WO2021004975A1

WO2021004975A1 - MODIFIED IgA ANTIBODY PROTEINS

Info

Publication number: WO2021004975A1
Application number: PCT/EP2020/068909
Authority: WO
Inventors: Jan Terje Andersen; Inger Sandlie; Simone MESTER; Stian FOSS; Algirdas GREVYS; Jeannette Henrica Wilhelmina LEUSEN; Saskia MEYER; Johannes Gerardus Maria EVERS
Original assignee: Universitetet I Oslo; Umc Utrecht Holding B.V.
Priority date: 2019-07-05
Filing date: 2020-07-03
Publication date: 2021-01-14
Also published as: GB201909710D0

Abstract

The present invention relates to a protein comprising at least two polypeptide chains that each comprise an IgA antibody constant domain, wherein two or more of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn at endosomal pH, wherein said fragment of human serum albumin is characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1; (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO:1; and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1. Compositions comprising said proteins, together with nucleic acid molecules encoding said proteins and host cells comprising said proteins are also provided.

Description

Modified IgA Antibody Proteins

This invention relates generally to the field of proteins, in particular proteins comprising IgA antibody constant domains, such as IgA antibodies or fragments thereof. The invention also relates to compositions comprising such proteins, constructs encoding such proteins, methods of producing such proteins, and methods and uses which employ such proteins.

Immunoglobulins (also known as antibodies) may provide several effects upon binding to a target cell. They may:

(1) induce apoptosis in target cells either directly or by ligand blockade; and/or

(2) lyse target cells by complement-dependent cytotoxicity (CDC); and/or

(3) recruit FcR bearing‘killer’ immune cells that can interact with the Fc- region of antibodies, leading to antibody-dependent cellular cytotoxicity (ADCC).

Immunoglobulin A (IgA) is believed to play a major role in a first line defence against invading pathogens. IgA is the most abundant antibody isotype in the body, comprising almost 70 percent of the body's total immunoglobulin by weight. The majority of IgA is found in the various mucous secretions, including saliva, milk, colostrum, tears, and secretions from the respiratory tract, genitourinary tract, and prostate. IgA is also the second most abundant isotype in the circulation, following immunoglobulin G (IgG).

IgA binding of FcaRI (which may also be referred to as CD89, or FcaR, or Fc-alpha receptor) expressed on polymorphonuclear leukocytes (PMNs) results in potent induction of ROS production, phagocytosis, NET formation, cytokine release and antibody-dependent cytotoxicity (ADCC). IgA is significantly more efficient in inducing these effector functions of PMNs than IgG. In normal blood, the most abundant PMNs are the neutrophils. The profound ability of IgA to activate PMNs, and in particular neutrophils, has been demonstrated for tumor relevant antigens such as CEA, CD20, EGFR and Her2. IgA antibodies are thus of therapeutic interest. However, the immunotherapeutic potential of IgA has not yet been explored in humans. One major obstacle is its short serum persistence (i.e. short half-life), meaning that multiple and frequent injections of large quantities of antibody would be required to reach an effective therapeutic concentration. Although IgA is produced in 5 times higher amounts than IgG, the serum concentration of IgA is 5 times lower than that of IgG, which is due to its rapid elimination from the circulation to the mucosa.

Meyer et ai. 2016 ( MAbs , 2016, 8:(1) pages 87-98), mentioned that an albumin-binding domain (ABD) can be attached to IgA in order to increase in vivo half-life. ABDs are small, 3-helical protein subunits expressed by various gram positive bacteria. In a xenograft mouse model, the modified lgA1 antibodies exhibited a slightly improved anti-tumor response compared to the unmodified antibody. Due to their bacterial origin, such ABDs may be generally undesirable, for example because of their potential to cause unwanted immunogenic responses.

Summary

The present inventors have found that modifying IgA antibodies such that they have attached thereto a mutated version of a fragment of Human Serum Albumin (HSA) increases the in vivo half-life of IgA antibodies. The inventors have observed that, surprisingly and advantageously, such modified IgA antibodies can maintain the ability to bind to their target antigen, can maintain the ability to interact with FcaRI, and can maintain ADCC activity. The inventors have also observed that in contrast to the increased in vivo half-life observed with the attachment of a mutated version of a domain III fragment of HSA to IgA antibodies, the attachment of an equivalent wild-type (or non-mutated) domain III fragment of HSA has no effect on the in vivo half-life of the molecule.

Thus, in a first aspect, the present invention provides a protein comprising at least two polypeptide chains that each comprise an IgA antibody constant domain, wherein one or more of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn, wherein said fragment of human serum albumin is characterized by comprising at least one mutant residue selected from the group consisting of (or comprising):

(i) a mutant residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 ;

(ii) a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO:1; and

(iii) a mutant residue at the position corresponding to amino acid position In further embodiments of the invention, the present invention provides a protein comprising at least two polypeptide chains that each comprise an IgA antibody constant domain, wherein one or more of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn, wherein said fragment of human serum albumin is characterized by comprising at least one mutant residue selected from the group consisting of (or comprising):

(ii) a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 ;

(iii) a mutant residue at the position corresponding to amino acid position

573 of SEQ ID NO:1 ; and

(iv) a mutant residue at the position corresponding to amino acid position

547 of SEQ ID NO:1.

In preferred embodiments, two or more (e.g. 2, 3 or 4, preferably 2 or 4) of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin in accordance with the invention.

In some preferred embodiments, two of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin in accordance with the invention.

In some preferred embodiments, four of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin in accordance with the invention.

As set out above, the fragment of human serum albumin is characterized by comprising at least one mutant residue as defined above (1 , 2, 3 or 4 of the mutant residues). In preferred embodiments, the fragment of human serum albumin is characterized by comprising at least 2 or 3 of the mutant residues as defined above, preferably all 3 or all 4 of the mutant residues. In some preferred embodiments, these 1 , 2, 3 or 4 mutant residues are the only mutant residues in the HSA fragment as compared to the corresponding sequence (or corresponding fragment) of wild- type HSA (SEQ ID NO:1).

By“mutant residue” is meant an amino acid residue other than the amino acid residue that appears at the stated corresponding position in SEQ ID NO:1. SEQ ID NO:1 is the amino acid sequence of human serum albumin (full-length human serum albumin or wild-type human serum albumin). Thus, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , the mutant residue is a residue other than glutamate (E) (i.e. is not E). If the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1 , the mutant residue is a residue other than threonine (T) (i.e. is not T). If the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 573 of SEQ ID NO: 1, the mutant residue is a residue other than lysine (K) (i.e. is not K). If the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 547 of SEQ ID NO:1 , the mutant residue is a residue other than valine (V) (i.e. is not V).

Preferably, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 , the mutant residue is glutamine (Q) or an amino acid residue that represents a conservative amino acid substitution with respect to Q.

Preferably, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1, the mutant residue is methionine (M) or an amino acid that represents a conservative amino acid substitution with respect to M. Preferably, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 573 of SEQ ID NO:1 , the mutant residue is proline (P) or an amino acid residue that represents a conservative amino acid substitution with respect to P. Preferably, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position

corresponding to amino acid position 547 of SEQ ID NO:1 , the mutant residue is alanine (A) or an amino acid residue that represents a conservative amino acid substitution with respect to A.

A "conservative amino acid substitution", as used herein, is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

Thus, amino acid residues that would represent a conservative amino acid substitution with respect to Q (glutamine) include those having uncharged polar side chains (e.g. asparagine, serine, threonine, tyrosine, cysteine). In some

embodiments, a preferred conservative amino acid substitution with respect to Q (glutamine) is asparagine. Thus, in some embodiments, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1, the mutant residue is glutamine (Q) or asparagine (N), preferably glutamine (Q).

Amino acid residues that would represent a conservative amino acid substitution with respect to M (methionine) include those having nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan).

Amino acid residues that would represent a conservative amino acid substitution with respect to P (proline) include those having nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan).

Amino acid residues that would represent a conservative amino acid substitution with respect to A (alanine) include those having nonpolar side chains (e.g. glycine, cysteine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan).

In one embodiment, there is also provided a protein comprising an Fc fragment of an IgA antibody, wherein both the C-terminal ends of the Fc fragment are attached to domain III of human serum albumin, wherein domain III comprises a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1.

In one embodiment, there is also provided a protein comprising an Fc fragment of an IgA antibody, wherein both the C-terminal ends of the Fc fragment are attached to domain III of human serum albumin, wherein domain III comprises an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO: 1 and/or a proline residue at the position corresponding to amino acid position 573 of SEQ I D NO: 1. The present disclosure also provides a small polypeptide unit able to interact with FcRn in a pH-dependent manner. Said unit can be defined as domain III (which is further defined below) comprising a glutamine residue in position 505, a methionine residue in position 527 and a proline residue in the position

corresponding position 573; all three positions relative to SEQ ID NO:1. One example of such polypeptide unit can be represented by SEQ ID NO:2 or 3.

Optionally such small polypeptide unit may also comprise an alanine residue in position 547 relative to SEQ ID NO:1. One example of such polypeptide unit can be represented by SEQ ID NO:8 or 9. Accordingly, as demonstrated herein, non-HSA proteins fused to said unit may acquire the ability to interact with FcRn in a pH- dependent manner and thus display increased half-life in vivo.

Preferably, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , the mutant residue is glutamine (Q). Preferably, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1 , the mutant residue is methionine (M). Preferably, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position

corresponding to amino acid position 573 of SEQ ID NO:1, the mutant residue is proline (P). Preferably, if the fragment of human serum albumin is characterized by comprising a mutant residue at the position corresponding to amino acid position 547 of SEQ ID NO:1 , the mutant residue is alanine (A).

In preferred embodiments, the fragment of human serum albumin is characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1. In other preferred embodiments, the fragment of human serum albumin is characterized by further comprising (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1

In a preferred embodiment, the present invention provides a protein comprising at least two polypeptide chains that each comprise an IgA antibody constant domain, wherein two or more of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn, wherein said fragment of human serum albumin is characterized by comprising (i) a glutamine or an asparagine residue at the position corresponding to amino acid position 505 of SEQ I D NO: 1 ;

(ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 ; and

(iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1.

In other preferred embodiments, the fragment of human serum albumin is characterized by further comprising (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1.

In a particularly preferred embodiment, the present invention provides a protein comprising at least two polypeptide chains that each comprise an IgA antibody constant domain, wherein two or more of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn, wherein said fragment of human serum albumin is characterized by comprising

(i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 ;

IgA antibodies comprise heavy chains (HC) and light chains (LC) that each comprise a constant region.

Heavy chain constant regions of an IgA antibody comprise (or consist of) CH1 (or C_a1 or constant heavy chain domain 1), CH2 (or C_a2 or constant heavy chain domain 2) and CH3 (or C_a3 or constant heavy chain domain 3) domains. The CH1 domain is positioned C-terminally to the heavy chain variable region (VH), the CH2 domain is positioned C-terminally to the CH1 domain, and the CH3 domain is positioned C-terminally to the CH2 domain. Thus, a constant region of an IgA antibody heavy chain comprises a CH1 domain, a CH2 domain and a CH3 domain. Light chain constant regions of an IgA antibody comprise (or consist of) a CL domain (constant light chain domain). The CL domain is positioned C-terminally to the light chain variable region (VL).

In accordance with the present invention, the protein comprises at least two polypeptide chains that each comprise an (e.g. at least one) IgA antibody constant domain. Preferably, the protein comprises two or four polypeptide chains that each comprise an IgA antibody constant domain. Amino acid sequences of IgA constant domains are well-known in the art.

In some embodiments, the protein comprises two polypeptide chains that each comprise an IgA antibody constant domain. In some embodiments, the protein has (has only) two polypeptide chains comprising an IgA antibody constant domain.

In some embodiments, the protein comprises four polypeptide chains that each comprise an IgA antibody constant domain. In some embodiments, the protein has (has only) four polypeptide chains comprising an IgA antibody constant domain.

In some embodiments, a polypeptide that comprises an IgA antibody constant domain comprises a CH1 , a CH2 or a CH3 domain.

In some embodiments, a polypeptide that comprises an IgA antibody constant domain comprises CH1 , CH2 and CH3 domains. Thus, in some embodiments, a polypeptide that comprises an IgA antibody constant domain may comprise an IgA heavy chain constant region (e.g. a full heavy chain constant region).

In some embodiments, a polypeptide that comprises an IgA antibody constant domain comprises the amino acid sequence of an Fc region (or Fc fragment) of an IgA antibody. An Fc region (or fragment crystallizable region) typically provides (or confers) an effector function (e.g. antibody dependent cellular cytotoxicity). Such Fc regions may typically comprise CH2 and CH3 domains of an IgA antibody.

In some embodiments, a polypeptide that comprises an IgA antibody constant domain that comprises CH1 , CH2 and/or CH3 domains of an IgA antibody, or that comprises a heavy chain constant region of an IgA antibody or that comprises an Fc fragment of an IgA antibody, has a modified (or mutated or inactivated or truncated) tailpiece, or does not comprise a tailpiece (or the tailpiece has been removed).

Thus, Fc fragments (or Fc regions) and heavy chain constant regions include those in which the tailpiece has been modified, mutated, inactivated, truncated or removed.

A tailpiece is typically an 18 amino acid region (or extension) at the C- terminal end of an IgA heavy chain constant region or Fc region that contains a cysteine residue that is essential for polymerization (e.g. formation of dimers).

In preferred embodiments, in proteins of the present invention (e.g. IgA antibodies or fragments thereof) the tailpiece has been modified, mutated, inactivated, truncated or removed in order to prevent polymerization of proteins (e.g. IgA antibodies) of the invention. In some such embodiments, the cysteine residue that is essential for polymerization has been modified, mutated, inactivated, truncated or removed.

The skilled person is familiar with IgA tailpieces (e.g. Janeway et ai, 2001 , Immunobiology: The Immune System in Health and Disease, 5^th Edition, New York: Garland Science). One example of an lgA1 tailpiece can be represented by the sequence PTHVNVSVVMAEVDGTCY (SEQ ID NO:6).

In some embodiments, a polypeptide that comprises an IgA antibody constant domain comprises a CL domain.

In some preferred embodiments, the polypeptide that comprises an IgA antibody constant domain is a heavy chain or a light chain of an IgA antibody.

Thus, in some preferred embodiments, a polypeptide that comprises an IgA antibody constant domain further comprises an antibody variable domain, e.g. a light chain variable region (VL) or a heavy chain variable region (VH). A heavy chain of an IgA antibody comprises a heavy chain constant region and heavy chain variable region (VH). A light chain of an IgA antibody comprises a light chain constant region and light chain variable region (VL).

In some embodiments in which the protein has (has only) four polypeptide chains, each comprising an IgA antibody constant domain, two of said polypeptide chains are IgA antibody heavy chains (e.g. as defined elsewhere herein) and two of said polypeptide chains are IgA antibody light chains (e.g. as defined elsewhere herein). Thus, in some embodiments in which the protein has (has only) four polypeptide chains, each comprising an IgA antibody constant domain, said protein is (or comprises) an IgA antibody (e.g. as defined elsewhere herein).

