C-terminal of the light chain. Specific examples of Fab-based CD16 binding fusion proteins includes, but are not limited to, fusion proteins comprising a CD16 binding Fab and one or more half-life extending polypeptide moieties, said moieties comprising a defined number of units (#) and having a defined location (# units HC/ # units LC): 16/18, 18/16, 15/19, 19/15, 14/20, 20/14, 13/21 , 21/13, 12/17, 17/12, 13/17, 17/13, 17/17, -/17, 171-, 29/7, 7/29, 17/34, 34/17, 34/-, -/34, 34/34, 51/-, -/51 , -/68 and 68/- units, where indicates no units.

Non-limiting examples of Fab-based CD16 binding fusion proteins are fusion proteins comprising at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 260, 262-355, and at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 261 , 356-401. For instance, CD16 binding fusion proteins may comprise a combination of polypeptides selected from the group consisting of: a combination of a polypeptide comprising SEQ ID NO: 263 and a polypeptide comprising SEQ ID NO: 261 ; a combination of a polypeptide comprising SEQ ID NO: 262 and a polypeptide comprising SEQ ID NO: 261 ; and a combination of a polypeptide comprising SEQ ID NO: 260 and a polypeptide comprising SEQ ID NO: 261. Reference is made to Example 14 describing production and characterization of such example fusion proteins.

In one embodiment, the biological activity of said antibody fragment is a binding activity, such as a P-selectin binding activity. Binding to P-selectin may provide P-selectin blockade. Blockade of P-selectin can reduce tethering and adhesion of red and white blood cells to endothelium. A fusion protein comprising an antibody fragment having binding activity to P-selectin is also referred to as a P-selectin binding fusion protein. One example of a biologic targeting P-selectin is a monoclonal antibody known as Inclazumab. An example of an antibody fragment useful for binding to P-selectin is thus an antibody fragment, such as a dAb, Fab or scFv, based on Inclazumab.

In one embodiment, said antibody fragment, or plurality of antibody fragments, is a Fab binding to P-selectin. In one example, such antibody fragment(s) binding to P-selectin may comprise at least one heavy chain having an amino acid sequence selected from SEQ ID NO: 522 and SEQ ID NO: 523, and optionally at least one light chain having an amino acid sequence as defined in SEQ ID NO: 521. It is to be understood that P-selectin binding fusion proteins may comprise such P-selectin binding Fab(s) and one or more half-life extending polypeptide moieties as disclosed herein. As an example, P-selectin binding fusion proteins may comprise in total 10 to 136 units, forming one or more half-life extending polypeptide moieties, such as in total at least 17, 29, 34, 36, 51 , or at least 68 units. For example, a fusion protein binding to P-selectin may comprise one or more half-life extending polypeptide moieties, each of said half-life extending moieties individually comprising at least 7 units, such as at least 8, 9, 10, 11 , 12, 13, 14, 15, 16,

17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 34 units.

Said half-life extending polypeptide moiety may be located at a C-terminal of a heavy chain or a light chain of said P-selectin binding Fab. Alternatively, one half-life extending polypeptide moiety may be located at a C-terminal of the heavy chain and one half-life extending polypeptide moiety may be located at a C-terminal of the light chain. Specific examples of Fab-based P- selectin binding fusion proteins includes, but are not limited to, fusion proteins comprising a P-selectin binding Fab and one or more half-life extending polypeptide moieties, said moieties comprising a defined number of units (#) and having a defined location (# units FIC/ # units LC): 16/18, 18/16, 15/19, 19/15, 14/20, 20/14, 13/21 , 21/13, 12/17, 17/12, 13/17, 17/13, 17/17, -/17,

171-, 29/7, 7/29, 17/34, 34/17, 34/-, -/34, 34/34, 51/-, -/51 , -/68 and 68/- units, where indicates no units.

Non-limiting examples of Fab-based P-selectin binding fusion proteins are fusion proteins comprising at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 522-524, 548- 593, and at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 521 , 525-547. Reference is made to Example 15 describing production and characterization of such example fusion proteins.

In addition, further non-limiting examples of Fab-based P-selectin binding fusion proteins include fusion proteins comprising a combination of

polypeptides selected from the group consisting of: a combination of a polypeptide comprising SEQ ID NO:548 and a polypeptide comprising SEQ ID NO:525; a combination of a polypeptide comprising SEQ ID NO:571 and a polypeptide comprising SEQ ID NO:525; a combination of a polypeptide comprising SEQ ID NO: 565 and a polypeptide comprising SEQ ID NO:525; a combination of a polypeptide comprising SEQ ID NO: 588 and a polypeptide comprising SEQ ID NO:525; a combination of a polypeptide comprising SEQ ID NO: 548 and a polypeptide comprising SEQ ID NO: 521 ; and a

combination of a polypeptide comprising SEQ ID NO: 524 and a polypeptide comprising SEQ ID NO:521.

The fusion protein may comprise a linker, typically a peptide linker, linking the antibody fragment to one or more half-life extending polypeptide moieties as described herein, or linking multiple antibody fragments to one another and/or to one or more half-life extending polypeptide moieties. Hence, in

embodiments of the invention the fusion protein further comprises a peptide linker positioned between an amino acid sequence of the antibody fragment and an amino acid sequence of the half-life extending polypeptide moiety. In one example, the fusion protein comprising a linker has the general structure: [antibody fragment]-[linker]-[half-life extending polypeptide moiety], wherein the antibody fragment, the linker and the one or more half-life extending polypeptide moieties are as disclosed herein. For example, the peptide linker may be selected from -GS-, -(G ₄ S) _n -, -(G ₄ A) _n -, -(GsV and -GSGAA-, wherein n is an integer from 1 to 5, typically from 1 to 3, from 1 to 2, or from 2 to 3.

The use of a linker may be advantageous in that it may reduce the

occurrence of, or, in the case of n being at least 2, prevent the formation of neo epitopes and subsequent binding of such neo epitopes by antigen- presenting cells of the immune system. Examples of linkers that may be used for linking different fragments and/or moieties in the fusion proteins as disclosed herein, are linkers comprising an amino acid sequence selected from the group consisting of GS and SEQ ID NO: 107, 608-617.

In addition, the linker may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 90-91 and 95-97, in order to incorporate a APC, thrombin, Factor Xla/FVIIa or Factor IXa cleavage site. Peptide linkers comprising such cleavage sites may in particular be useful in FIX/FIXa binding fusion proteins as disclosed herein.