In some embodiments in which the protein has (has only) two polypeptide chains, each comprising an IgA antibody constant domain, said protein is (or comprises) an Fc fragment (or Fc region) of an IgA antibody.

In some embodiments in which the protein has (has only) two polypeptide chains, each comprising an IgA antibody constant domain, said protein is (or comprises) a Fab fragment (or Fab region) of an IgA antibody.

Typically, there is at least one bond (preferably at least one disulphide bond) between at least two polypeptide chains comprising IgA antibody constant regions. For example, in whole IgA antibodies there are typically disulphide bonds between the CL domains and the CH1 domains and there are typically disulphide bonds between the IgA antibody heavy chains.

The wild type sequences encoding constant regions of IgA antibodies are well known. For example, the gene encoding human Immunoglobulin heavy constant alpha 1 is known as IGHA1 and the gene encoding human Immunoglobulin heavy constant alpha 2 is known as IGHA2. Engineered variants with modified glycosylation pattern and/or stability are also well known. An exemplary lgA1 heavy chain constant region amino acid sequence is set forth herein as SEQ ID NO:7.

In some embodiments of the present invention that comprise an IgA heavy chain constant region or an Fc region, the final (C-terminal-most) amino acid residue of the IgA heavy chain constant region or Fc region is the residue corresponding to position 453 by Bur numbering (e.g. the G at position 453 by Bur numbering in SEQ ID NO:7). The Bur numbering scheme is described by Liu et al. (Science. 1976 Sep 10; 193(4257): 1017-20).

In preferred embodiments of the present invention, the protein comprises (or is) is an antibody (or immunoglobulin), or a fragment thereof. Antibodies (or fragments thereof) of the invention may be considered“modified” or“variant” antibodies (or fragments thereof), with a modification or variation being that they have attached thereto a fragment of human serum albumin in accordance with the invention.

Particularly preferred are IgA antibodies, or fragments thereof. In some embodiments, the IgA antibody is an lgA1 antibody. In some embodiments, the IgA antibody is an lgA2 antibody. The subunit structures and three-dimensional configurations of different classes of antibodies are well known.

In some embodiments, wherein said IgA antibody or said IgA antibody constant domain is an lgA1 antibody, the fragments of HSA as described herein are attached to the light chain constant domains or heavy chain constant domains of said lgA1 antibody. In some embodiments, wherein said IgA antibody or said IgA antibody constant domain is an lgA1 antibody, the fragments of HSA as described herein are attached to the light chain constant domains of said lgA1 antibody. In some embodiments, wherein said IgA antibody or said IgA antibody constant domain is an lgA2 antibody, the fragments of HSA as described herein are attached to the light chain constant domains or heavy chain constant domains of said lgA2 antibody. In some embodiments, wherein said IgA antibody or said IgA antibody constant domain is an lgA2 antibody, the fragments of HSA as described herein are attached to the heavy chain constant domains of said lgA2 antibody.

The terms "antibody" and "immunoglobulin", as used herein, refer broadly to any immunological binding agent that comprises an antigen binding domain. This term includes antibody fragments that comprise an antigen binding domain.

As will be understood by those in the art, the immunological binding reagents encompassed by the term "antibody" includes or extends to all antibodies and antigen binding fragments thereof, including whole antibodies, dimeric, trimeric and multimeric antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies, and fragments thereof.

Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.

Monoclonal antibodies are particularly preferred.

Preferably, the antibody or antibody fragment comprises an antibody light chain variable region (VL) that comprises three CDR domains and an antibody heavy chain variable region (VH) that comprises three CDR domains. Said VL and VH generally form the antigen binding site.

The term "heavy chain complementarity determining region" ("heavy chain CDR") as used herein refers to regions of hypervariability within the heavy chain variable region (VH domain) of an antibody molecule. The heavy chain variable region has three CDRs termed heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxy terminus. The heavy chain variable region also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.

The term "heavy chain variable region" (VH domain) as used herein refers to the variable region of a heavy chain of an antibody molecule.

The term "light chain complementarity determining region" ("light chain

CDR") as used herein refers to regions of hypervariability within the light chain variable region (VL domain) of an antibody molecule. Light chain variable regions have three CDRs termed light chain CDR1 , light chain CDR2 and light chain CDR3 from the amino terminus to the carboxy terminus. The light chain variable region also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.

The term "light chain variable region" (VL domain) as used herein refers to the variable region of a light chain of an antibody molecule.

The CDRs of the antibodies of the invention are preferably separated by appropriate framework regions such as those found in naturally occurring antibodies and/or effective engineered antibodies. Thus, the CDR sequences are preferably provided within or incorporated into an appropriate framework or scaffold to enable antigen binding. Such framework sequences or regions may correspond to naturally occurring framework regions, FR1 , FR2, FR3 and/or FR4, as appropriate to form an appropriate scaffold, or may correspond to consensus framework regions, for example identified by comparing various naturally occurring framework regions. Alternatively, non-antibody scaffolds or frameworks, e.g. T cell receptor frameworks can be used.

An "Fv" fragment is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region has a dimer of one heavy chain variable region and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions (CDRs) confer antigen- binding specificity to the antibody.

Antibodies may comprise (or consist of) two heavy chains (HC) and two light chains (LC). The light chains may be k (kappa) or l (lambda) light chains. Heavy chains and light chains each comprise a variable region and a constant region. As described above, the constant region of a heavy chain comprises three constant domains CH1 , CH2 and CH3, and the constant region of light chain comprises a CL constant domain.

As discussed elsewhere herein, in preferred embodiments the antibody or antibody fragment of the present invention comprises all or a portion of IgA heavy chain constant region. Furthermore, the antibody or antibody fragment can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region, or a portion thereof. Appropriate sequences for such constant regions are well known and documented in the art. All or part of such constant regions may be produced naturally or may be wholly or partially synthetic.

When a full complement of constant regions from the heavy and light chains is included in the antibodies of the invention, such antibodies are typically referred to herein as "full length" antibodies or "whole" antibodies. In preferred

embodiments, the protein is (or comprises) a whole (or full-length) antibody, preferably a whole (or full-length) IgA antibody. “Whole” or“full-length” antibodies comprising two heavy chains and two light chains are preferred antibodies.

However, in some embodiments, the protein does not comprise a whole or full-length antibody. In some embodiments, the protein comprises a fragment of an antibody, preferably a fragment of an IgA antibody. Such a fragment may be an Fc fragment (or Fc region). Other fragments include antigen-binding fragments, for example Fab fragments.

Thus, in some embodiments, the protein is characterized in that the antibody-based (or antibody-derived) moiety (or component) is (or comprises) an IgA Fc fragment.

In some embodiments, the protein is characterized in that the antibody- based (or antibody-derived) moiety (or component) is (or comprises) an antigen binding fragment of an antibody, e.g. a Fab fragment (or Fab region).

An Fc fragment, e.g. an IgA Fc fragment, typically comprises two identical polypeptide chains, each chain containing (or comprising or consisting of) amino acid sequences of the CH2 and CH3 domains. The two chains of the Fc fragment are connected to each other by at least one cysteine bridge (disulphide bond between cysteine residues).

A Fab fragment comprises the region of an antibody that binds to antigens and has two chains, (i) an antibody light chain having a VL domain and a CL constant domain, and (ii) a portion (or fragment) of the antibody heavy chain that has a VH domain and a CH1 domain. In a Fab fragment, the two chains are connected via a disulphide bond between cysteine residues of the CL and CH1 constant domains. The enzyme papain can be used to cleave an immunoglobulin (antibody) monomer into two Fab fragments and an Fc fragment.

IgA antibodies may be monomeric IgA antibodies or dimeric IgA antibodies.

Monomeric antibodies (or fragments thereof), preferably monomeric IgA antibodies (or fragments thereof), are typically preferred. Preferred monomeric antibodies, e.g. preferred monomeric IgA antibodies, have two heavy chains (HC) and two light chains (LC). Preferred monomeric antibodies, e.g. preferred monomeric IgA antibodies, have two identical Fab regions which are capable of binding to a target antigen. In whole (or full-length) antibodies, the Fab regions are linked through the hinge regions to the Fc fragment. Monomeric antibodies may thus have the canonical Ύ-shaped” antibody structure. Without wishing to be bound by theory, monomeric IgA antibodies are particularly preferred as they have a superior ability to be retained in the circulation as compared to dimeric, secretory, IgA antibodies.

Dimeric IgA antibodies may also be referred to as secretory IgA antibodies (slgA or secretory form of IgA). Dimeric IgA antibodies comprise two monomeric IgAs, a J-chain and a secretory component.

In accordance with the present invention, antibodies, preferably IgA antibodies, may comprise an antigen binding domain that binds to any target protein (target antigen) of interest.

In some embodiments, antibodies of the present invention comprising an antigen binding domain bind (or specifically bind) to a therapeutically (or clinically) relevant target protein (or target antigen). Thus, in some embodiments, antibodies of the present invention comprising an antigen binding domain bind to a disease- associated target protein (or target antigen). A disease-associated target protein (or target antigen) may be a target protein whose expression (e.g. unwanted

expression or aberrant expression or overexpression) is associated with a disease. In some embodiments, the disease is cancer (or a tumour), for example breast cancer.

In some embodiments, antibodies of the present invention comprising an antigen binding domain bind to a given cancer (or tumour) protein or antigen (e.g. a cancer specific protein or antigen, or a cancer associated protein or antigen, or a tumour specific protein or antigen, or a tumour associated protein or antigen). In some embodiments the cancer is breast cancer.

In some embodiments, antibodies of the present invention comprising an antigen binding domain bind to a member of the human epidermal growth factor receptor. In some embodiments, antibodies of the present invention comprising an antigen binding domain bind to HER2. Over-expression of the protein HER2 can play an important role in the development and progression of certain breast cancers.

In some embodiments, antibodies of the present invention comprise the antigen binding domain of the antibody Trastuzumab.

In some embodiments, the antibody has been reformatted (e.g. from an IgG antibody or other class of antibody) into the IgA format (e.g. the lgA1 or lgA2 format). Thus, in some embodiments, the antibody is an IgA antibody comprising an antigen binding domain of a non-IgA antibody (e.g. from or derived from or based upon an antigen binding domain of a non-IgA antibody, e.g. of an IgG antibody). Methods of reformatting antibodies into the IgA format are well-known in the art and the skilled person will be familiar with such methods.

In some embodiments, antibodies of the present invention comprise an antigen binding domain of a therapeutically effective antibody. In some such embodiments, the antibody is an IgA antibody comprising an antigen binding domain of a therapeutically effective non-IgA antibody (e.g. from or derived from or based upon an antigen binding domain of a therapeutically effective non-IgA antibody).

In some embodiments, the antibody (IgA) is modified (or mutated) in order to keep it in monomeric form (e.g. to prevent it dimerizing).

In some embodiments, the antibody (IgA) has a modified (or mutated or inactivated or truncated) tailpiece, or does not comprise a tailpiece (or the tailpiece has been removed). The tailpiece (or tailpiece region) confers the ability to form multimers (e.g. dimers). Without wishing to be bound by theory, the absence of a tailpiece (or the mutation or inactivation or truncation of the tailpiece) allows the IgA antibody to be in monomeric form, or assists in keeping the IgA antibody in monomeric form (as opposed to forming slgA dimers).

In some preferred embodiments, the present invention provides an IgA antibody, preferably a monomeric IgA (lgA1 or lgA2) antibody (having two heavy chains and two light chains), that has attached to the C-terminal end of each of its two light chains and/or to each of its two heavy chains a HSA fragment in accordance with the invention.

In some preferred embodiments, the present invention provides an IgA antibody, preferably a monomeric IgA (lgA1 or lgA2) antibody (having two heavy chains and two light chains), that has attached to the C-terminal end of each of its two light chains a HSA fragment in accordance with the invention.

In some preferred embodiments, the present invention provides an IgA antibody, preferably a monomeric IgA (lgA1 or lgA2) antibody (having two heavy chains and two light chains), that has attached to the C-terminal end of each of its two heavy chains a HSA fragment in accordance with the invention.

In some preferred embodiments, the present invention provides an IgA antibody, preferably a monomeric IgA (lgA1 or lgA2) antibody (having two heavy chains and two light chains), that has attached to the C-terminal end of each of its two heavy chains and to the C-terminal end of each of its two light chains a HSA fragment in accordance with the invention.

In some preferred embodiments, the present invention provides an Fc fragment of an IgA antibody (e.g. lgA1 or lgA2), preferably a monomeric Fc fragment of an IgA antibody (e.g. lgA1 or lgA2), that has attached to the C-terminal end of each of the two polypeptide chains thereof a HSA fragment in accordance with the invention.

In some preferred embodiments, the present invention provides a Fab fragment of an IgA antibody (lgA1 or lgA2) that has attached to the C-terminal end of each of the two polypeptide chains thereof a HSA fragment in accordance with the invention.

In preferred embodiments, the antibodies (or fragments thereof) in accordance with the invention are human antibodies (or fragments thereof). In this regard, human antibodies generally have potential advantages for use in human therapy, for example the human immune system should not recognize the antibody as foreign.

The term "human" as used herein in connection with antibody molecules and binding proteins refers to antibodies and binding proteins having variable regions (e.g., VH, VL, CDR or FR regions) and, preferably, constant antibody regions, isolated or derived from a human repertoire or derived from or corresponding to sequences found in humans or a human repertoire, e.g., in the human germline or somatic cells.

"Human" antibodies (or fragments thereof) in accordance with the invention may further include amino acid residues not encoded by human sequences, e.g., mutations introduced by random or site directed mutations in vitro, for example mutations introduced by in vitro cloning or PCR. Particular examples of such mutations are mutations that involve conservative substitutions or other mutations in a small number of residues of the protein, e.g., in up to 5, 4, 3, 2 or 1 of the residues of the antibody (or fragment thereof). Certain examples of such "human" antibodies include antibodies and variable regions that have been subjected to standard modification techniques to reduce the amount of potentially immunogenic sites. Thus, "human" antibodies (and fragments thereof) of the invention include sequences derived from and related to sequences found in humans, but which may not naturally exist within humans, e.g. within the antibody germline repertoire in vivo.

In some embodiments, the discussion herein in relation to antibodies of the invention may be applied, mutatis mutandis, to proteins of the invention.

Human serum albumin (HSA) comprises three domains (domains (D) I, II and III), of which domain I and domain III are known to interact with the neonatal Fc receptor (FcRn) in vivo. These interactions are pH-dependent and allow rescue of HSA from lysosomal degradation based on low affinity binding at neutral pH and high affinity binding at endosomal pH (which is more acidic). FcRn binds HSA in a strictly acidic pH-dependent manner, with negligible binding at neutral pH. FcRn is predominantly located within acidic endosomes where, without wishing to be bound by theory, HSA may be taken up (i.e. into endosomes) and recycled to the cell surface upon exposure to the physiological (neutral) pH. By this mechanism, HSA may avoid lysosomal degradation. This is believed to be responsible for the long half-life of HSA in the circulation.

The amino acid sequence of HSA (full-length HSA or wild-type HSA or mature HSA) is set forth herein as SEQ ID NO:1.

In accordance with the present invention, the proteins comprise“a fragment of human serum albumin that is capable of binding to FcRn” that is characterized by comprising certain amino acid residues at positions corresponding to certain amino acid positions in SEQ ID NO: 1 , as described above.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to or is) domain III (Dill) of human serum albumin and is further characterized by comprising at least one mutant residue selected from the group consisting of (or comprising) (i) a mutant residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , (ii) a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1 and (iii) a mutant residue at the position corresponding to amino acid position 573 of SEQ ID NO:1.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to or is) domain III (Dill) of human serum albumin and is further characterized by comprising at least one mutant residue selected from the group consisting of (or comprising) (i) a mutant residue at the position corresponding to amino acid position 505 of SEQ I D NO: 1 , (ii) a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 , (iii) a mutant residue at the position corresponding to amino acid position 573 of SEQ ID NO:1 , and (iv) a mutant residue at the position corresponding to amino acid position 547 of SEQ ID NO: 1.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to) domain III (Dill) of human serum albumin and is further

characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1.

characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 , (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1 , and (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1.

Polypeptides comprising a glutamine residue at the position corresponding to position 505, a methionine residue at the position corresponding to position 527 and a proline residue at the position corresponding to position 573; all three positions relative to SEQ ID NO:1 ; are also referred to as E505Q/T527M/K573P, or QMP polypeptides, e.g. DIII-QMP polypeptides. Polypeptides which in addition comprise an alanine residue at the position corresponding to position 547 of SEQ ID NO:1 are also referred to as E505Q/T527M/V547A/K573P or QUAD polypeptides, e.g. DIN-QUAD polypeptides. Such QMP polypeptides, DIII-QMP polypeptides, QUAD polypeptides, and DIII-QUAD polypeptides are preferred for use in the present invention.

As used herein, domain III means a polypeptide comprising (or consisting of) 180 to 300 amino acid residues (i.e. the length is 180 to 300 amino acid residues) wherein the polypeptide comprises SEQ ID NO:5 or a sequence with at least 95% sequence identity thereto and wherein the polypeptide has the ability to bind FcRn at endosomal pH. In some embodiments, domain III is a polypeptide comprising (or consisting of) 186 to 300 amino acid residues (i.e. the length is 186 to 300 amino acid residues) wherein the polypeptide comprises SEQ ID NO:5 or a sequence with at least 95% sequence identity thereto and wherein the polypeptide has the ability to bind FcRn at endosomal pH.

However, more preferably domain III is a polypeptide comprising (or consisting of) 180 to 220 amino acid residues (i.e. the length is 180 to 220 amino acid residues) wherein the polypeptide comprises SEQ ID NO:5 or a sequence with at least 95% sequence identity thereto and wherein the polypeptide has the ability to bind FcRn at endosomal pH. In some embodiments, domain III is a polypeptide comprising (or consisting of) 186 to 220 amino acid residues (i.e. the length is 186 to 220 amino acid residues) wherein the polypeptide comprises SEQ ID NO:5 or a sequence with at least 95% sequence identity thereto and wherein the polypeptide has the ability to bind FcRn at endosomal pH. As used herein, endosomal pH is in the range of pH 5.0 to 6.0.

In some preferred embodiments, domain III of HSA (wild-type HSA) may be characterized as comprising (or consisting of) amino acids 381-585 of SEQ ID NO:1. In some embodiments, domain III of HSA (wild-type HSA) may be characterized as comprising (or consisting of) amino acids 400-585 of SEQ ID NO:1.

In some embodiments, domain III of HSA (wild-type HSA) may be characterized as comprising (or consisting of) amino acids 382-585 of SEQ ID NO:1. In some embodiments, domain III of HSA (wild-type HSA) may be

characterized as comprising (or consisting of) amino acids 383-585 of SEQ ID NO: 1. In some embodiments, domain III of HSA (wild-type HSA) may be

characterized as comprising (or consisting of) amino acids 384-585 of SEQ ID NO: 1. In some embodiments, domain III of HSA (wild-type HSA) may be characterized as comprising (or consisting of) amino acids 385-585 of SEQ ID NO: 1.

In some embodiments, the HSA fragment in accordance with the invention comprises (or consists of) a mutated version of domain III of HSA. Exemplary mutations are defined elsewhere herein and in particular include one or more, or all, of the described mutations at positions 505, 527 and 573 of SEQ ID NO: 1 , or one or more, or all, of the described mutations at positions 505, 527, 547 and 573 of SEQ ID NO:1.

Thus, in some embodiments, a HSA fragment in accordance with the invention is a polypeptide comprising (or consisting of) 180 to 300 amino acid residues (i.e. the length is 180 to 300 amino acid residues) wherein the polypeptide comprises SEQ ID NO:3 or a sequence with at least 95% sequence identity thereto and wherein the polypeptide has the ability to bind FcRn at endosomal pH. In some embodiments, a HSA fragment in accordance with the invention is a polypeptide comprising (or consisting of) 186 to 300 amino acid residues (i.e. the length is 186 to 300 amino acid residues) wherein the polypeptide comprises SEQ ID NO:3 or a sequence with at least 95% sequence identity thereto and wherein the polypeptide has the ability to bind FcRn at endosomal pH.

Thus, in some embodiments, a HSA fragment in accordance with the invention is a polypeptide comprising (or consisting of) 180 to 220 amino acid residues (i.e. the length is 180 to 220 amino acid residues) wherein the polypeptide comprises SEQ ID NO:3 or a sequence with at least 95% sequence identity thereto and wherein the polypeptide has the ability to bind FcRn at endosomal pH. In some embodiments, a HSA fragment in accordance with the invention is a polypeptide comprising (or consisting of) 186 to 220 amino acid residues (i.e. the length is 186 to 220 amino acid residues) wherein the polypeptide comprises SEQ ID NO:3 or a sequence with at least 95% sequence identity thereto and wherein the polypeptide has the ability to bind FcRn at endosomal pH.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to) amino acids 381-585 of human serum albumin (SEQ ID NO:1) and is further characterized by comprising at least one mutant residue selected from the group consisting of (or comprising) (i) a mutant residue at the position

corresponding to amino acid position 505 of SEQ ID NO: 1 , (ii) a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 , (iii) a mutant residue at the position corresponding to amino acid position 573 of SEQ ID NO:1 , and (iv) a mutant residue at the position corresponding to amino acid position 547 of SEQ ID NO:1.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to) amino acids 381-585 of human serum albumin (SEQ ID NO:1) and is further characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO: 1. In some such embodiments the HSA fragment further comprises (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to) amino acids 400-585 of human serum albumin (SEQ ID NO:1) and is further characterized by comprising at least one mutant residue selected from the group consisting of (or comprising) (i) a mutant residue at the position

In some embodiments, the HSA fragment comprises (or consists of or corresponds to) amino acids 400-585 of human serum albumin (SEQ ID NO:1) and is further characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1. In some such embodiments the HSA fragment further comprises (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to) the amino acid sequence of SEQ ID NO:2, or a sequence substantially homologous thereto (e.g. having at least 80%, at least 85%, at least 95%, at least 98% or at least 99% sequence identity thereto) that comprises a glutamine residue at position 125 of SEQ ID NO:2 (or at the position corresponding to position 125 of SEQ ID NO:2), a methionine residue at position 147 of SEQ ID NO:2 (or at the position corresponding to position 147 of SEQ ID NO:2) and a proline residue at position 193 of SEQ ID NO:2 (or at the position corresponding to position 193 of SEQ ID NO:2).

In some embodiments, the HSA fragment comprises the amino acid sequence of SEQ ID NO:2.

In some embodiments, the HSA fragment consists of the amino acid sequence of SEQ ID NO:2.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to) the amino acid sequence of SEQ ID NO:3, or a sequence substantially homologous thereto (e.g. having at least 80%, at least 85%, at least 95%, at least 98% or at least 99% sequence identity thereto) that comprises a glutamine residue at position 106 of SEQ ID NO:3 (or at the position corresponding to position 106 of SEQ ID NO:3), a methionine residue at position 128 of SEQ ID NO:3 (or at the position corresponding to position 128 of SEQ ID NO:3) and a proline residue at position 174 of SEQ ID NO:3 (or at the position corresponding to position 174 of SEQ ID NO:3).

In some embodiments, the HSA fragment comprises the amino acid sequence of SEQ ID NO:3.

In some embodiments, the HSA fragment consists of the amino acid sequence of SEQ ID NO:3.

In some embodiments, the HSA fragment comprises (or consists of or corresponds to) the amino acid sequence of SEQ ID NO:8, or a sequence substantially homologous thereto (e.g. having at least 80%, at least 85%, at least 95%, at least 98% or at least 99% sequence identity thereto) that comprises a glutamine residue at position 125 of SEQ ID NO:8 (or at the position corresponding to position 125 of SEQ ID NO:8), a methionine residue at position 147 of SEQ ID NO:8 (or at the position corresponding to position 147 of SEQ ID NO:8), a proline residue at position 193 of SEQ ID NO:8 (or at the position corresponding to position 193 of SEQ ID NO:8) and an alanine residue at position 167 of SEQ ID NO:8 (or at the position corresponding to position 167 of SEQ ID NO:8).

In some embodiments, the HSA fragment comprises the amino acid sequence of SEQ ID NO:8.

In some embodiments, the HSA fragment consists of the amino acid sequence of SEQ ID NO:8. In some embodiments, the HSA fragment comprises (or consists of or corresponds to) the amino acid sequence of SEQ ID NO:9, or a sequence substantially homologous thereto (e.g. having at least 80%, at least 85%, at least 95%, at least 98% or at least 99% sequence identity thereto) that comprises a glutamine residue at position 106 of SEQ ID NO:9 (or at the position corresponding to position 106 of SEQ ID NO:9), a methionine residue at position 128 of SEQ ID NO:9 (or at the position corresponding to position 128 of SEQ ID NO:9), a proline residue at position 174 of SEQ ID NO:9 (or at the position corresponding to position 174 of SEQ ID NO:9) and an alanine residue at position 148 of SEQ ID NO:9 (or at the position corresponding to position 148 of SEQ ID NO: 9).

In some embodiments, the HSA fragment comprises the amino acid sequence of SEQ ID NO:9.

In some embodiments, the HSA fragment consists of the amino acid sequence of SEQ ID NO:9.

In some embodiments, the HSA fragment comprises (or consists of) an amino acid sequence that is substantially homologous to the amino acid sequence of a HSA fragment as described above. For example, such a substantially homologous sequence may have one or more (e.g. 1 , 2, 3, 4 or 5) additional mutant residues (or substitutions) as compared to the corresponding sequence of SEQ ID NO: 1 , i.e. that are additional to the mutant residue(s) (or substitution(s)) that are characteristic of the HSA fragment in accordance with the invention. A substantially homologous sequence may have one or more (e.g. 1 , 2, 3, 4 or 5) additional residues and/or deleted residues as compared to the fragments as defined herein.

A substantially homologous sequence may have at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence of a HSA fragment as defined herein. For the avoidance of doubt, any substantially homologous sequence must have (or retain) the mutant residue(s) (or substitution(s)) that are characteristic of the HSA fragment in accordance with the invention.

In some embodiments, a HSA fragment in accordance with the present invention is characterized by comprising at least one mutant residue selected from the group consisting of (or comprising) (i) a mutant residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1, (ii) a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 , (iii) a mutant residue at the position corresponding to amino acid position 573 of SEQ ID NO:1 , and (iv) a mutant residue at the position corresponding to amino acid position 547 of SEQ ID NO: 1 , by having no further mutant residues as compared to the

corresponding sequence of SEQ I D NO: 1. Put another way, in some embodiments, the above mutant residues in the HSA fragment are the only mutant residues as compared to the corresponding sequence of SEQ ID NO:1.

In some embodiments, a HSA fragment in accordance with the present invention is characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1, (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1 and by having no further mutant residues as compared to the corresponding sequence of SEQ ID NO:1. In some such embodiments the HSA fragment further comprises (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1. Put another way, in some embodiments, the above residues in the HSA fragment are the only mutant residues as compared to the corresponding sequence of SEQ ID NO:1.

In some embodiments, the HSA fragment is at least 150 amino acids in length, at least 155 amino acids in length, at least 160 amino acids in length, at least 165 amino acids in length, at least 170 amino acids in length, at least 175 amino acids in length, preferably at least 185 amino acids in length (e.g. at least 186 amino acids in length or at least 205 amino acids in length). In some embodiments, the HSA fragment is 150-250 amino acids in length, or 175-225 amino acids in length, or 185-215 amino acids in length, or 180-300 amino acids in length, or 180- 220 amino acids in length, or 186-300 amino acids in length, or 186-220 amino acids in length. In some embodiments, the HSA fragment is 186 amino acids in length (e.g. SEQ ID NO:3 or 9). In some embodiments the HSA fragment is 205 amino acids in length (e.g. SEQ ID NO:2 or 8).

As discussed above, the HSA fragment may comprise an amino acid sequence or consist of an amino acid sequence of a HSA fragment as described herein. In some preferred embodiments, the HSA fragment consists of an amino acid sequence of a HSA fragment as described herein.

For the avoidance of doubt, HSA fragments in accordance with the present invention do not include fragments of wild-type HSA (SEQ ID NO:1). For example, SEQ ID NO:5 itself is not a HSA fragment in accordance with the invention. Put another way, in accordance with the present invention HSA fragments comprise at least one mutant residue as compared to the corresponding sequence in wild-type HSA (SEQ I D NO: 1), as described elsewhere herein.

In accordance with the present invention, the fragment of human serum albumin that is capable of binding to FcRn is characterized by comprising one or more mutant residues at certain positions“corresponding to amino acid position XXX of SEQ ID NO: 1”. For example, in some embodiments there is a mutant residue (e.g. Q) at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , a mutant residue (e.g. M) at the position corresponding to amino acid position 527 of SEQ ID NO: 1 and/or a mutant residue (e.g. P) at the position corresponding to amino acid position 573 of SEQ ID NO: 1. In some embodiments there is a mutant residue (e.g. A) at the position corresponding to amino acid position 547 of SEQ ID NO: 1. SEQ ID NO: 1 is the amino acid sequence of human serum albumin, HSA (full-length HSA or wild-type HSA). Fragments of HSA are, by definition, shorter than the full-length (or wild-type) HSA protein. As a result, and for example, the amino acid position (the number of the position) of a given amino acid residue in a fragment will not necessarily be the same as in the full-length sequence. For example, the amino acid position (the number of the position) of a given amino acid residue in a fragment of HSA that is N-terminally truncated with respect to the full- length (or wild-type) HSA sequence will be lower than the position (the number of the position will be lower) in the corresponding (or counterpart) full-length (or wild- type) HSA sequence. Thus, SEQ ID NO: 1 acts as a reference sequence

(corresponding to full-length or wild-type HSA), and referring to the amino acid positions in the fragments by reference to the corresponding position in the full- length (or wild-type) HSA provides a useful means to define certain positions in fragments of full-length (or wild-type) HSA.