The fusion protein may in some embodiments comprise further, or additional, C-terminal or N-terminal amino acid residues. For example, one or more of said one or more half-life extending polypeptide moieties may comprise further C- or N-terminal amino acid residues. Each additional amino acid residue may individually or collectively be added in order to, for example, improve production, purification, stabilization in vivo or in vitro, coupling, or detection of the polypeptide.

For example, one or more amino acids may be added at the N- or C-terminal of the amino acid sequence in order to allow, or improve, expression in a cell. One example is a methionine residue, added at the N-terminal of the fusion protein, such as at the N-terminal of the antibody fragment, in order to express the fusion protein, or part of the fusion protein, in a bacterial cell line such as E. coli. Following expression of a fusion protein in E. coli, the methionine residue may optionally be removed. Thus, fusion proteins as disclosed herein may in some instances comprise an N-terminal methionine residue.

Another example is additional amino acid residues that provide a“tag” for, inter alia, purification or detection of the fusion protein. Alternatively, a“tag” may be provided by replacement of terminal amino acid residues within e.g. one of said one or more half-life extending polypeptide moieties. Thus, in one embodiment, at least one of said one or more half-life extending polypeptides comprises a C-terminal unit comprising a tag, wherein said tag optionally is a peptide tag. This means that at least one of said one or more half-life extending polypeptide moieties, wherein each moiety comprises 2 to 136 units, comprises a C-terminal unit including a tag. Such a tag may, as set out above, for example improve purification or detection of the fusion protein. Further, said tag may constitute a replacement of a terminal part of said C- terminal unit, or may constitute a C-terminal addition to said C-terminal unit. When said tag constitutes a replacement, it may consist of 2-4 amino acid residues.

Non-limiting examples of C-terminal units comprising a tag includes C- terminal units having an amino acid sequence independently selected from the group consisting of all amino acid sequences according to

a) X1 -X2-X3-X4-X5-X6-D-E-P-E-A (SEQ ID NO:597),

wherein each of X1 -X6 are as defined in claim 1 ;

b) X1 -X2-X3-X4-X5-X6-D-X8-X9-E-P-E-A (SEQ ID NO:598), wherein each of X1 -X6 and X8-X9 are as defined in claim 1 ;

c) X1 -X2-X3-X4-X5-X6-D-X8-X9-X10-X11 -E-P-E-A (SEQ ID NO:599) wherein each of X1 -X11 are as defined in claim 1 , and

d) X1 -X2-X3-X4-X5-X6-D-X8-X9-X10-X11 -L f-E-P-E-A (SEQ ID NO:600) wherein each of X1 -X11 are as defined in claim 1 , and L1 is a linker, said linker optionally being selected from -GS-, -(G ₄ S) _n -, -(G ₄ A) _n -, -(GsV and - GSGAA-, wherein n is an integer from 1 to 5, typically from 1 to 3, from 1 to 2, or from 2 to 3.

In particular, said C-terminal unit may have an amino acid sequence independently selected from:

The fusion proteins described herein can be produced by recombinant techniques using prokaryotic or eukaryotic, such as mammalian, expression systems, using conventional methods known to persons of skill in the art. Example 2 below describes cloning and production of fusion proteins in which half-life extending polypeptide moieties are fused to antibody fragments. It should be noted that the invention is by no means limited to use of those strains and cell types of Example 2; in contrast, suitable cell lines for production of fusion proteins are known to persons of skill in the art, and examples include E. coli, Pichia pastoris, Saccharomyces cerevisiae, algae, moss cells, plant cells such as carrot cells, and mammalian cells such as CHO, HEK-293, and HT1080.

The fusion protein according to the invention has an increased hydrodynamic radius and apparent size in solution compared to the size of the antibody fragment alone. As a consequence, at least in part of reduced renal clearance due to the size increase, the pharmacokinetic properties of the fusion protein are altered. Most notably, the biological half-life is extended, as demonstrated in Examples 6 below.

Preferably, the half-life extending polypeptide moiety extends the biological half-life of the antibody fragment by a factor of at least 2 in at least one species, typically humans. In other words, the fusion protein preferably has a biological half-life that is at least 2 times that of the antibody fragment alone. For example, the fusion protein may extend the biological half-life of the antibody fragment by a factor of at least 3, at least 5, at least 10, at least 20, or at least 50. From a pharmacokinetic perspective, it may be desirable to extend the biological half-life as much as possible. Flowever, as the half-life extending effect has been shown to be proportional to the size of the half-life extending moiety, and very large half-life extending polypeptide moieties may be undesirable for various reasons, such as feasibility of production or impediment of the biological activity of the antibody fragment, the half-life extension for a given antibody fragment may have to be balanced against other requirements, and the optimum half-life extension may thus be less than the theoretical maximum half-life extension achievable by the present invention. For instance, it may be desirable to use no more than two moieties (e.g. one at the N-terminal and one at the C-terminal) of 68 units each.

Furthermore, the half-life extending polypeptide moiety, may provide increased solubility to the fusion protein. In particular, the hydrophilic nature of the half-life extending polypeptide moiety, may be beneficial in that it may increase the bioavailability of a fusion protein that is administered

subcutaneously, relative to the bioavailability of the antibody fragment alone.

In such cases, the increased solubility of the fusion protein may promote transfer to the blood stream rather than remaining in the tissue extracellular matrix after injection. This could mean that for some antibody fragments that otherwise require intravenous administration due to limited bioavailability, subcutaneous administration may be an option if the antibody fragments are fused to one or more half-life extending polypeptide moieties as described herein.

Thus, the one or more half-life extending polypeptide moieties used in the present invention may be used as a means of extending the biological half-life of an antibody fragment and possibly of adapting other pharmacokinetic properties thereof.

The fusion protein of the invention may be formulated as a pharmaceutical composition, for use in therapy and/or prevention of a condition, disorder or disease. The term "composition" as used herein should be understood as encompassing solid and liquid forms. A composition may preferably be a pharmaceutical composition, suitable for administration to a patient (e.g. a mammal) for example by injection or orally. The pharmaceutical composition typically includes the fusion protein according to the invention and at least one pharmaceutically acceptable carrier or substituent. The pharmaceutical composition may for instance comprise any one of a salt, a pH regulator, an oil, a preservative, an osmotically active agent, and any combination thereof.