Thus, referring to“the position corresponding to amino acid position XXX of SEQ ID NO: 1” identifies the amino acid residue in the fragment that corresponds to (or aligns with) the stated position (the corresponding position) in a reference sequence (SEQ ID NO: 1). The skilled person will appreciate that this can readily be done by aligning a given sequence (amino acid sequence of a fragment) with the reference sequence and identifying the amino acid residue in the fragment that aligns with the particular position (corresponding position or counterpart position) in the reference sequence (SEQ ID NO: 1). For example, in order to identify the amino acid residue in a given fragment that corresponds to position 573 in SEQ ID NO: 1 (HSA), the fragment’s amino acid sequence can be aligned with SEQ ID NO: 1 (HSA) and the amino acid that aligns with position 573 of SEQ ID NO: 1 (HSA) (e.g. under best alignment) is identified as the amino acid in the given fragment that corresponds to position 573 of SEQ ID NO:1. Methods of sequence alignment are well-known in the art and the skilled person will be familiar with such methods (and suitable alignment methods are discussed elsewhere herein.

Accordingly, the“position corresponding to amino acid position XXX of SEQ ID NO:1” may be considered the position of an amino acid in a fragment of human serum albumin in accordance with the invention that corresponds to (or aligns with or matches) the stated amino acid position (or reference amino acid position) in SEQ ID NO:1.

In accordance with the present invention, the fragment of HSA is“capable of binding to FcRn”. This means that the fragment of HSA is able to bind to (e.g. specifically bind to), or interact with the FcRn receptor (neonatal Fc receptor), preferably in a pH-dependent manner (preferably is able to bind to the FcRn receptor at endosomal pH). In this regard, preferably the HSA fragment is capable of binding to FcRn at acidic pH (e.g. pH 5.5 or endosomal pH) but is not capable of binding to FcRn (or has no significant ability to bind to FcRn or significantly less or reduced ability or a weak ability or low affinity to bind to FcRn) at neutral pH (e.g. pH

7.4). As used herein, endosomal pH is in the range of pH 5.0 to 6.0.

The ability to bind to FcRn, e.g. in a pH-dependent manner, may be as assessed (or determined) by any appropriate assay or method and the skilled person will be familiar with suitable assays and methods. Preferably an ELISA may be used. Parallel ELISA assays may be performed, with one at neutral pH (e.g. pH

7.4) and another at an acidic pH (e.g. pH 5.5), in order to assess the pH- dependency of FcRn binding. Binding at acidic pH, with no significant binding (or no binding or reduced binding or significantly reduced or a weak binding or low affinity binding) at neutral pH, would be indicative of pH-dependent binding of the fragment of HSA to FcRn. A particularly preferred ELISA may comprise assessing (or determining) the capability of a fragment of HSA to bind to FcRn, e.g. in a pH- dependent manner, when the HSA fragment is attached to a protein in accordance with the invention. Such ELISA assays are described elsewhere herein. Another assay that may be used to assess the capability of a fragment of HSA to bind to FcRn (e.g. when the HSA fragment is attached to a protein in accordance with the invention) is the human endothelial cell-based recycling assay (HERA). HERAs are described elsewhere herein. In some embodiments, the FcRn is human FcRn. In some embodiments, the FcRn is recombinant FcRn (e.g. GST-tagged human FcRn). In some embodiments, the FcRn is recombinant human FcRn (e.g. GST-tagged recombinant human FcRn, e.g. as described in Example 2 herein).

In some embodiments, the FcRn is FcRn (preferably human FcRn) that is expressed by (or in or on) an endothelial cell (e.g. a human endothelial cell such as a HMEC1 cell as described in Example 3 herein). In such embodiments the FcRn is thus on (or in) a cell (e.g. in the endosome of a cell), as opposed to being isolated (e.g. recombinant) FcRn.

In accordance with the present invention, a fragment of human serum albumin in accordance with the invention is attached to the“C-terminal end” of a polypeptide chain. At its broadest,“C-terminal end” means the C-terminal portion of the polypeptide chain. For example, in some embodiments the C-terminal end may be defined as the 50 C-terminal (50 C-terminal-most) amino acids, 40 C-terminal (40 C-terminal-most) amino acids, 30 C-terminal (30 C-terminal-most) amino acids, 20 C-terminal (20 C-terminal-most) amino acids, 10 C-terminal (10 C-terminal-most) amino acids, 5 C-terminal (5 C-terminal-most) amino acids, 2 C-terminal (2 C- terminal-most) amino acids, or the (final) C-terminal (C-terminal-most) residue of the polypeptide chain. In preferred embodiments, the“C-terminal end” of the polypeptide chain is the C-terminal amino acid (i.e. the final C-terminal amino acid or the amino acid located at the C-terminus).

Any suitable means of attachment (or conjugation or linking) may be used and skilled person will be familiar with suitable means of attachment.

Typically and preferably, a fragment of human serum albumin in accordance with the invention is attached to (or conjugated to or linked to or fused to) the C- terminal end of a polypeptide chain by (or via) a covalent bond.

In preferred embodiments, a fragment of human serum albumin in accordance with the invention is attached to the C-terminal end of a polypeptide chain comprising an IgA constant domain by virtue of being present on (i.e.

expressed on) the same polypeptide chain, i.e. as a fusion polypeptide. Thus, in some embodiments, a fragment of human serum albumin in accordance with the invention is attached to the C-terminal end of the polypeptide chain comprising an IgA constant domain as (or to form) a fusion polypeptide (fusion polypeptide chain). For the avoidance of doubt, in the context of the present invention a fusion polypeptide is a single polypeptide chain comprising a fragment of human serum albumin in accordance with the invention and a polypeptide comprising an IgA constant domain in accordance with the invention, wherein the fragment of human serum albumin is positioned (or located) C-terminally relative to the polypeptide comprising an IgA constant domain.

Thus, in some embodiments, the polypeptide chains (preferably two or more polypeptide chains) having attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn are fusion polypeptides, wherein each fusion polypeptide comprises a polypeptide chain comprising an IgA antibody constant domain and said fragment of human serum albumin that is capable of binding to FcRn, said fragment of human serum albumin that is capable of binding to FcRn being fused to the C-terminal end of said polypeptide chain comprising an IgA antibody constant domain, optionally via a linker.

Thus, in some embodiments, the protein comprises two or more (e.g. 2 or 4) fusion polypeptides (or fusion polypeptide chains). In such embodiments, each fusion polypeptide comprises (or consists of) (i) a HSA fragment in accordance with the invention, and (ii) a polypeptide chain comprising an IgA constant domain in accordance with the invention, wherein the HSA fragment is positioned (or located) C-terminally relative to the polypeptide chain in accordance with the invention.

The skilled person is familiar with methods of generating fusion

polypeptides, e.g. by expressing a nucleic acid molecule encoding a fusion polypeptide (e.g. in a host cell). Such a nucleic acid molecule typically comprises a contiguous nucleotide sequence encoding, in frame, the various components of the fusion polypeptide. In the present case, such a nucleic acid molecule may comprise a contiguous nucleotide sequence comprising a nucleotide sequence encoding a HSA fragment in accordance with the invention and a nucleotide sequence encoding a polypeptide chain in accordance with the invention, wherein the nucleotide sequence encoding the HSA fragment is located at the 3’-end of the nucleic acid molecule relative to the nucleotide sequence encoding the polypeptide chain in accordance with the invention.

In other embodiments, a fragment of human serum albumin in accordance with the invention is attached to the C-terminal end of the polypeptide chain by a cross-linker (e.g. a chemical cross-linker or cross-linking agent). Suitable cross- linking agents and methods for attaching (or joining or linking or conjugating) different proteins or polypeptides together are known in the art.

In some embodiments, a HSA fragment in accordance with the invention may be attached to (or joined to or linked to or conjugated to) a polypeptide chain in accordance with the invention by (or via) a thioether bond. Such a bond may, for example, be formed with (or using) the reagent sulfo-SMCC (sulfo-succinimidyl-4- (N-maleimidomethyl) cyclohexane- 1-carboxylate) or the reagent SMCC

(succinimidyl-4-(N-maleimidomethyl) cyclohexane- 1-carboxylate). Such methods of conjugation are well-known in the art.

In some embodiments, polypeptide chains in accordance with the invention (or IgA antibodies of the invention or fragments thereof) are produced separately and then subsequently attached together (or joined together or linked together or conjugated together), e.g. as discussed above.

The attachment in accordance with the invention may be a direct attachment or an indirect attachment.

For example, the attachment may be via a linker, meaning that the attachment is an indirect attachment. Thus, in some embodiments, a HSA fragment in accordance with the invention is attached via a linker to a polypeptide chain comprising an IgA constant domain in accordance with the invention (i.e. there may be a linker between the HSA fragment and the polypeptide chain).

Linkers comprising (or consisting of) amino acids (e.g. peptide linkers) are particularly preferred. In some such embodiments, a 1-30 amino acid (e.g. 5-30 or 8-30) linker, a 1-20 amino acid (e.g. 5-20 or 8-20) linker, a 1-10 amino acid (e.g. 5- 10) linker or a 1-5 amino acid linker. In one preferred embodiment the linker consists of 8 amino acids.

In some embodiments, the linker comprises (or consists of) glycine and/or serine residues. In one preferred embodiment the linker is ((GGS)4GG) (SEQ ID NO:4).

Linkers (amino acid linkers or peptide linkers) are most preferably used in the context of fusion polypeptides, with the linker being positioned (or located) between a HSA fragment in accordance with the invention and a polypeptide chain in accordance with the invention. In such embodiments, polypeptide chains (fusion polypeptides) typically comprise (or consist of), from the N-terminal end to the C- terminal end, a polypeptide comprising an IgA constant domain in accordance with the invention, an amino acid linker, and a HSA fragment in accordance with the invention.

In some embodiments, no linker is present. Thus, for example, in some embodiments a HSA fragment in accordance with the invention is directly attached to a polypeptide chain comprising an IgA constant domain in accordance with the invention. With such a direct attachment, in the context of a fusion polypeptide, the first (i.e. N-terminal) amino acid of the HSA fragment may be fused directly to (via a peptide bond) the final (i.e. C-terminal) amino acid of the polypeptide chain (i.e. with no linker in between).

In some preferred embodiments, two polypeptide chains have attached to the C-terminal ends thereof a fragment of human serum albumin in accordance with the present invention. In some such embodiments, the protein has four polypeptide chains each comprising an IgA constant domain, and two of said polypeptide chains have attached to the C-terminal ends thereof a fragment of human serum albumin in accordance with the present invention.

In some preferred embodiments, four polypeptide chains have attached to the C-terminal ends thereof a fragment of human serum albumin in accordance with the present invention. In some such embodiments, the protein has four polypeptide chains each comprising an IgA constant domain, and four of said polypeptide chains have attached to the C-terminal ends thereof a fragment of human serum albumin in accordance with the present invention.

In some embodiments, the protein has two polypeptide chains each comprising an IgA constant domain, and two (i.e. both) of said polypeptide chains have attached to the C-terminal ends thereof a fragment of human serum albumin in accordance with the present invention.

In some preferred embodiments, the present invention provides an IgA antibody, preferably a monomeric IgA (lgA1 or lgA2) antibody (having two heavy chains and two light chains), that has attached to the C-terminal end of each of its two light chains a HSA fragment in accordance with the invention. Preferably, the HSA fragment is characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1 , and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1, and optionally, (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1. Preferred HSA fragments in accordance with the invention are described elsewhere herein. In some embodiments, the HSA fragment comprises (or consists of) SEQ ID NO:2 or 8. In some embodiments, HSA fragments in accordance with the present invention are attached to the C-terminal ends of the light chains via an amino acid linker, preferably a glycine-serine linker (such as (GGS)4GG). Thus, in some embodiments, a HSA fragment in accordance with the present invention and the light chain are provided on (present on) the same polypeptide chain (as a fusion polypeptide). In some such embodiments, the antibody is an IgA version (or Ig format) of the antibody Trastuzumab. In some embodiments, the protein of the invention is the hlgA1-LC(DIII-QMP)₂ antibody or the hlgA2-LC(DIII-QMP)₂ antibody as described in the Example section herein (which is also schematically depicted in Figure 1). In some embodiments, the protein of the invention is the hlgA1-LC(DIII-QUAD)₂ antibody or the hlgA2-LC(DI 11- QUAD)2 antibody, e.g. as described in the Example section (h = human). In some embodiments, the IgA antibody is an lgA1 antibody.

In some preferred embodiments, the present invention provides an IgA antibody, preferably a monomeric IgA (lgA1 or lgA2) antibody (having two heavy chains and two light chains), that has attached to the C-terminal end of each of its two heavy chains a HSA fragment in accordance with the invention. Preferably, the HSA fragment is characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1 , and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO: 1, and optionally, (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1. Preferred HSA fragments in accordance with the invention are described elsewhere herein. In some embodiments, the HSA fragment comprises (or consists of) SEQ ID NO:2 or 8. In some embodiments, HSA fragments in accordance with the present invention are attached to the C-terminal ends of the heavy chains via an amino acid linker, preferably a glycine-serine linker (such as (GGS)4GG). Thus, in some embodiments, a HSA fragment in accordance with the present invention and the heavy chain are provided on (present on) the same polypeptide chain (as a fusion polypeptide). In some such embodiments, the antibody is an IgA version (or Ig format) of the antibody Trastuzumab. In some embodiments, the protein of the invention is the hlgA1-HC(DIII-QMP)₂ antibody or the hlgA2-HC(DIII-QMP)₂ antibody as described in the Example section herein (which is also schematically depicted in Figure 1). In some embodiments, the protein of the invention is the hlgA1-HC(DIII-QUAD)₂ antibody or the hlgA2-HC(DI II- QUAD)2 antibody (h = human), e.g. as described in the Example section. In some embodiments, the IgA antibody is an lgA2 antibody.

In some preferred embodiments, the present invention provides an IgA antibody, preferably a monomeric IgA (lgA1 or lgA2) antibody (having two heavy chains and two light chains), that has attached to the C-terminal end of each of its two heavy chains and to the C-terminal end of each of its two light chains a HSA fragment in accordance with the invention. Preferably, the HSA fragment is characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1 , and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO: 1 , and optionally, (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO: 1. Preferred HSA fragments in accordance with the invention are described elsewhere herein. In some embodiments, the HSA fragment comprises (or consists of) SEQ ID NO:2 or 8. In some embodiments, HSA fragments in accordance with the present invention are attached to the C-terminal ends of the heavy chains and light chains via an amino acid linker, preferably a glycine-serine linker (such as (GGS)4GG). Thus, in some embodiments, a HSA fragment in accordance with the present invention and the heavy chain are provided on (present on) the same polypeptide chain (as a fusion polypeptide) and a HSA fragment in accordance with the present invention and the light chain are provided on the same polypeptide chain (as a fusion polypeptide). In some such

embodiments, the antibody is an IgA version (or Ig format) of the antibody

Trastuzumab.

In some preferred embodiments, the present invention provides an Fc fragment of an IgA antibody that has attached to the C-terminal end of each of the two polypeptide chains thereof a HSA fragment in accordance with the invention. Preferably, the HSA fragment is characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1 , and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO: 1 , and optionally, (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO: 1. Preferred HSA fragments in accordance with the invention are described elsewhere herein. In some embodiments, the HSA fragment comprises (or consists of) SEQ ID NO:2 or 8. In some embodiments, HSA fragments in accordance with the present invention are attached to the C-terminal end of each of the two chains of the Fc fragment via an amino acid linker, preferably a glycine-serine linker (such as (GGS)4GG). Thus, in some embodiments, each chain of the Fc fragment is provided on (present on) the same polypeptide chain a HSA fragment in accordance with the present invention (as a fusion polypeptide).

In some preferred embodiments, the present invention provides a Fab fragment of an IgA antibody that has attached to the C-terminal end of each of the two polypeptide chains thereof a HSA fragment in accordance with the invention. Preferably, the HSA fragment is characterized by comprising (i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO: 1 , (ii) a methionine residue at the position corresponding to amino acid position 527 of SEQ ID NO: 1 , and (iii) a proline residue at the position corresponding to amino acid position 573 of SEQ ID NO:1 , and optionally, (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO: 1. Preferred HSA fragments in accordance with the invention are described elsewhere herein. In some embodiments, the HSA fragment comprises (or consists of) SEQ ID NO:2 or 8. In some embodiments, HSA fragments in accordance with the present invention are attached to the C-terminal end of each of the two chains of the Fab fragment via an amino acid linker, preferably a glycine-serine linker (such as (GGS)4GG). Thus, in some embodiments, each chain of the Fab fragment is provided on (present on) the same polypeptide chain a HSA fragment in accordance with the present invention (as a fusion polypeptide). In some such embodiments, the Fab fragment is (or corresponds to or is based on) the Fab fragment of the antibody Trastuzumab.

In preferred embodiments, proteins in accordance with the present invention, and in particular proteins of the present invention comprising an IgA Fc fragment (or Fc region), for example proteins comprising IgA antibodies (e.g. lgA1 or lgA2 antibodies), have antibody dependent cellular cytotoxicity (ADCC) activity. ADCC is an important effector function conferred by the Fc fragment (Fc region) and thus ADCC activity a desirable and advantageous property of proteins of the invention. The inventors have evidenced herein (see Example 2) that the attachment of fragments of human serum albumin in accordance with the invention to IgA antibodies does not eliminate ADCC activity and significant levels of ADCC activity are maintained or retained or present.

In some embodiments, a protein of the present invention (e.g. an IgA antibody) has at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the ADCC activity of a control protein (e.g. 50%- 200% or 50%-150% or 50%-100% or 80%-200% or 80%-150% or 80%-100%), for example a control protein that comprises an IgA Fc fragment (e.g. an IgA antibody such as an lgA1 or lgA2 antibody) but that does not comprise a fragment of HSA (e.g. does not comprise a fragment of HSA in accordance with the invention). In some embodiments, this may be as determined when using a protein of the invention (and control protein) at a concentration of about 5nM (e.g. 5.12nM).

In some embodiments, ADCC activity of a protein of the present invention is not significantly altered (e.g. not significantly lower than) as compared to the ADCC activity of a control protein, for example a control protein that comprises an IgA Fc fragment (e.g. an IgA antibody such as an lgA1 or lgA2 antibody) but that does not comprise a fragment of HSA (e.g. does not comprise a fragment of HSA in accordance with the invention).

In some embodiments, ADCC activity of a protein of the present invention is altered (e.g. lowered) by no more than 75%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10% or no more than 5% as compared to the ADCC activity of a control protein (e.g. as described above). In some embodiments, this may be as determined when using a protein of the invention (and control protein) at a concentration of about 5nM (e.g. 5.12nM).

Preferably, a control protein is substantially identical to (or identical to) the protein of the invention with which the comparison is being made, with the exception (or difference) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). In some embodiments, the protein of the invention comprises an IgA antibody and the control protein comprises (or consists of) the same IgA antibody with the sole difference (as compared to the protein of the invention) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention).

Typically and preferably, ADCC activity may be expressed in terms of the amount of specific lysis or specific cell lysis (e.g. % specific lysis or % specific cell lysis). Any appropriate ADCC assay may be used and the skilled person is familiar with appropriate assays. The discussion and values above may be as determined by (or as assessed by) any appropriate ADCC assay. A particularly preferred ADCC assay is a chromium-release assay and in some embodiments the discussion and values above in relation to ADCC activity relate to ADCC activity as determined using a chromium-release assay.

In a preferred such assay, cells such as cancer cells (e.g. SKBR 3 cells) are labeled with Chromium-51 and added to proteins of the invention (e.g. to titrated amounts of such proteins), e.g. in round-bottomed microtiter plates.

Polymorphonuclear leukocytes (PM Ns) (which may be isolated from the blood of healthy donors e.g. by ficoll-histopaque density gradient) are added to the mixture of labeled cells/proteins of the invention (e.g. with an effector-to-target ratio (E:T) of 40: 1 , e.g. in a final volume of 200pl/well of the microtiter plate). The cells are then incubated, e.g. for 4h at 37°C/5%C0₂. The supernatant is then analysed, for example using a liquid scintillation counter. The % lysis (% specific lysis) may be calculated using the following formula: % lysis = ((counts of sample - minimal release)/(maximum release - minimum release))*100. Culture medium can be used to determine minimal release and 3% TritonX-100 can be used to determine maximum release. A particularly preferred ADCC chromium release assay is described in Example 2 herein.

Preferably, proteins in accordance with the present invention, in particular proteins of the present invention comprising an antigen binding domain, for example proteins comprising a Fab fragment (or Fab region) e.g. IgA antibodies (e.g. lgA1 or lgA2 antibodies), have the ability to bind to (e.g. specifically bind to) their target antigen (e.g. human target antigen). In some embodiments, proteins of the invention have the ability to bind to a recombinant version of the target antigen (e.g. recombinant human target antigen). It is of course important that the attachment (inclusion) of a human serum albumin fragment in accordance with the invention does not significantly affect an antibody’s (or antigen binding fragment’s) ability to recognize and bind to its target antigen. The inventors have reported herein (see Example 2) that the attachment of fragments of human serum albumin in

accordance with the invention to IgA antibodies does not affect antigen binding.

Thus, in some embodiments, a protein of the present invention comprising an antigen binding domain, for example proteins comprising a Fab fragment (or Fab region) e.g. IgA antibodies (e.g. lgA1 or lgA2 antibodies), has an ability to bind to its target antigen that is not significantly altered (or not altered) or is substantially equivalent to (or equivalent to or comparable to) the ability of a control protein comprising an (e.g. the same) antigen binding domain to bind to the antigen.

Preferably, a control protein is substantially identical to (or identical to) the protein of the invention with which the comparison is being made, with the exception (or difference) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). In some embodiments, the protein of the invention comprises an Fab fragment (or Fab region), e.g. comprises (or is) an IgA antibody and the control protein comprises (or consists of) the same protein with the sole difference (as compared to the protein of the invention) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention).

The ability of a protein of the invention comprising an antigen binding domain to bind to its target antigen may be assessed by any appropriate method or assay. The discussion above may be in relation to target antigen binding as determined by (or as assessed by) any appropriate assay and the skilled person is familiar with appropriate assays. Typically and preferably, an ELISA assay is used.

In a preferred such ELISA assay, wells of ELISA plates (e.g. 96-well plates) are coated with (e.g. 1.0 mg/ml) recombinant antigen (e.g. diluted in PBS), and incubated (e.g. ON at 4°C). Plates are then blocked (e.g. with PBS containing 4% skimmed milk (S)) for e.g. 1 hour at room temperature (RT), and washed (e.g. 4 times) (e.g. with PBS containing 0.05% Tween 20 (T)). Titrated amounts (e.g. 100 pi thereof) of a purified protein of the invention comprising an antigen binding domain (or control protein) (e.g. diluted in PBS/S/T), are added to the plates and incubated (e.g. for 1 hour on a shaker at RT). The plates are washed as before, and an alkaline phosphatase (ALP) conjugated anti-human IgA (a-chain specific) antibody produced in goat (e.g. 100 mI thereof) is added (e.g. 1 :2000) and incubated (e.g. for 1 hour at RT). The plates are washed as above, and bound proteins are visualized by adding (e.g. 100 mI) of ALP substrate (e.g. 1 mg/ml phosphate in diethanolamine buffer). Absorbance is measured at 405 nm (e.g. with a Sunrise spectrophotometer, TECAN). A particularly preferred assay for determining antigen binding ability of proteins of the invention is described in Example 2 herein.

Preferably, proteins in accordance with the present invention, in particular proteins of the present invention comprising an IgA Fc fragment (or Fc region) for example proteins comprising IgA antibodies (e.g. lgA1 or lgA2 antibodies), have the ability to bind to FcaR (e.g. human FcaR). FcaR may also be referred to as FcaRI or CD89. In some embodiments, proteins of the invention have the ability to bind to a recombinant version of FcaR (e.g. recombinant human FcaR). IgA binding to (or cross-linking with) FcaR expressed on polymorphonuclear leukocytes (PM Ns) may result in potent induction of ROS production, phagocytosis, NET formation, cytokine release and antibody-dependent cytotoxicity (ADCC), so it is preferable that the attachment (inclusion) of a human serum albumin fragment in accordance with the invention does not significantly affect a protein of the invention’s ability to bind to FcaR. The inventors have reported herein (see Example 2) that the attachment of fragments of human serum albumin in accordance with the invention to IgA antibodies does not affect the ability to bind to FcaR.

Thus, in some embodiments, a protein of the present invention comprising an IgA Fc fragment (or Fc region), for example a protein comprising an IgA antibody (e.g. lgA1 or lgA2 antibody), has an ability to bind to FcaR that is not significantly altered (or not altered) or is substantially equivalent to (or equivalent to or comparable to) the ability of a control protein to bind to FcaR, for example a control protein that comprises an IgA Fc fragment (e.g. an IgA antibody such as an lgA1 or lgA2 antibody) but that does not comprise a fragment of HSA (e.g. does not comprise a fragment of HSA in accordance with the invention). Preferably, a control protein is substantially identical to (or identical to) the protein of the invention with which the comparison is being made, with the exception (or difference) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). In some embodiments, the protein of the invention comprises an IgA antibody and the control protein comprises (or consists of) the same IgA antibody with the sole difference (as compared to the protein of the invention) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention).

The ability of a protein of the invention to bind to FcaR may be assessed by any appropriate method or assay and the skilled person is familiar with appropriate assays. The discussion above may be in relation to FcaR binding as determined by (or as assessed by) any appropriate assay. Typically and preferably, an ELISA assay is used.

In a preferred ELISA assay, wells of ELISA plates (e.g. 96-well plates) are coated and incubated with a protein of the invention (or a control protein), followed by adding (e.g. 100 pi) of His-tagged human FcaR, e.g. at a concentration of 2 pg/ml, and incubated (e.g. for 1 hour at RT). The plates are washed before ALP conjugated anti-His-tag antibody is added (e.g. 1 :5000) (e.g. 100 mI thereof) and incubated (e.g. for 1 hour at RT). The plates are washed and bound proteins were visualized by adding 100 mI of ALP substrate. Visualisation may be done by measuring absorbance at 405 nm (e.g. with a Sunrise spectrophotometer, TECAN). A particularly preferred assay for determining FcaR binding of proteins of the invention is described in Example 2 herein.

Preferably, proteins in accordance with the present invention, have the ability to bind to FcRn (e.g. human FcRn). In some embodiments, proteins of the invention have the ability to bind to a recombinant version of FcRn (e.g.

recombinant human FcRn). As discussed elsewhere herein, human serum albumin (HSA) binds FcRn and is rescued from degradation by a pH dependent mechanism. As also discussed elsewhere herein, the present inventors have identified mutant fragments of HSA that, when attached to IgA-based proteins in accordance with the invention, can extend the half-life of IgA-based proteins in vivo. It is believed that this half-life extension is a result of the protein being rescued from degradation by virtue of the interaction between the HSA fragment in accordance with the invention and FcRn. The inventors have reported herein (see Example 2 and 6) that in an ELISA, proteins in accordance with the invention bind to (or interact) with FcRn in a pH-dependent manner, with binding at acidic pH (e.g. pH 5.5) but with no (or negligible) or significantly reduced (or weak or low affinity) binding at neutral pH (e.g. pH 7.4). The inventors have reported that in such an ELISA control proteins lacking the HSA fragment are not able to bind to FcRn (or have negligible binding to FcRn).

Thus, in some embodiments, a protein of the present invention has the ability to bind to FcRn, preferably in a pH-dependent manner. More specifically, preferably a protein of the present invention has an ability to bind to FcRn at acidic pH (e.g. pH 5.5) but has no significant ability (or no ability) to bind to (or shows no or no significant binding to or significantly reduced binding to or a weak or low affinity binding to) FcRn at neutral pH (e.g. pH 7.4). This pH-dependent binding to FcRn may be contrasted with a control protein that does not have the ability to bind to FcRn (e.g. shows no binding or no significant binding to FcRn). Preferably, a control protein is substantially identical to (or identical to) the protein of the invention with which the comparison is being made, with the exception (or difference) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). In some embodiments, the protein of the invention comprises (or is) an IgA antibody and the control protein comprises (or consists of) the same IgA antibody with the sole difference (as compared to the protein of the invention) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). For example, the inventors have shown that a fragment of HSA in accordance with the invention has an ability to bind FcRn at acidic pH (e.g. pH 5.5) compared to an equivalent (or the same) IgA molecule with no HSA attached (which shows no significant ability to bind FcRn at acidic pH (e.g. pH 5.5)), or compared to an equivalent (or the same) IgA molecule with an equivalent non-mutated HSA fragment attached, e.g. a wild-type or non- mutated fragment, e.g. a wild-type or non-mutated domain III fragment as described herein (which shows no significant ability to bind FcRn at acidic pH (e.g. pH 5.5)).

The ability of a protein of the invention to bind to FcRn in a pH-dependent manner may be assessed by any appropriate method or assay and the skilled person is familiar with appropriate assays and methods. The discussion above may be in relation to FcRn binding as determined by (or as assessed by) any appropriate assay. Typically and preferably, an ELISA assay is used. Typically and preferably, parallel ELISA assays may be performed, one at neutral pH (e.g. pH 7.4) and one at an acidic pH (e.g. pH 5.5) in order to assess the pH-dependency of FcRn binding.

In a preferred ELISA assay, wells of ELISA plates (e.g. 96-well plates) are coated and incubated with a protein of the invention (or a control protein). Wells are washed with either a buffer of pH 5.5 (e.g. PBS/T pH 5.5) or a buffer of pH 7.4 (e.g. PBS/T pH 7.4) before GST-fused soluble recombinant human FcRn (e.g. 2 pg/ml thereof), diluted in a buffer of pH 5.5 (e.g. PBS/S/T pH 5.5) or in a buffer of pH 7.4 (e.g. PBS/S/T pH 7.4), was added and incubated (e.g. for 1 hour at RT). The plates are washed at either pH 5.5 or pH 7.4, and bound receptors (FcRn) are detected using horseradish peroxidase (HRP) conjugated goat anti-GST antibody, diluted (e.g. 1 :8000) in a buffer (e.g. PBS/S/T) with pH 5.5 or 7.4, and incubated (e.g. 1 hour at RT). The plates are washed as before and 100 pi of 3, 3', 5,5'- tetramethybenzidine (TMB) substrate solution is then added to the wells.