The pharmaceutical composition may be formulated for any route of administration, including intravenous, subcutaneous, nasal, oral, and topical administration. For example, the composition may be formulated for intravenous or subcutaneous administration.

The condition, disorder or disease to be treated is not limited by the half-life extending polypeptide; rather, suitability of the fusion protein for treatment of a particular condition, disorder or disease may be determined solely by the antibody fragment, which may be correspond to or be based on an existing biopharmaceutical. Examples of suitable antibody fragments that may benefit from fusion with the half-life extending polypeptide moiety are described herein.

The fusion protein of the invention may be used in a method of treatment of a condition, disorder or disease, comprising the step of administering to a patient suffering from said condition, disorder or disease a fusion protein comprising an antibody fragment useful for treatment of said condition, disorder or disease, fused to a half-life extending polypeptide moiety as described herein. The patient is typically a mammal, such as a human. In this method, administration may occur less frequently compared to a treatment regimen involving administration of the biologically active antibody fragment alone.

The invention will be further described in the following examples.

Examples

Example 1: Identification of repeating units of human origin

A blast search was performed with the catalytic domain of Bile salt-stimulated lipase (BSSL) versus the non-redundant protein sequence database at the National Institute of Health (NIH), USA and identified 10 reported protein sequences for the protein of human origin that contained the whole or part of the C-terminal repetitive unstructured domain.

Material and methods

Blast at NIH was used to search for proteins of human origin that match the catalytic domain of Bile salt stimulated lipase with UniProt ID P19835

(Accession number CELJHUMAN).

Results

The BLAST search resulted in finding 10 entries that contained both a significant portion of the catalytic domain and the C-terminal repetitive unstructured domain. The number of the repeating units in the domains differed and some variability among the sequence of the repeating units was noted, see Table 1 for the different hits. Each repeating domain is initiated by a truncated sequence of 9 residues, while the most prevalent repeating units are 11 residues long. In the table below, the repeating units are separated by sign for clarity. In the enclosed sequence listing, the repetitive portions are represented by SEQ ID NOs:12-19. 3

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Table 2. Units corresponding to repeating units found in human BSSL-CTD. Potential glycosylation site is underlined.

Hence, there exists a variety of lengths of the C-terminal domain in the human population. Furthermore, the order of the repeating units can vary in the human population. This could imply that variations in the order of the repeating units and the length of the entire domain motifs are allowed. Each unit carries one site that may be O-glycosylated. The most prevalent human form is made up of the combination of the following sequence of repeating units:

[SEQ ID NO:2] - [SEQ ID NO:3] - [SEQ ID NO:4] - [SEQ ID NO:5] - [SEQ ID NO:5] - [SEQ ID NO:5] - [SEQ ID NO:5] - [SEQ ID NO:5] - [SEQ ID NO:5] - [SEQ ID NO:5] - [SEQ ID NO:5] - [SEQ ID NO:6] - [SEQ ID NO:5] - [SEQ ID NO:5] - [SEQ ID NO:7] - [SEQ ID NO:5] - [SEQ ID NO:8] - [SEQ ID NO:9]

Expressed differently:

[SEQ ID NO:2] - [SEQ ID NO:3] - [SEQ ID NO:4] - [SEQ ID NO:5]x8 - [SEQ ID NO:6] - [SEQ ID NO:5]x2 - [SEQ ID NO:7] - [SEQ ID NO:5] - [SEQ ID NO:8] - [SEQ ID NO:9] Example 2: Cloning and production of antibody fragment based fusion proteins - CD40L

This Example describes the general methods for cloning and production of fusion proteins of Ruplizumab antibody fragment sequences with half-life extending polypeptides as disclosed herein. Half-life extending polypeptides were fused to either the light chain (LC) or the heavy chain (HC) of the antibody fragments. The fusion proteins produced in this Example were characterized and tested as described in Example 3 - 6 below.

Materials and methods

DNA constructions: DNA sequences encoding a set of antibody fragments with and without half-life extending polypeptides were codon optimized for expression in E. coli or CHO cells and synthesized by the Invitrogen GeneArt Gene Synthesis service at Thermo Fisher Scientific. The genes were cloned in expression vectors for subsequent expression in E. coli, Expi293 cells or ExpiCHO cells. For PSI0716, PSI0717, PSI0762 and PSI0761 a bicistronic vector was used to incorporate both the nucleotide sequences for the heavy and the light chain.

Cultivation and purification: E. coli cells were transformed with expression vectors containing the gene fragments encoding the recombinant antibody fragments or fusion proteins and then cultivated in bioreactors using fed-batch techniques or in shake flasks, followed by protein expression and harvest of cells by centrifugation. Cell pellets were stored at -20 °C or directly subjected to osmotic shock, released proteins were clarified by centrifugation and stored at -20 °C. Expression of recombinant antibody fragments or fusion proteins was performed using the Expi293 and ExpiCHO expression systems (Thermo Fisher Scientific), essentially according to the manufacturer’s protocol.

Supernatants were harvested by centrifugation 6 days after transfection of expression vectors and stored at -70 °C.

Frozen E. coli cell pellets were resuspended and then disrupted by sonication and the cell debris subsequently removed by centrifugation followed by filtration (0.22 pm). Osmotic shock samples and supernatants from the ExpiCHO and the Expi293 cultures were thawed and filtered (0.22 pm) before purification. Each supernatant, containing the recombinant antibody fragment or fusion proteins was purified using conventional chromatography methods. Recombinant antibody fragment or fusion proteins for use in animal studies were also subjected to an endotoxin removal purification using Detoxi-Gel Endotoxin Removing Columns (Pierce, cat.no. 20344). Purified antibody fragment or fusion proteins were buffer exchanged to PBS and, unless otherwise stated, PBS was also the formulation buffer used in subsequent experiments. The purity of the fusion proteins was analyzed by SDS-PAGE stained with Coomassie Blue and the molecular weight of each protein was analyzed using mass spectrometry (HPLC/MS or MALDI-TOF/MS).

Table 3 below lists the amino acid sequences of the produced fusion proteins. A half-life extending polypeptide was fused to the C terminal of either the light chain (LC in the table below) or heavy chain (HC in the table below), or both of the light chain and the heavy chain of the Ruplizumab Fab. A peptide linker was used for fusing the FIC/LC chain to the half-life extending polypeptide. Fusion proteins without linkers were also produced, as indicated in the Table below. Moreover, fusion proteins with heavy chains without the five amino acid sequences of the hinge region were produced as indicated in the Table below.

Results

Purification resulted in protein preparations with high purity, which was analyzed by SDS-PAGE stained with Coomassie Blue. The correct identity and molecular weight of each fusion protein were confirmed by mass spectrometry analysis.

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Conclusions

Fusion proteins containing antibody fragments and half-life extending polypeptides of various lengths can be produced by constructing synthetic genes followed by expression in mammalian or bacterial systems and purified to high purity using conventional techniques.

Example 3: Biophysical characterization of fusion proteins

This Example describes the characterization of fusion proteins of Ruplizumab Fab and half-life extending polypeptides, using unfused proteins and

PEGylated proteins as references, with respect to biophysical characteristics such as apparent size and molecular weight (MW) in solution and

determination of hydrodynamic radius in solution by size exclusion

chromatography (SEC) and column calibration and Multi Angle Light

Scattering (MALS).

Material and methods

The size of the fusion proteins, unfused proteins and PEGylated proteins in solution, was assessed by analytical gel filtration on an AKTA Micro (GE Healthcare Life Sciences) using a calibrated column Superdex 200 Increase 3.2/300 (GE Healthcare Life Sciences). The column was calibrated with Gel Filtration Calibration Kit LMW (code no. 28-4038-41 , GE Healthcare Life Sciences) and Calibration Kit HMW (code no. 28-4038-42, GE Healthcare Life Sciences), containing 8 globular proteins in the size range of 6 to 669 kDa and Blue Dextran 2000, using a running buffer of 25 mM NaP and 125 mM NaCI pH 7.0 with a flow rate of 75 mI/min at a temperature of 25°C. The corresponding size and hydrodynamic radius in solution can be calculated from the elution volume of a protein on a calibrated column by the methods described in appendix 10 of Handbook of Size Exclusion Chromatography Principles and Methods (order no 18-1022-18, GE Healthcare Life Sciences. The molecular weight of the proteins was determined by a connected MALS- Rl system: Static light scattering detector miniDawn Tristar and Differential refractometer Optilab rEX, and the Astra V software (Wyatt Technology Europe, Germany). The proteins of interest were analyzed under the same conditions as during the calibration. Results

Table 4 presents the results for the fusion proteins and reference proteins.

Table 4. Characterization of Ruplizumab Fab based fusion proteins and reference proteins

Conclusions

A correlation of total length of the half-life extending polypeptide comprised in the fusion protein and the fusion protein’s size in solution was observed. The size in solution did not depend upon the positioning of the half-life extension polypeptide, since the size of the different fusion proteins was similar if all units of the half-life extending polypeptide were fused to the heavy chain (HC), to the light chain (LC), or if the same number of units was split between the heavy and the light chain of the Fab. Similar effect on increase of size in solution is noted for material with half-life extending polypeptides produced in different production systems, such as ExpiCHO, Expi293 and E.coli.

It has been noted that the hydrodynamic radius or Stokes radius of albumin, which is above the size limit of renal clearance, is 3.8 nm. This could serve as a limit of minimal size required to avoid renal clearance. All the above tested fusion proteins had a size above that of albumin’s.

Example 4: Binding to human CD40

This Example describes the binding characteristics of fusion proteins of Ruplizumab Fab and half-life extending polypeptides, wherein Ruplizumab, unfused Fab protein and another Fab targeting human CD40L were used as reference proteins.

Material and methods

The binding affinities of the fusion proteins of Ruplizumab Fab and half-life extending polypeptides for human CD40 ligand (CD40L or CD154) were analyzed using an OctetRED96 instrument (Pall/ForteBio). Polypeptides, immobilized using anti-human Fab-CFI1 2 ^nd generation (FAB2G,

Pall/ForteBio) sensors, were tested for binding to the extracellular part of human CD40L (aa 108-261 recombinantly produced in E. coli ,) typically over a concentration range from 2.5 to 80 nM in 1 :2 step increments.

Typically, association for each concentration of CD40L was monitored for 180s followed by a dissociation of 600 s. The sensors were regenerated by 3 x 10s pulses at pH 2 between each cycle and data for each sensor were referenced against buffer exposure. Kinetic constants were calculated from the sensorgrams using the Langmuir 1 :1 analyte model (Global fit) of the software“Octet System Data Analysis, Release 10.0 - kinetics module” (ForteBio, Pall Life Sciences). Results

The resulting KD values are tabulated in Table 5. When dissociation was below 5 % over the 600 s monitoring time (kd < 1 e ^-4 ), this value was used as dissociation constant.

Table 5. Binding of immobilized human Fab fusion protein to human CD40L

Conclusions

The fusion of the Fab to the half-life extending polypeptide has no

measurable influence on affinity of said Fab to the soluble part of human CD40L. The affinities of all tested fusion proteins for human CD40L were comparable to the control proteins Ruplizumab and Dapirolizumab Fab. Production system do not influence affinity of the fusion proteins. Example 5: In vitro and in silico immunogenic propensity investigation

This Example aims to identify potentially immunogenic regions present in PSI0699 (SEQ ID NO:37 / SEQ ID NO:38), a fusion protein comprising a CD40L binding Fab and two half-life extending polypeptide moieties. The Prolmmune ProPresent® Antigen Presentation assay was performed by Prolmmune (UK). Immunogenic regions were determined by identifying peptides that would be naturally processed by monocyte derived dendritic cells and consequently presented by the MHC antigen presentation system. Detection of putative immunogenic peptides was performed utilizing mass spectrometry LC/MS/MS-based analysis.

Material and methods

The Prolmmune ProPresent® Antigen Presentation assay was used to identify potentially immunogenic regions present in PSI0699. They were determined by identifying peptides naturally processed by monocyte-derived dendritic cells, and consequently presented by Class II MHC (HLA-DR) molecules. Dendritic cells used in this assay were isolated from 11 normal healthy blood donors that had an adequate coverage of the HLA types present in the human population. Putative immunogenic peptides were identified by LC/MS/MS-based analysis sequencing mass spectrometry.

The in silico immunogenicity analysis was performed using the software TEPredict (Antonets & Maksyutov TEpredict: Software for T-Cell Epitope Prediction Molecular Biology, 2010, Vol. 44, No. 1 , pp. 119-127).