Absorbance is measured at 620 nm with a Sunrise spectrophotometer (TECAN).

The reaction is stopped (e.g. by adding 50 mI 1 M HCI), and then measured at 450 nm. A particularly preferred assay for determining pH-dependent binding of proteins of the invention to FcRn is described in Example 2 herein.

Typically and preferably, proteins in accordance with the present invention may be recycled and rescued from intracellular degradation (e.g. from lysosomal degradation) in an FcRn-dependent (or FcRn-mediated) manner. As discussed elsewhere herein, IgA antibodies typically suffer from having a short in vivo half-life (e.g. a half-life of around 1 day). The inventors have demonstrated herein that the attachment of fragments of human serum albumin in accordance with the invention to IgA antibodies can confer upon IgA antibodies the ability (or enhance the ability or increase the ability) to be recycled and rescued via an FcRn-dependent mechanism. Without wishing to be bound by theory, this ability may be responsible for the increased in vivo half-life observed for proteins in accordance with the invention (as discussed elsewhere herein).

In preferred embodiments, the ability of a protein of the invention to be recycled and rescued via an FcRn-dependent mechanism is increased (preferably significantly increased) as compared to the ability of a control protein to be recycled and rescued via an FcRn-dependent mechanism.

In some embodiments, the ability of a protein of the invention to be recycled and rescued via an FcRn-dependent mechanism is increased, for example, by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold, as compared to the ability of a control protein to be recycled and rescued via a FcRn-dependent mechanism (e.g. there is an up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold, up to 7-fold, up to 8-fold, up to 9-fold, up to 10-fold, up to 50-fold or up to 100-fold increase, such as a 2-fold to 100-fold increase, 2-fold to 50-fold, 2-fold to 20-fold increase, 2-fold to 10-fold increase, 4-fold to 100-fold increase, 4-fold to 50- fold increase, 4-fold to 20-fold increase, 4-fold to 10-fold increase, 5-fold to 100-fold increase, 5-fold to 50-fold, 5-fold to 20-fold or a 5-fold to 10-fold increase).

In some embodiments, the ability of a protein of the invention to be recycled and rescued via an FcRn-dependent mechanism is increased, for example, by at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900% or at least 1000% as compared to the ability of a control protein to be recycled and rescued via an FcRn-dependent mechanism (e.g. there is an up to 50% increase, up to 100% increase, up to 200% increase, up to 300% increase, up to 400% increase, up to 500% increase, up to 600% increase, up to 700% increase, up to 800% increase, up to 900% increase, up to 1000% increase or up to 2000% increase, such as 50% to 100% increase, 50% to 500% increase, a 50% to 1000% increase, a 50% to 2000% increase, a 100% to 500% increase, a 100% to 1000% increase, a 100% to 2000% increase, a 500% to 1000% increase or a 500% to 2000% increase).

Preferably, a control protein is substantially identical to (or identical to) the protein of the invention with which the comparison is being made, with the exception (or difference) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). In some embodiments, the protein of the invention comprises (or is) an IgA antibody and the control protein comprises (or consists of) the same IgA antibody with the sole difference (as compared to the protein of the invention) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention).

The ability of a protein of the invention to be recycled and rescued via an FcRn-dependent mechanism may be as determined by any suitable assay or method and a skilled person will be familiar with suitable assays or methods. The discussion above may be in relation recycling and rescuing via an FcRn-dependent mechanism as determined by (or as assessed by) any appropriate assay. In some embodiments, the ability of a protein of the invention to be recycled and rescued via an FcRn-dependent mechanism is as determined in (or as assessed in) a human endothelial cell-based recycling assay (HERA). Suitable HERAs are known in the art (e.g. Grevys et ai, 2018, Nat. Commun., 9(1):621).

In a preferred HERA, human endothelial cells (e.g. HMEC1 cells) stably expressing HA-hFcRn-EGFP are seeded into plates (e.g. 24-well plates), e.g.

7.5 10⁴ cells per well of a 24-well plate, and cultured for one day in growth medium. The cells are washed and starved (e.g. for 1 h) in Hank’s balanced salt solution (HBSS). Proteins of the invention (or control proteins) (e.g. 400 nM thereof) diluted in (e.g. 250 pi) HBSS (pH 7.4) are added to the cells followed by incubation (e.g. for 4h). The medium is removed and the cells are washed with ice cold HBSS (pH 7.4) before fresh warm HBSS (pH 7.4) or growth medium without FCS and supplemented with MEM non-essential amino acids is added. Samples of the growth medium are collected the next day. The amounts of a protein of the invention in the growth medium are quantified using an ELISA, e.g. in the case of proteins having an antigen binding domain a target antigen ELISA as described elsewhere herein. A particularly preferred HERA for determining the ability of a protein of the invention to be recycled and rescued via an FcRn-dependent mechanism is described in Example 3 herein.

Typically and preferably, proteins in accordance with the present invention, for example proteins comprising IgA antibodies (e.g. lgA1 or lgA2 antibodies), have a therapeutically useful in vivo half-life (e.g. a therapeutically useful half-life in the mammalian, preferably human, circulation or a therapeutically useful serum half-life or a therapeutically useful plasma half-life). IgA antibodies typically suffer from having a short in vivo half-life (e.g. a half-life of around 1 day). The inventors have demonstrated herein that the attachment of fragments of human serum albumin in accordance with the invention to IgA antibodies can increase the in vivo half-life of the antibodies (see Example 4 and 5). This is an advantageous property which means that IgA antibodies which otherwise may be of no (or sub-optimal) in vivo (e.g. therapeutic) use due to their short half-life can be modified via the attachment of a fragment of human serum albumin in accordance with the invention to extend their half-life.

In some embodiments, a protein of the present invention has an in vivo half- life of at least 2 days or at least 3 days or at least 4 days (e.g. up to 3 days or up to 4 days or up to 5 days or up to 6 days, up to 7 days, for example 2-3 days or 2-4 days or 2-5 days or 2-6 days or 2-7 days or 3-4 days or 3-5 or 3-6 days or 3-7 days). In preferred embodiments, the in vivo half-life is the in vivo half-life in a mammal, e.g. as assessed in an experimental animal such as a mouse, or in a human. The in vivo half-life may be the serum half-life or plasma half-life (or half-life in the circulation or in the bloodstream) in an experimental animal such as a mouse. In some embodiments, the in vivo half-life is the b-phase half-life.

In some preferred embodiments, proteins of the present invention, for example proteins comprising IgA antibodies (e.g. lgA1 or lgA2 antibodies), have an in vivo half-life that is increased (preferably significantly increased) in comparison to the in vivo half-life of a control protein.

In some embodiments, the in vivo half-life of a protein in accordance with the present invention (e.g. an IgA antibody or fragment thereof), is increased by at least 1.5-fold, at least 2-fold or at least 3-fold as compared to the in vivo half-life of a control protein (e.g. there is an up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold or up to 7-fold increase, such as a 1.5-fold to 3-fold increase, a 1.5-fold to 5-fold increase, a 1.5 fold to 7-fold increase, 2-fold to 3-fold increase, a 2-fold to 5- fold increase, a 2 fold to 7-fold increase, a 3-fold to 5-fold increase or a 3 fold to 7- fold increase), for example as compared to the in vivo half-life of a control protein (e.g. an IgA antibody or fragment thereof) that does not comprise a fragment of HSA (e.g. does not comprise a fragment of HSA in accordance with the invention).

In some embodiments, the in vivo half-life of a protein (e.g. an IgA antibody or fragment thereof) in accordance with the present invention is increased by at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or at least 600% (e.g. there is an up to 50% increase, up to 100% increase, up to 200% increase, up to 300% increase, up to 400% increase, up to 500% increase or up to 600% increase, such as 50% to 300% increase, a 50% to 400% increase, a 50% to 600% increase, 100% to 200% increase, a 100% to 300% increase, a 100% to 600% increase, a 200% to 300% increase, a 200% to 600% increase or a 300% to 600% increase, for example as compared to the in vivo half-life of a control protein (e.g. an IgA antibody or fragment thereof) that does not comprise a fragment of HSA (e.g. does not comprise a fragment of HSA in accordance with the invention).

Preferably, a control protein is substantially identical to (or identical to) the protein of the invention with which the comparison is being made, with the exception (or difference) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). In some embodiments, the protein of the invention comprises (or is) an IgA antibody and the control protein comprises (or consists of) the same IgA antibody with the sole difference (as compared to the protein of the invention) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). For example, the inventors have shown that a fragment of HSA in accordance with the invention can result in a significant increase in in vivo half-life compared to an equivalent (or the same) IgA molecule with no HSA fragment attached, or compared to an equivalent (or the same) IgA molecule with an equivalent non-mutated HSA fragment attached, e.g. a wild-type or non-mutated fragment, e.g. a wild-type or non-mutated domain III fragment as described herein.

In vivo half-life may be as determined by any suitable method and a skilled person will be familiar with suitable methods. In some embodiments, in vivo half-life is as determined in an experimental animal (e.g. an experimental mouse) and in some embodiments the discussion and values above in relation to in vivo half-life relate to in vivo half-life as determined in a mouse model (e.g. as described elsewhere herein).

In a preferred such method for determining in vivo half-life, mice are used to determine the half-life of proteins in accordance with the invention, e.g. mice which express human FcRn, but not mouse albumin and not mouse FcRn (such as homozygote Tg32 mice (B6.Cg-Fcgrftm1 Dcr Tg(FCGR7)32Dcr/DcrJ; The Jackson Laboratory)). Preferred mice are described in the Examples (e.g. Example 4). In some embodiments, mice (e.g. male mice), e.g. aged 7-9 weeks, and e.g. weighing between 17 and 27 g, receive e.g. equimolar amounts (e.g. 1.3 mg/kg for constructs containing IgA-DIII and 1 mg/kg for IgA-WT constructs) or 5 mg/kg of proteins in accordance with the invention (or control protein) diluted in an acceptable diluent (e.g. PBS) by intravenous injections. Blood samples (e.g. 25 pi) are drawn (e.g. from the retro-orbital sinus) at e.g. days 1 , 2, 3, 4, 5, 7, 10, 12, 16, 19, 23, 30 and 37 after injection. The blood samples are immediately mixed with an anti-coagulant (e.g. 1 mI 1% K3-EDTA to prevent coagulation) and then centrifuged (e.g. at 17,000 ^c g for 5 min at 4°C). Plasma is then isolated and diluted (e.g. 1 :10 in 50% glycerol/PBS solution) and then optionally stored (e.g. at -20°C) until analysis, e.g. by ELISA. Plasma samples may be diluted, e.g. 1 :400, in for example PBS/S/T, and e.g. 100 mI may be added per ELISA well. The ELISA can be used to establish the

concentration of the protein of the invention (or control protein) in the sample. The plasma concentration of proteins of the invention may be presented as percentage remaining in the circulation at time points post injection compared to the

concentration on day 1. Nonlinear regression analysis may be performed to fit a straight line through the data (e.g. using the Prism 7 software). The half-life (b- phase half-life) can be calculated using the formula: t1/2 = log 0.5/(log Ae/AO) ^c t, where t1/2 is the half-life of the protein of the invention, Ae is the amount of protein of the invention remaining, A0 is the amount of protein of the invention on day 1 and t is the elapsed time. A particularly preferred method for determining in vivo half- life is described in Example 4 herein.

Preferably, the proteins of the invention, for example proteins comprising IgA antibodies (e.g. lgA1 or lgA2 antibodies) have one or more, and preferably all of the functional properties described herein.

Preferably, the above described abilities and properties are observed at a measurable or significant level and more preferably at a statistically significant level, when compared to appropriate controls (e.g. control proteins). In any statistical analysis referred to herein, preferably the statistically significant difference over a relevant control or other comparative entity or measurement has a probability value of < 0.1 , preferably < 0.05. Appropriate methods of determining statistical significance are well known and documented in the art and any of these may be used.

Reference is made herein to certain“substantially homologous” sequences. The term "substantially homologous" as used herein in connection with an amino acid or nucleic acid sequence includes sequences having at least 65%, 70% or 75%, preferably at least 80%, and even more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid or nucleic acid sequence disclosed. Substantially homologous sequences of the invention thus include single or multiple base or amino acid alterations (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level preferred substantially homologous sequences contain up to 5, e.g. only 1 , 2, 3, 4 or 5, preferably 1 , 2 or 3, more preferably 1 or 2, altered amino acids. Said alterations can be with conservative or non-conservative amino acids. Preferably, said alterations are conservative amino acid substitutions.

The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the present invention that perform substantially the same function as the proteins or nucleic acid molecules of the invention in substantially the same way. Preferably, any substantially homologous protein should retain one or more (or all) of the functional capabilities of the starting protein.

Methods of carrying out manipulation of amino acids and protein domains (e.g. to generate substantially homologous sequences) are well known to a person skilled in the art. For example, said manipulations could conveniently be carried out by genetic engineering at the nucleic acid level wherein nucleic acid molecules encoding appropriate proteins and domains thereof are modified such that the amino acid sequence of the resulting expressed protein is in turn modified in the appropriate way.

Homology may be assessed by any convenient method. However, for determining the degree of homology between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W

(Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used together with BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1 , so that the highest order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment. Other methods that may be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Other methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Upton (Carillo and Upton, SIAM J. Applied Math., 48:1073, 1988) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.

Generally, computer programs will be employed for such calculations.

Programs that compare and align pairs of sequences, like ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990) and gapped BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387,

1984) are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998).

By way of providing a reference point, sequences according to the present invention having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, sequence identity etc. may be determined using the ALIGN program with default parameters (for instance available on Internet at the GENESTREAM network server, IGH, Montpellier, France).

Where the terms“comprise”,“comprises”,“has” or“having”, or other equivalent terms are used herein, then in some more specific embodiments these terms include the term“consists of” or“consists essentially of”, or other equivalent terms.

In another aspect, the present invention provides a protein comprising at least two polypeptide chains that each comprise an IgA antibody constant domain, wherein two or more of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn, wherein said fragment of human serum albumin is characterized by comprising

(i) a glutamine residue at the position corresponding to amino acid position 505 of SEQ ID NO:1;

In other embodiments, the fragment of human serum albumin is

characterized by further comprising (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1.

Discussion of various features of the proteins and antibodies of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention.

In another aspect, the present invention provides an IgA antibody (or fragment thereof), wherein one or more of the polypeptide chains of said antibody have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn, wherein said fragment of human serum albumin is characterized by comprising at least one mutant residue selected from the group consisting of (or comprising): (i) a mutant residue at the position corresponding to amino acid position 505 of SEQ ID NO:1 ;

(ii) a mutant residue at the position corresponding to amino acid position 527 of SEQ ID NO:1 ; and

(iii) a mutant residue at the position corresponding to amino acid position

573 of SEQ ID NO:1.

In this aspect, optionally (or alternatively) the fragment of human serum albumin is characterized by comprising (iv) a mutant residue at the position corresponding to amino acid position 547 of SEQ ID NO: 1.

In another aspect, the present invention provides an IgA antibody (or fragment thereof), wherein two or more of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn, wherein said fragment of human serum albumin is characterized by comprising

In other embodiments, the fragment of human serum albumin is

characterized by further comprising (iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO: 1.

In another aspect, the present invention provides a polypeptide (or a small polypeptide unit) able to interact with FcRn in a pH-dependent manner. Said polypeptide (or unit) can be defined as domain III (which is further defined elsewhere herein) comprising one or more, preferably all, of a glutamine residue at the position corresponding to position 505 of SEQ ID NO: 1 , a methionine residue at the position corresponding to position 527 of SEQ ID NO: 1 and a proline residue at the position corresponding to position 573 of SEQ ID NO: 1. In other embodiments, the domain III is characterized by further comprising an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1.

Discussion of various features of the proteins and HSA fragments of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention. One example of such a polypeptide (or polypeptide unit) can be represented by SEQ ID NO:2 or 8.

In another aspect, the present invention provides a fusion protein or a fusion polypeptide (or a protein comprising a fusion polypeptide) which comprises a polypeptide of interest (or protein of interest) fused to a polypeptide unit as defined above. In some embodiments, the polypeptide unit is attached to the C-terminal end of the polypeptide or protein of interest. Discussion of various features of the proteins and HSA fragments of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention. As

demonstrated herein, non-HSA proteins (e.g. IgAs, IgA fragments, IgEs, IgE fragments, interleukins, interferons etc.) fused to said unit may acquire the ability to interact with FcRn in a pH-dependent manner and thus display increased half-life in vivo. In some embodiments, the polypeptide of interest (or protein of interest) is selected from the group consisting of (or comprising) an IgA, an IgA fragment, an IgE, an IgE fragment, an interleukin and an interferon.

In another aspect, the present invention provides a method of extending the in vivo half-life (e.g. serum half-life) of a protein (preferably an IgA antibody or fragment thereof), said method comprising attaching a fragment of human serum albumin in accordance with the invention (or attaching a polypeptide unit as described above) to said protein (preferably to the C-terminal end of said protein). Discussion of various features of the proteins and HSA fragments of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention.

In another aspect, the present invention provides a method of transmucosal delivery (or increasing or improving transmucosal delivery, or increasing or improving transport across a mucosal barrier or membrane, e.g. the mucosal lung barrier or membrane) of a protein (preferably an IgA antibody or fragment thereof), said method comprising attaching a fragment of human serum albumin in accordance with the invention (or attaching a polypeptide unit as described above) to said protein (preferably to the C-terminal end of said protein). Such transmucosal delivery as enabled by a fragment of human serum albumin in accordance with the invention is shown in Example X. Thus, viewed alternatively this aspect can be seen as a method of transmucosal delivery of a protein of the invention. Such transmucosal delivery can also result in the transport or delivery (or increased or improved transport or delivery) of a protein of the invention into the serum or blood of a subject (e.g. resulting in increased levels, preferably significantly increased levels, in the serum or blood of the subject), which means that this mode of delivery has advantages.

In this embodiment said fragment of HSA as described herein is preferably attached to the C-terminal end of an IgA antibody constant domain as described elsewhere herein. In some embodiments, wherein said IgA antibody or said IgA antibody constant domain is an lgA1 or lgA2 antibody, the fragments of HSA as described herein can be attached to the light chain constant domains or heavy chain constant domains of said lgA1 or lgA2 antibody as described elsewhere herein. In some embodiments a heavy chain constant domain is used. In some embodiments said IgA antibody or said IgA antibody constant domain is an lgA2 antibody.

In some embodiments, the transmucosal delivery of a protein in accordance with the present invention (e.g. an IgA antibody or fragment thereof), is increased (preferably significantly increased) in comparison to the transmucosal delivery of a control protein.

In some embodiments the transmucosal delivery (e.g. transmucosal delivery to blood or serum) of a protein in accordance with the present invention (e.g. an IgA antibody or fragment thereof), is increased by at least 1.5-fold, at least 2-fold, or at least 3 fold, as compared to the transmucosal delivery of a control protein (e.g. there is an up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold or up to 7- fold increase, such as a 1.5-fold to 3-fold increase, a 1.5-fold to 5-fold increase, a 1.5 fold to 7-fold increase, 2-fold to 3-fold increase, a 2-fold to 5-fold increase, a 2 fold to 7-fold increase, a 3-fold to 5-fold increase or a 3 fold to 7-fold increase), for example as compared to the transmucosal delivery of a control protein (e.g. an IgA antibody or fragment thereof) that does not comprise a fragment of HSA (e.g. does not comprise a fragment of HSA in accordance with the invention).

In some embodiments, the transmucosal delivery of a protein (e.g. an IgA antibody or fragment thereof) in accordance with the present invention is increased by at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or at least 600% (e.g. there is an up to 50% increase, up to 100% increase, up to 200% increase, up to 300% increase, up to 400% increase, up to 500% increase or up to 600% increase, such as a 50% to 300% increase, a 50% to 400% increase, a 50% to 600% increase, a 100% to 200% increase, a 100% to 300% increase, a 100% to 600% increase, a 200% to 300% increase, a 200% to 600% increase or a 300% to 600% increase, for example as compared to the transmucosal delivery of a control protein (e.g. an IgA antibody or fragment thereof) that does not comprise a fragment of HSA (e.g. does not comprise a fragment of HSA in accordance with the invention).

Preferably, a control protein is substantially identical to (or identical to) the protein of the invention with which the comparison is being made, with the exception (or difference) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention). In some embodiments, the protein of the invention comprises (or is) an IgA antibody and the control protein comprises (or consists of) the same IgA antibody with the sole difference (as compared to the protein of the invention) being that the control protein does not comprise a fragment of human serum albumin (e.g. does not comprise a fragment of HSA in accordance with the invention, e.g. is a naked or wild type IgA).

Transmucosal delivery may be as determined by any suitable method and a skilled person will be familiar with suitable methods. For example, as such transmucosal delivery can result in the transport or delivery of the protein into the serum or blood, then levels of the protein of the invention in such fluids can readily be monitored. In some embodiments, transmucosal delivery is as determined in an experimental animal (e.g. an experimental mouse) and in some embodiments the discussion and values above in relation to transmucosal delivery relate to in vivo transmucosal delivery as determined in a mouse model (e.g. as described elsewhere herein, e.g. in Example 7). A particularly preferred method for determining transmucosal delivery is described in Example 7 herein.

Discussion of various features of the proteins and HSA fragments of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention.

As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. Therefore, a "protein", as used herein, means "at least a first protein". The operable limits and parameters of

combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present disclosure.

Nucleic acid molecules comprising nucleotide sequences that encode the proteins (e.g. antibodies or fragments thereof) of the present invention as defined herein or parts or fragments thereof, or nucleic acid molecules substantially homologous thereto, form yet further aspects of the invention.

Thus, in one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a protein of the invention.

In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide chain in accordance with the invention. Thus, the invention also provides a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide chain which comprises an IgA antibody constant domain having attached to the C-terminal end thereof a fragment of human serum albumin in accordance with the invention.

In another aspect, the present invention provides a set (or plurality) of nucleic acid molecules each comprising a nucleotide sequence, wherein said set of nucleic acid molecules together (or collectively) encode a protein (e.g. an antibody such as a whole antibody) in accordance with the invention. Such a set of nucleic acid molecules may be characterised in that when the set is expressed (i.e.

expressed together) (e.g. in a host cell) a protein (an entire protein e.g. a whole antibody) of the present invention is expressed and preferably assembled.

Nucleic acid molecules encoding proteins or polypeptides of the invention can be derived or produced by any appropriate method, e.g. by cloning or synthesis.

The term "nucleic acid sequence" or "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid molecules may be double stranded or single stranded. The nucleic acid molecules may be wholly or partially synthetic or recombinant.

The proteins and nucleic acid molecules of the invention are generally "isolated" or "purified". The term "isolated" or "purified" typically refers to a protein or nucleic acid that is substantially free of cellular material or other proteins (or other nucleic acids) from the source from which it is derived or produced. In some embodiments, particularly where the protein is to be administered to humans or animals, such isolated or purified proteins are substantially free of culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

A person skilled in the art will appreciate that the proteins of the invention (e.g. IgA antibodies or antibody fragments or immunoconjugates), may be prepared in any of several ways well known and described in the art, but are most preferably prepared using recombinant methods. Proteins of the present invention can be produced in vitro or in vivo.

Once nucleic acid molecules (or a set of nucleic acid molecules) encoding the proteins of the invention have been obtained, these fragments can be further manipulated by standard recombinant DNA techniques. For example, the nucleic acid molecules (or a set of nucleic acid molecules) encoding proteins of the invention are generally incorporated into one or more appropriate expression vectors (or into a set of expression vectors) in order to facilitate production of the proteins of the invention.

Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used. The expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the invention (or a set of nucleic acid molecules) and regulatory sequences selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner that allows expression of the nucleic acid.

The invention therefore provides an expression vector or construct (e.g. a recombinant expression vector or recombinant expression construct) containing a nucleic acid molecule of the invention and the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention. Also provided is a set of expression vectors (or a set of expression constructs) which, together (collectively), encode a protein of the invention. Such a set of expression vectors may be characterised in that when the set is expressed (i.e. expressed together) (e.g. in a host cell) a protein (an entire protein e.g. a whole antibody) of the present invention is expressed and preferably assembled.

Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes and are well known in the art. Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.

The recombinant expression vectors of the invention may also contain a selectable marker gene that facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.

The recombinant expression vectors may also contain genes that encode a fusion moiety that provides increased expression of the recombinant protein;

increased solubility of the recombinant protein; and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification (for example appropriate "tags" to enable purification and/or identification may be present, e.g., His tags or myc tags).

Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to encompass introduction of nucleic acid (e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming and transfecting host cells can be found in Sambrook et a!., 1989 (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other laboratory textbooks.

Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells. For example, proteins of the invention may be expressed in yeast cells or mammalian cells (e.g. HEK293E cells). In addition, proteins of the invention may be expressed in prokaryotic cells, such as Escherichia coli. Given the teachings provided herein, promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished.

Alternatively, the proteins of the invention may also be expressed in non human transgenic animals such as, rats, rabbits, sheep and pigs.

The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis.

N-terminal or C-terminal fusion proteins comprising the proteins of the invention conjugated to other molecules, such as proteins, may be prepared by fusing through recombinant techniques. The resultant fusion proteins contain an antibody or protein of the invention fused to the selected protein or marker protein, or tag protein as described herein. The antibodies and proteins of the invention may also be conjugated to other proteins by known techniques. For example, the proteins may be coupled using heterobifunctional thiol-containing linkers as described in WO 90/10457, N-succinimidyl-3-(2-pyridyldithio-proprionate) or N- succinimidyl-5 thioacetate.

A yet further aspect provides an expression construct or expression vector comprising one or more of the nucleic acid molecules of the invention. Preferably the expression constructs or vectors are recombinant. Preferably said constructs or vectors further comprise the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention. Sets of expression vectors are also provided (e.g. as discussed elsewhere herein).

A yet further aspect provides a host cell or virus comprising one or more expression constructs or expression vectors of the invention, or comprising a set of expression vectors (as discussed elsewhere herein). Also provided are host cells or viruses comprising one or more of the nucleic acid molecules of the invention or a set of nucleic acid molecules (as discussed elsewhere herein). A host cell (e.g. a mammalian host cell) or virus expressing a protein of the invention forms a yet further aspect.

A yet further aspect provides a cell (e.g. a host cell) that has been transduced with a protein or nucleic molecule or expression vector (or with a set of nucleic molecules or a set of expression vectors) in accordance with the invention.

A yet further aspect of the invention provides a method of producing (or manufacturing) a protein (e.g. an IgA antibody or fragment thereof) of the present invention comprising a step of culturing the host cells of the invention. Preferred methods comprise the steps of (i) culturing a host cell comprising one or more of the recombinant expression vectors or one or more of the nucleic acid molecules of the invention (or a set of nucleic acid molecules or a set of expression vectors of the invention) under conditions suitable for the expression of the encoded protein; and optionally (ii) isolating or obtaining the protein from the host cell or from the growth medium/supernatant. Such methods of production (or manufacture) may also comprise a step of purification of the protein product and/or formulating the protein product into a composition including at least one additional component, such as a pharmaceutically acceptable carrier or excipient.

Proteins in accordance with the invention comprise at least two polypeptide chains and as such all the polypeptides (polypeptide chains) are preferably expressed in the host cell, either from the same expression vector or from different expression vectors, so that the complete proteins can assemble in the host cell and be isolated or purified therefrom. In some cases, the HSA fragment in accordance with the invention is part of (i.e. expressed on) one or more of the polypeptides expressed in the host cell (e.g. as a fusion polypeptide). In some other cases, the HSA fragment in accordance with the invention may be attached subsequently (e.g. by cross-linking, as discussed elsewhere herein).

The invention also provides a range of conjugated proteins and fragments thereof in which the protein of the invention is operatively attached to at least one other agent (e.g. therapeutic agent). The term "immunoconjugate" is broadly used to define the operative association of the protein with another effective agent (e.g. therapeutic agent) and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical "conjugation". Recombinant fusion proteins are particularly contemplated. So long as the delivery or targeting agent is able to bind to the target and the agent is sufficiently functional upon delivery, the mode of attachment will be suitable.

In some embodiments, antibodies of the invention are used (e.g. used therapeutically) in their "naked" unconjugated form.

Compositions comprising at least a first protein of the invention or an immunoconjugate thereof constitute a further aspect of the present invention.

Formulations (compositions) comprising one or more proteins of the invention in admixture with a suitable diluent, carrier or excipient constitute a preferred embodiment of the present invention. Such formulations may be for pharmaceutical use and thus compositions of the invention are preferably pharmaceutically acceptable. Suitable diluents, excipients and carriers are known to the skilled man.

The compositions according to the invention may be presented, for example, in a form suitable for oral, nasal (intranasal), pulmonal (intrapulmonal), parenteral, intravenal, topical or rectal administration. In a preferred embodiment, compositions according to the invention are presented in a form suitable for intravenal

administration. In some embodiments, compositions according to the invention are presented in a form suitable for injection into a vein.

The active compounds defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, pulmonal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms.

Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as

p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions may then be filled into injection vials or ampoules.

Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers, either with an aerosol propellant or provided with means for manual compression.

The pharmaceutical compositions (formulations) of the present invention are preferably administered parenterally. Intravenous administration is preferred.

Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of the antibody in the form of a nasal spray or pulmonal spray (e.g. by inhalation), for example to enable transmucosal delivery of the proteins of the invention. As a still further option, the proteins of the invention can also be administered transdermally, e.g. from a patch, optionally an iontophoretic patch, or transmucosally, e.g. bucally. Indeed, as described elsewhere herein, the inventors have shown that transmucosal delivery (e.g. by nasal delivery or pulmonary delivery) is particularly efficient for the proteins of the invention. Thus, in certain embodiments of the invention administration in a form and by a route suitable for transmucosal delivery (e.g. nasal or pulmonary spray) is preferred. This feature of the proteins of the invention to allow needle-free administration of an active agent, e.g. IgA antibodies or fragments thereof, or other proteins as described herein (e.g. non-HSA proteins attached to the HSA fragments described herein), is extremely advantageous.