Results

Overall there were 4 potentially immunogenic peptides identified in the assay, 1 was from the heavy chain of the Fab of PSI0699 and 3 were from the light chain of the Fab. Out of these, 2 were previously published as a potential Tregitope sequence, termed Treg 134, which encompasses both of these peptides. All peptides originate from constant regions of the antibody derived portion of the molecule. No immunogenic peptides derived from the half-life extending polypeptide were presented in the assay.

Moreover, the in silico evaluation did not predict any peptide from the half-life extending polypeptide to have propensity to bind to any MHC class of molecules. However, the in silico analysis predicted that further peptides from the Fab portion are likely to bind to various MHC molecules, including peptides from the variable regions inferring target specificity of the Fab.

Conclusions As no peptides were presented from the half-life extending polypeptide in the current assay setup, the potential for immunogenicity of the half-life extending polypeptide is judged to be low. The overall immunogenic potential is also judged to be low as only regions that are common to many antibodies are presented in the assay. The presentation of a previously published Tregitope peptide also suggests a low response.

Example 6: Comparative study of pharmacokinetic properties of Fab based fusion proteins - CD40L

In this Example, the intravenous and subcutaneous pharmacokinetic properties (PK) of PSI0699 (SEQ ID NO:37 / SEQ ID NO:38) and PSI0701 (SEQ ID NO:41 / SEQ ID NO:36), including unfused CD40L Fab as a control (PSI0698, Ruplizumab Fab, SEQ ID NO:35 / SEQ ID NO:36).

Materials and methods

The study followed the same general design, with a single intravenous (IV) or subcutaneous (SC) dose in male Sprague-Dawley rat (N=3 per administration route and protein), for both PSI0699 and PSI0701. For PSI0698 only the IV portion of the experiment were performed.

For PSI0699 and PSI0701 , the dose and timepoints for IV experiment were as follows: 2 mg/kg, 5 and 20 min and 1 , 4, 8, 24, 48, 72, 96 and 120 hours. For the SC experiments a dose of 4 mg/kg was used and blood samples were taken at these time points: 20 min and 1 , 4, 8, 24, 48, 72, 96, 120 and 168 hours. For the IV experiment of PSI0698 a dose of 13 mg/kg was used and blood was withdrawn at the following timepoints: 5 and 20 min and 1 , 2, 4, 8, 24, 30 and 48 hours. PSI0698, PSI0699 and PSI0701 serum concentrations were determined by a sandwich assay on the Meso Scale Discovery platform (Meso Scale Diagnostics). Active drug was captured using biotinylated CD40L and detected using a Rutenium conjugated anti-human IgG (Fab specific) antibody produced in Goat (I5260, Sigma-Aldrich). Individual concentration versus time profiles were compiled from the actual serum concentration measurements and nominal time points. The maximum

PSI0698, PSI0699 and PSI0701 concentration in serum, Cmax, and the time to reach this maximum serum concentration following administration, t _max , were determined from individual data. Other exposure and pharmacokinetic parameter estimates were determined profiles by Non-Compartmental Analysis (using Phoenix WinNonlin 8.0); i.e. AUC (area under the plasma serum concentration-time curve from time zero to infinity), CL (clearance), CL/F, (clearance following SC administration), V _ss (apparent volume of distribution at steady-state), MRT (mean residence time) and ti _/ 2 _Z (terminal half-life). The subcutaneous bioavailability, F, was calculated based on individual AUC/Dose (SC) divided by the median AUC/Dose (IV).

Results

The results are summarized in Table 6 for the IV experiment and Table 7 for the SC experiment.

Table 6. Median (range) PK parameter estimates following an intravenous single dose.

Table 7. Median (range) PK parameter estimates following a subcutaneous single dose

Conclusions The clearance of PSI0699 and PSI0701 was more than 100 times lower than the CL of the CD40L fab. The intravenous PK of PSI0699 and PSI0701 , respectively, was characterized by a low clearance and a small volume of distribution.

Both PSI0699 and PSI0701 showed a relatively high SC bioavailability, with Cmax levels observed at 24 or 48 hrs after dose, and then declining monophasically with a ti _/ 2 _Z in the order of 32-55 hrs. Based on median estimates, PSI0699 showed a somewhat higher bioavailability (68 vs. 50%), longer elimination half-life (53 vs. 40 hrs) and C168h/Dose levels (1.6 vs. 0.96), compared to PSI0701.

Example 7; Cloning and production of anti-FIXa binding antibody fragment (FVIII mimetic)

This Example describes the general strategies for cloning and production of fusion proteins of anti-FIXa binding antibody fragment with half-life extending polypeptides as disclosed herein. The fusion proteins produced in this Example were used in Examples 8 - 10 below.

Materials and methods

DNA constructions: DNA sequences encoding a set of fusion proteins including half-life extending polypeptides were codon optimized for expression in E. coli or CHO cells and synthesized by the Invitrogen GeneArt Gene Synthesis service at Thermo Fisher Scientific. The genes were cloned in expression vectors for subsequent expression in E. coli, Expi293 cells or ExpiCHO cells.

Cultivation and purification: E. coli cells were transformed with expression vectors containing the gene fragments encoding the recombinant fusion proteins and then cultivated in bioreactors using fed-batch techniques or in shake flasks, followed by protein expression and harvest of cells by centrifugation. Cell pellets were stored at -20 °C or directly subjected to osmotic shock, released proteins were clarified by centrifugation and stored at -20 °C. Expression of recombinant fusion proteins was performed using the Expi293 and ExpiCHO expression systems (Thermo Fisher Scientific), essentially according to the manufacturer’s protocol. Supernatants were harvested by centrifugation 6 days after transfection of expression vectors and stored at -70 °C.

Frozen E. coli cell pellets were resuspended and then disrupted by sonication and the cell debris subsequently removed by centrifugation followed by filtration (0.22 pm). Osmotic shock samples and supernatants from the ExpiCHO and the Expi293 cultures were thawed and filtered (0.22 pm) before purification. Each supernatant, containing the recombinant fusion proteins was purified using conventional chromatography methods. Purified fusion proteins were buffer exchanged to PBS and, unless otherwise stated, PBS was also the formulation buffer used in subsequent experiments. The purity of the fusion proteins was analyzed by SDS-PAGE stained with Coomassie Blue and the molecular weight of each protein was analyzed using mass

spectrometry (LC/MS or MALDI-TOF/MS).

Table 8 lists the encoded protein sequences.

Results

Purification resulted in protein preparations with high purity, which were analyzed by SDS-PAGE stained with Coomassie Blue. The correct identity and molecular weight (MW) of each fusion protein were confirmed by mass spectrometry analysis.

Table 8. Description, expression system and SEQ ID Nos of proteins produced.

Conclusions

Fusion proteins containing antibody fragments and half-life extending polypeptides of various lengths can be produced by constructing synthetic genes followed by expression in mammalian or bacterial systems and purified to high purity using conventional techniques.

Example 8: Biophysical characterization of fusion proteins (FVIII mimetic) This Example describes the characterization of fusion proteins of anti-FIXa antibody fragments and half-life extending polypeptides, using unfused anti- FIXa antibody fragments and PEGylated proteins as references, with respect to biophysical characteristics such as apparent size and molecular weight (MW) in solution and determination of hydrodynamic radius in solution by size exclusion chromatography (SEC) and column calibration and Multi Angle Light Scattering (MALS).

Material and methods

The size of the fusion proteins, unfused proteins and PEGylated proteins in solution, was assessed by analytical gel filtration on an AKTA Micro (GE Healthcare Life Sciences) using a calibrated column Superdex 200 Increase 3.2/300 (GE Healthcare Life Sciences). The column was calibrated with Gel Filtration Calibration Kit LMW (code no. 28-4038-41 , GE Healthcare Life Sciences) and Calibration Kit HMW (code no. 28-4038-42, GE Healthcare Life Sciences), containing 8 globular proteins in the size range of 6 to 669 kDa and Blue Dextran 2000, using a running buffer of 25 mM NaP and 125 mM NaCI pH 7.0 with a flow rate of 75 mI/min at a temperature of 25°C. The corresponding size and hydrodynamic radius in solution calculated as set out in Example 3. The molecular weight of the proteins was determined by a connected MALS-RI system: Static light scattering detector miniDawn Tristar and Differential refractometer Optilab rEX, and the Astra V software (Wyatt Technology Europe, Germany).

The proteins of interest were analyzed under the same conditions as during the calibration. Results

Table 9 presents the results for the fusion proteins and reference proteins.

Table 9. Characterization of Anti-FIXa antibody fragment based fusion proteins and reference proteins

Conclusions

The size of the ScFv and the Fab antibody fragments in this series closely matched the sizes shown in Example 3. The effect of size for the fusion proteins that incorporate a half-life extending polypeptide followed the same trend noted for those fusion proteins.

It has been noted that the hydrodynamic radius or Stokes radius of albumin, which is above the size limit of renal clearance, is 3.8 nm. This may serve as a limit of minimal size required to avoid renal clearance. Example 9: Binding to human FIX (FVIII mimetic)

This Example describes the binding characteristics of fusion proteins of FIX antibody fragments and half-life extending polypeptides. Emicizumab and anti-FIX ScFv were used as reference proteins.

Material and methods

The binding affinities of fusion proteins of FIX antibody fragments and half-life extending polypeptides for human FIX were analyzed using an Biacore T200 instrument (GE Flealthcare). Human FIX was immobilized by amine coupling to a CM5 chip Series S (#BR-1005-30). Emicizumab was tested in the concentration range 40.6 nM to 650 nM. SEQ ID NO:56 and SEQ ID NO:57 were tested in the concentration ranges 1.6 to 25 nM and 3.1 to 50 nM, respectively.

Typically, association for each concentration was monitored for 120 s followed by a dissociation of 1200 s but only around 150 s was used for 1.1 fit. Kinetic constants were calculated from the sensorgrams using the

Langmuir 1 :1 analyte model.

Results

The resulting K _D values are tabulated in Table 10.

Conclusions

The tested ScFv antibody fragments showed an increased affinity towards FIX compared to the bispecific antibody Emicizumab. The affinity found for Emicizumab matches literature reported values.

Example 10: Chromogenic assay activity measurement In this Example, the possibility of the Fab and ScFv vs FIX to induce an effect in a chromogenic assay were assessed. As control, Factor VIII (FVIII) and Emicizumab was included in the assay.

Material and methods

The activity of anti-FIX ScFv and Fab and fusion proteins were assessed with Biophen Factor VIII:C chromogenic assay (Flyphen BioMed, 221402-RUO, lot F1700013P5) for measuring Factor VIII in plasma or concentrates, essentially as described by the producer, with the exception that the proteins were measured in buffer instead of plasma. Emicizumab were measured at 10 and 50 nM, the antibody fragments were measured at 50 and 200 nM.

Results

In the chromogenic assay the response of ScFv SEQ ID NO: 56 and Fab SEQ ID NO: 55 was 50 times lower than the response for the same molar equivalents of Emicizumab. The response of SEQ ID NO: 58 (FIX ScFv(VL- VH)-linker-FIX ScFv(VL-VFI)) was 7 times lower than for Emicizumab. This is also the case (7 times lower) for the variant with shorter inter-ScFv linker (SEQ ID NO: 621 ), the variants with thrombin and APC cleavage sites in the linker (SEQ ID NO: 623 and SEQ ID NO: 624) and for the protein with the PS- binding peptide at the C-terminus (SEQ ID NO: 626).

The aFIX-5GS-aFIX-[half-life extending polypeptide moiety]-CTag construct (SEQ ID: 625) showed a 5 times lower activity compared to unfused SEQ ID NO: 58.

Conclusion

The FIX binding arm is alone enough to induce a coagulation response judged by the chromogenic assay and the effect was enhanced upon placing them one after another, to form a tandem ScFv. The presence of a half-life extending polypeptide at the C-terminus, decrease the activity of the construct compared the unfused form, thereby showing the potential for activation by proteolytical processing in constructs, such as SEQ ID NO: 98, 630 and 633- 636, which contain a thrombin cleavage site.

Example 11: Cloning, production and characterization of antibody fragment based fusion proteins - CD28

This Example describes the general methods for cloning and production of fusion proteins of CD28 blocking antibody fragments with half-life extending polypeptides as described herein. The CD28 targeting moiety is based on Lulizumab, a domain antibody based on a VL domain. Material and methods

The proteins are produced essentially as in Example 2. The sequences of the proteins are listed in Table 11.

Results

All proteins are produced at high purity. Conclusion

CD28 blocking antibody fragment sequences based fusion proteins can be produced and exhibit an increased size.

Example 12: Cloning, production and characterization of antibody fragment based fusion proteins - CD25

This Example describes the general methods for cloning and production of fusion proteins based on CD25 (IL-2 receptor alpha chain) blocking antibody fragment with half-life extending polypeptides as described herein. The CD25 targeting moiety is based on Basiliximab, a chimeric mouse/human antibody.

Material and methods

The proteins are produced essentially as in Example 2. The sequences of the proteins are listed in Table 12. A full Fab will be assembled by the

combination of one HC and one LC.

Results

All proteins are produced at high purity.

Conclusion

CD25 blocking antibody fragment based fusion proteins can be produced and exhibit an increased size.

Example 13: Cloning, production and characterization of antibody fragment based fusion proteins - VEGF

This example describes the general methods for cloning and production of fusion proteins of an antibody fragment binding human VEGF and a half-life extending polypeptide disclosed herein.

Materials and methods

Cloning, expression and characterization were performed essentially as described in examples 2, 3 and 4. The VEGF binding antibody fragment (Fab) is known in the art as Ranibizumab (Table 13, SEQ ID NO: 402 and 403). Fusion of the half-life extending polypeptide was made to the C-terminus of the heavy chain polypeptide of the Fab and constructs were expressed in ExpiCFIO cells.

Results

Fusion proteins were isolated at high homogeneity using conventional purification steps. Apparent molecular weights were determined by SEC and MALS, and a significant size increase due to fusion of the half-life extending polypeptide was apparent. Antibody fragment based fusion proteins showed no significant reduction in affinity towards VEGF as compared to the affinity of the antibody fragment alone. The affinity was studied using an OctetRED96 instrument (Pall/ForteBio). No dissociation of VEGF was detected from the biosensor over a 15 min time period, and the off rate constant koff was therefore determined to be at less than 5.7x1 O ⁵ s ¹ for the proteins

characterized, resulting in dissociation constants below 0.3 nM.

Table 13. Characterization of Ranibizumab Fab based fusion proteins and ^: erence proteins

Conclusions

A correlation of total length of the half-life extending polypeptide comprised in the fusion protein and the fusion protein’s size in solution was observed. It was noted that the hydrodynamic radius or Stokes radius of albumin, which is above the size limit of renal clearance, is 3.8 nm. This could serve as a limit of minimal size required to avoid renal clearance. The two above tested fusion proteins had a size (Stokes radius) above that of albumin.

Example 14: Cloning, production and characterization of antibody fragment based fusion proteins - CD16 This example describes the general methods for cloning and production of fusion proteins of an antibody fragment binding human CD16 and a half-life extending polypeptide disclosed herein.

Methods

Cloning, expression and characterization were performed essentially as described in examples 2, 3 and 4. The CD16 binding antibody fragment (Table 14: SEQ ID NO: 263 and 261 ) originates from the monoclonal antibody known in the art as GMA-161. Fusion of half-life extending polypeptide was made to the C-terminus of the heavy chain polypeptide of the Fab and constructs were expressed in ExpiCFIO. Results

Fusion proteins were isolated at high homogeneity using conventional purification steps. Apparent molecular weights were determined by SEC and MALS, and a significant size increase due to fusion of the half-life extending polypeptide was apparent. Antibody fragment based fusion proteins showed only a minor reduction in affinity towards CD16 as compared to the affinity of the antibody fragment alone. The affinity was studied using an OctetRED96 instrument (Pall/ForteBio).

Table 14. Characterization of anti-CD16 Fab based fusion proteins and reference proteins

Conclusions

A correlation of total length of the half-life extending polypeptide comprised in the fusion protein and the fusion protein’s size in solution was observed. It was noted that the hydrodynamic radius or Stokes radius of albumin, which is above the size limit of renal clearance, is 3.8 nm. This could serve as a limit of minimal size required to avoid renal clearance. The two above tested fusion proteins had a size above that of albumin.

Example 15: Cloning, production and characterization of antibody fragment based fusion proteins - P-selectin

This example describes the general methods for cloning and production of fusion proteins of P-selectin binding antibody fragment based fusion proteins and a half-life extending polypeptide disclosed herein.

Methods

Cloning, expression and characterization was performed essentially as described in examples 2, 3 and 4. The P-selectin binding antibody fragment (Table 15, SEQ ID NO: 522 and 521 ) originates from the monoclonal antibody known in the art as Inclazumab. Fusion of half-life extending polypeptide was made to the C-terminus of the heavy chain polypeptide of a Fab fragment of Inclazumab. Results

Fusion proteins were isolated at high homogeneity using conventional purification steps and apparent molecular weights were determined by SEC and MALS. An antibody fragment based fusion protein had retained affinity towards P-selectin as illustrated using methods described in Example 4.

Table 15. anti-P-selectin Fab based fusion proteins

Conclusions

Anti-P-selectin Fab based fusion proteins were produced and exhibited an increased size. Increased hydrodynamic radiuses along with retained P- selectin binding properties may permit long retention in the blood

compartment which may make such anti-P-selectin Fab based fusion proteins suitable for prevention of cellular adhesion events. Example 16: Cloning, production and characterization of antibody fragment based fusion proteins - CD40L

This example describes the general methods for cloning and production of fusion proteins of anti-CD40L antibody fragments with half-life extending polypeptides as described herein.

Material and methods

Cloning, expression and characterization were performed essentially as described in examples 2, 3 and 4. By pairing a heavy chain (FIC) containing a sequence selected from SEQ ID NO: 35, 37, 39, 41 -42, 52, 99, 101 , 106, 127-171 , 216-237, with a LC containing sequence selected from 36, 38, 40, 53, 100, 172-215, new anti-CD40L Fab based fusion proteins are produced.

Table 16 below lists the amino acid sequences of the fusion proteins to be produced. A half-life extending polypeptide is fused to the C terminal of either the light chain (LC in the table below) or heavy chain (HC in the table below), or both of the light chain and the heavy chain of the Ruplizumab Fab with an intervening linker, marked with -linker-, or without such linker. Furthermore, the FIC may contain the first five residues of the hinge region, shown as HC (hinge) in the table and the sequence of the protein is shown in SEQ ID NO: 35. Alternatively, the HC does not contain the first five residues of the hinge region, indicated by“HC” in the table and the sequence of that protein is shown in SEQ ID NO: 106. Results

Purification resulted in protein preparations of high purity, according to SDS- PAGE. The correct identity and molecular weight of each fusion protein was confirmed by mass spectrometry analysis.

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Conclusions

Fusion proteins containing antibody fragments and half-life extending polypeptides of various lengths is produced by constructing synthetic genes followed by expression in mammalian or bacterial systems and purified to high purity, using conventional techniques.

Example 17: Additional study of pharmacokinetic (PK) properties of Fab based fusion proteins - CD40L In this Example, the intravenous and subcutaneous pharmacokinetic properties (PK) of PSI0762 (SEQ ID NO: 40 / SEQ ID NO:36) and PSI0773 (SEQ ID NO:39 / SEQ ID NO:36) were tested.

Materials and methods

The study was carried out with a single intravenous (IV) or subcutaneous (SC) dose in male Sprague-Dawley rat (N=3 per administration route and protein), for both PSI0762 and PSI0773, respectively.

For PSI0762 and PSI0773, the doses and timepoints for IV experiments were as follows: 2 mg/kg, 1 5mg/kg, 5 and 20 min and 1 , 4, 8, 24, 48, 72, 96 and 120 hours. For the SC experiments doses of 4 and 3 mg/kg were used, and blood samples were taken at the following time points: 20 min and 1 , 4, 8, 24, 48, 72, 96, 120 and 168 hours. The experiments were performed as described in Example 6.

Results

The results are summarized in Table 17 for the IV experiment and Table 18 for the SC experiment.

Table 17. Median PK parameter estimates following an intravenous single dose.

Table 18. Median PK parameter estimates following a subcutaneous single dose

Conclusions

The clearance of PSI0762 and PSI0773, with 34 and 17 repeats of half-life extending polypeptide at the heavy chain only, was higher than PSI0699 and PSI0701 as provided in example 6, and it was inversely correlated to the length of the half-life extending polypeptide.

PSI0699 with half-life extending polypeptides fused to both chains of the Fab has the lowest clearance, the longest terminal half-life and the highest bioavailability. Example 18: Proteolytic inactivation of anti-FIX ScFv

This example describes how proteolytic processing deactivates anti-FIX ScFv of the invention.

Materials and methods

Supernatants from the cultivation of SEQ ID NO: 624 and SEQ ID NO: 625 were incubated in with 1 :500 volume ratio of Thrombin (Sigma-Aldrich,

T9326) with a concentration of 0.32 mg/mL, respectively and a 1 :10 ratio of Activated Protein C (APC, Sigma-Aldrich, P2200) and supernatants of cultivation. As activated Factor X (ThermoFisher Scientific, RP-43114, concentration 5.8 mg/mL) is known to also cleave FVIII at the APC site, it was used to cleave SEQ ID NO: 629 in a 1 :20 volume ratio. The proteolytic processing was followed by SDS-PAGE and the activity of the cleavage products were tested in the chromogenic assay as described in example 8.

Results

SEQ ID NO: 624 were totally processed after 2 hours according the SDS- PAGE, were the original band on the gel had disappeared. SEQ ID NO: 625 followed a longer time to inactivation where it was almost completely degraded after 22 hours according to SDS-PAGE. SEQ ID NO: 629 was processed at the first time-point and after 4 hours a mojority of the protein was degraded, and at the final time-point at 18 hours nothing of the original band remained.

SEQ ID NO: 624 showed a 2-fold decrease in activity in the chromogenic assay after proteolytic processing.

Conclusions

The introduced proteolytical sites were functional as seen by the degradation of the protein as seen on SDS-PAGE. The activity of SEQ ID NO: 624 decreased to a level that corresponded to the individual ScFv that were released by the cleavage, indicating that the introduced sites were functional. Example 19: Design of repeating units that do not contain any O-glycosylation sites and production and characterization of fusion proteins containing the same This example describes sequences of repeating units without O-glycan sites in the sequence and describes the general methods for cloning and production of fusion proteins of Ruplizumab antibody fragment sequences with afforementioned half-life extending polypeptides without O-glycan sites. Half-life extending polypeptides were fused to the light chain (LC) or the heavy chain (HC) of the antibody fragments.

Materials and methods

By utilizing the variable positions in SEQ ID NO: 1 the following sequences were designed that lacked serine or threonine that could be O-glycosylated during cultivation in eukaryotic expression systems such as CHO, HEK or yeast. The sequences are listed in Table 19 below.

Table 19. Units corresponding to the general formula in SEQ ID NO: 1 without serine or threonine present.

The final repeat is in some instances modified to incorporate a purification tag, as in SEQ ID NO: 597-607. In the same manner, a modified version of those can be designed to be O-glycan free and is assigned SEQ ID NO: 644 (PVPPVDDAKEPEA). These units (SEQ ID NO: 644) can be assembled into 16 repeats ended by a single C-terminal to form a half-life extending fusion partner (SEQ ID NO: 645) that is almost identical in length as the most abundant natural form provided in SEQ ID NO: 20.

Other possible units that lack O-glycan site are listed in Table 20 below. Table 20. Units corresponding to the general formula in SEQ ID NO: 1 without serine or threonine present.

DNA constructions: DNA sequences encoding the heavy and light chain of the Ruplizumab Fab with half-life extending polypeptides (SEQ ID NOs: 646 and 647) were codon-optimized for expression in CFIO cells and synthesized by the Invitrogen GeneArt Gene Synthesis service at Thermo Fisher

Scientific. The genes were cloned in expression vectors for subsequent expression in ExpiCFIO cells.

Cultivation and purification: Expression of recombinant antibody fragments or fusion proteins was performed using the ExpiCHO expression systems (Thermo Fisher Scientific), essentially according to the manufacturer’s protocol. Supernatants were harvested by centrifugation 6 days after transfection of expression vectors and stored at -70 °C. Each supernatant, containing the recombinant antibody fragment or fusion proteins was purified using conventional chromatography methods.

Characterization: The size of the fusion protein was assessed as in example 3 and data is included in the results (PSI0698 and PSI0699).

Results

The cultivations resulted in protein expression with the same (or higher level) as for the glycosylated counterparts, which was analyzed by SDS-PAGE stained with Coomassie Blue.

Table 21 presents the results for the fusion protein for the units without O- glycan site compared to the data from example 3, i.e. Fab PSI0698 (SEQ ID NO:35 / SEQ ID NO:36) and fusion protein PSI0699 HC17/LC17 (SEQ ID NO:37 / SEQ ID NO:38).

Table 21. Characterization of Ruplizumab Fab based fusion proteins and reference proteins

Conclusions

The fusion of the Fab to the half-life extending polypeptide without O-glycan sites behave as the fusion proteins based on the units of the half-life extending polypeptide present in the human genome in terms of expression and biophysical properties.