Suitable dosage units can be determined by a person skilled in the art.

The pharmaceutical compositions may additionally comprise further active ingredients (e.g. as described elsewhere herein) in the context of co-administration regimens or combined regimens.

A further aspect of the present invention provides the proteins (e.g. IgA antibodies or fragments thereof) of the invention for use in therapy. Therapy includes treatment or prophylaxis.

In some embodiments, the present invention provides proteins of the invention that comprise an antigen binding domain (e.g. IgA antibodies or fragments thereof) that bind to a given antigen for use in the treatment of a disease that is characterized by (or associated with) the expression of said antigen (e.g. unwanted or aberrant expression of said antigen), for example on the cell surface.

For example, in some embodiments, the invention provides proteins of the invention that comprise an antigen binding domain (e.g. IgA antibodies) that bind to a given cancer (or tumour) antigen (e.g. cancer specific antigen or cancer associated antigen or tumour specific antigen or tumour associated antigen) for use in the treatment of cancer (or a tumour).

In some embodiments, the present invention provides proteins of the invention that comprise an antigen binding domain (e.g. IgA antibodies) that bind to the protein Her2 for use in the treatment of a Her2 positive cancer, e.g. a Her2 positive breast cancer. In some such embodiments, the antigen binding domain (the VL and VH domains) is, or is based on, the antibody Trastuzumab.

In other embodiments, the present invention provides proteins of the invention for use in transmucosal delivery of said protein, for example the therapeutic uses as described herein are carried out by transmucosal delivery of a protein of the invention by an appropriate administration route, e.g. by intranasal or pulmonary administration.

In another aspect, the present invention provides immunoconjugates of the invention for use in therapy, e.g. therapies as discussed elsewhere herein.

The in vivo methods and uses as described herein (e.g. the therapeutic uses) are generally carried out in a mammal. Any mammal may be treated, for example humans and any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, cows and monkey. Preferably, however, the mammal is a human.

Thus, the term "animal" or "patient" as used herein includes any mammal, for example humans and any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, cows and monkey. Preferably, however, the animal or patient is a human subject. Thus, subjects or patients treated in accordance with the present invention will preferably be humans.

Alternatively viewed, the present invention provides a method of treating a disease that is characterized by (or associated with) expression of a given antigen (e.g. unwanted or aberrant expression of said antigen) which method comprises administering to a patient in need thereof a therapeutically effective amount of a protein of the invention that comprises an antigen binding domain (e.g. an IgA antibody) that binds to said antigen. Embodiments of the therapeutic uses of the invention described herein (e.g. in the treatment of cancer) apply, mutatis mutandis, to this aspect of the invention. For example, in other embodiments, the present invention provides a method of transmucosal delivery of the proteins of the invention which method comprises administering to a patient in need thereof a therapeutically effective amount of a protein of the invention. Alternatively, for example, the methods of treatment as described herein are carried out by transmucosal delivery of a protein of the invention by an appropriate administration route, e.g. by intranasal or pulmonary administration.

A therapeutically effective amount will be determined based on the clinical assessment and can be readily monitored. Preferred therapies are as described elsewhere herein.

Further alternatively viewed, the present invention provides the use of a protein of the invention in the manufacture of a medicament for use in therapy. In some embodiments, the present invention provides the use of a protein of the invention that comprises an antigen binding domain (e.g. an IgA antibody) that binds to a given antigen in the manufacture of a medicament for use in the treatment of a disease that is characterized by (or associated with) the expression of said antigen (e.g. unwanted or aberrant expression of said antigen), for example on the cell surface. Embodiments of the therapeutic uses of the invention described herein (e.g. in the treatment of cancer) apply, mutatis mutandis, to this aspect of the invention. For example, in other embodiments, the present invention provides the use of a protein of the invention in the manufacture of a medicament for use in a method of transmucosal delivery of said protein. Alternatively, for example, the uses as described herein are carried out by transmucosal delivery of a protein of the invention by an appropriate administration route, e.g. by intranasal or pulmonary administration. In another aspect, the present invention provides a method of inducing ADCC in a subject, which method comprises administering to a patient in need thereof an effective amount (e.g. a therapeutically effective amount) of a protein of the invention (e.g. an IgA antibody or fragment thereof).

The proteins and compositions and methods and uses of the present invention may be used in combination with other therapeutics. In terms of biological agents, preferably therapeutic agents, for use "in combination" with a protein in accordance with the present invention, the term "in combination" is succinctly used to cover a range of embodiments. The "in combination" terminology, unless otherwise specifically stated or made clear from the scientific terminology, thus applies to various formats of combined compositions, pharmaceuticals, cocktails, kits, methods, and first and second medical uses.

The "combined" embodiments of the invention thus include, for example, where a protein of the invention is a naked antibody (or fragment) and is used in combination with an agent or therapeutic agent that is not operatively attached thereto. In other "combined" embodiments of the invention, a protein of the invention is an immunoconjugate wherein the antibody (or fragment) is itself operatively associated or combined with the agent or therapeutic agent. The operative attachment includes all forms of direct and indirect attachment as described herein and known in the art.

The invention therefore provides compositions, pharmaceutical

compositions, therapeutic kits and medicinal cocktails comprising, optionally in at least a first composition or container, a biologically effective amount of at least a first protein of the invention (or immunoconjugate thereof) and a biologically effective amount of at least a second biological agent, component or system. The "at least a second biological agent, component or system" will often be a therapeutic agent, component or system, but it need not be.

Where therapeutic agents are included as the at least a second biological agent, component or system, such therapeutics will typically be those for use in connection with the treatment of one or more of the disorders defined above.

Thus, in certain embodiments "at least a second therapeutic agent" will be included in the therapeutic kit or cocktail. The term is chosen in reference to the protein of the invention being the first therapeutic agent. In certain embodiments of the present invention, the second therapeutic agent may be a radiotherapeutic agent, chemotherapeutic agent, anti-angiogenic agent, apoptosis-inducing agent, anti-tubulin drug, anti-cellular or cytotoxic agent, steroid, cytokine antagonist, cytokine expression inhibitor, chemokine antagonist, chemokine expression inhibitor, ATPase inhibitor, anti-inflammatory agent, signalling pathway inhibitor, checkpoint inhibitor, anti-cancer agent, other antibodies or coagulant.

In terms of compositions, kits and/or medicaments of the invention, the combined effective amounts of the therapeutic agents may be comprised within a single container or container means, or comprised within distinct containers or container means. The cocktails will generally be admixed together for combined use. Agents formulated for intravenous administration will often be preferred.

Imaging components may also be included. The kits may also comprise instructions for using the at least a first protein of the invention and the one or more other biological agents included.

Speaking generally, the at least a second therapeutic agent may be administered to the animal or patient substantially simultaneously with the protein of the invention; such as from a single pharmaceutical composition or from two pharmaceutical compositions administered closely together.

Alternatively, the at least a second therapeutic agent may be administered to the animal or patient at a time sequential to the administration of the protein of the invention. "At a time sequential", as used herein, means "staggered", such that the at least a second therapeutic agent is administered to the animal or patient at a time distinct to the administration of the protein of the invention. Generally, the two agents are administered at times effectively spaced apart to allow the two agents to exert their respective therapeutic effects, i.e. , they are administered at "biologically effective time intervals". The at least a second therapeutic agent may be administered to the animal or patient at a biologically effective time prior to the protein of the invention, or at a biologically effective time subsequent to the administration of the protein of the invention.

The invention further includes kits comprising one or more of the proteins of the invention (e.g. antibodies or fragments thereof or immunoconjugates) or compositions of the invention, or one or more of the nucleic acid molecules encoding the proteins of the invention, or one or more recombinant expression vectors comprising the nucleic acid sequences of the invention, or one or more host cells or viruses comprising the recombinant expression vectors or nucleic acid sequences of the invention. Preferably said kits are for use in the methods and uses as described herein, e.g. the therapeutic methods as described herein, or are for use in in vitro assays or methods e.g. as described herein. The protein in such kits may be a protein (e.g. antibody) conjugate as described elsewhere herein. Preferably said kits comprise instructions for use of the kit components. Preferably said kits are for treating diseases as described elsewhere herein, and optionally comprise instructions for use of the kit components to treat such diseases.

The proteins of the invention as defined herein may also be used as molecular tools for in vitro or in vivo applications and assays. Preferred proteins of the invention (e.g. antibodies) have an antigen binding site, and these can function as members of specific binding pairs and these molecules can be used in any assay where the particular binding pair member is required.

Thus, yet further aspects of the invention provide a reagent that comprises protein (e.g. an antibody) of the invention as defined herein and the use of such antibodies as molecular tools, for example in in vitro or in vivo assays.

LIST OF AMINO ACID SEQUENCES DISCLOSED HEREIN AND THEIR SEQUENCE IDENTIFIERS (SEQ ID NOs)

SEQ ID NO: 1 fwild-tvpe HSA)

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLWEVTEFAKTCVADESAENCDKSLHT LFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKAS SAKQRLKCASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI CENQDS I S SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPD YSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLI KQNCELFEQLGEYKFQNALL VRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTPVSDRVTKC CTESLWRRPCFSALEVDETYVPKEFNAETFTFHADI CTLSEKERQI KKQTALVELVKHKPKATKEQL KAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO: 2 (HSA fragment in accordance with the invention that was used in the Examples herein)

VEEPQNLI KQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPC

AEDYLSWLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAQTFTFHADI CTL

SEKERQI KKQMALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAALG

L

SEQ ID NO: 3 (short form of a HSA fragment in accordance with the invention)

EYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKT

PVSDRVTKCCTESLWRRPCFSALEVDETYVPKEFNAQTFTFHADI CTLSEKERQI KKQMALVELVKH

KPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL

SEQ ID NO: 4 (linker) GGSGGSGGSGGSGG

SEQ ID NO: 5 (wild-type sequence corresponding to SEQ ID NO:3, i.e. SEQ ID

NO:3 without mutations)

EYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKT PVSDRVTKCCTESLWRRPCFSALEVDETYVPKEFNAETFTFHADI CTLSEKERQI KKQTALVELVKH KPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO: 6 (an example of an lqA1 tailpiece amino acid sequence)

PTHVNVSVVMAEVDGTCY

SEQ ID NO: 7 (Exemplary lqA1 heavy chain constant region amino acid sequence. The tailpiece sequence is underlined: the P in bold is (corresponds to) position 440 by Bur numbering; the G in bold is (corresponds to) position 453 by Bur numbering)

ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTC HVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAV QGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPK DVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNV SVVMAEVDGTCY

SEQ ID NO: 8 (HSA fragment in accordance with the invention that was used in the Examples herein)

VEEPQNLI KQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPE

AKRMPCAEDYLSWLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAQTFTFH ADI CTLSEKERQI KKQMALVELVKHKPKATKEQLKAAMDDFAAFVEKCCKADDKETCFAEEGPKLVAA SQAALGL

SEQ ID NO: 9 (short form of a HSA fragment in accordance with the invention)

EYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKT PVSDRVTKCCTESLWRRPCFSALEVDETYVPKEFNAQTFTFHADI CTLSEKERQI KKQMALVELVKH KPKATKEQLKAAMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL

SEQ ID NO:1Q (wild type HSA fragment corresponding to SEQ ID NO:2, i.e. SEQ ID NO:2 without mutations, that was used as a control/comparator in the

Examples herein) VEEPQNLI KQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPC

AEDYLSWLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADI CTL

SEKERQI KKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALG

L

The invention will now be further described in the following non-limiting Examples with reference to the following drawings.

In the Figures and Examples herein, lgA-HC(DI 11)2 or lgA-HC(DIII-QMP)2 (sometimes referred to as hlgA-HC(DIII)2 or hlgA-HC(DIII-QMP)2) represents an IgA wherein both heavy chains are truncated at position 453 (According to the myeloma lgA1 Bur Numbering scheme, see Science. 1976 Sep 10; 193(4257): 1017-20 by Liu et al) to remove the tailpiece; and genetically fused to Dill QMP via a linker represented by SEQ ID NO:4. Dill QMP is a HSA fragment represented by SEQ ID NO: 2. One example of lgA-HC(DI 11)₂ or lgA-HC(DIII-QMP)₂ is lgA1-HC(DI ll)₂ or lgA1-HC(DIII-QMP)₂.

In the Figures and Examples herein, lgA-LC(DIII)2 or lgA-LC(DIII-QMP)2 (sometimes referred to as hlgA-LC(DI 11)2 or hlgA-LC(DIII-QMP)2) represents an IgA wherein both heavy chains are truncated at position 453 (According to the myeloma lgA1 Bur Numbering scheme, see Science. 1976 Sep 10; 193(4257): 1017-20 by Liu et al) to remove the tailpiece; and wherein both light chains are genetically fused to Dill QMP via a linker represented by SEQ ID NO:4. Dill QMP is a HSA fragment represented by SEQ ID NO: 2. One example of IgA- LC(DI 11)2 or lgA-LC(DIII-QMP)2 is lgA1-LC(DIII)₂ or lgA1-LC(DIII-QMP)₂.

In the Figures and Examples herein, lgA-HC(DIII-WT)2 represents an IgA wherein both heavy chains are truncated at position 453 (According to the myeloma lgA1 Bur Numbering scheme, see Science. 1976 Sep 10; 193(4257): 1017-20 by Liu et al) to remove the tailpiece; and wherein both heavy chains (HC) are genetically fused to wild type Dill (DIII-WT) via a linker represented by SEQ ID NO:4. DIII-WT is a HSA fragment represented by SEQ ID NO: 10. One example of lgA-HC(DIII-WT)2 is lgA1-HC(DIII-WT)₂ or lgA2-HC(DIII-WT)₂.

In the Figures and Examples herein, lgA-LC(DIII-WT)2 represents an IgA wherein both heavy chains are truncated at position 453 (According to the myeloma lgA1 Bur Numbering scheme, see Science. 1976 Sep 10; 193(4257): 1017-20 by Liu et al) to remove the tailpiece; and wherein both light chains (LC) are genetically fused to wild type Dill (DIII-WT) via a linker represented by SEQ ID NO:4. DIII-WT is a HSA fragment represented by SEQ ID NO: 10. One example of lgA-LC(DIII-WT)2 is lgA1-LC(DIII-WT)₂ or lgA2-LC(DIII-WT)₂.

In the Figures and Examples herein, IgA-WT (e.g. lgA1-WT or lgA2-WT) represents an IgA (e.g. lgA1 or lgA2, as appropriate) wherein both heavy chains are truncated at position 453 (According to the myeloma lgA1 Bur Numbering scheme, see Science. 1976 Sep 10; 193(4257): 1017-20 by Liu et al) to remove the tailpiece.

In the Figures and Examples herein, lgA1-LC(DIII-QUAD)₂ or lgA2-LC(DI 11- QUAD)₂ represents an lgA1 or lgA2 wherein both heavy chains are truncated at position 453 (According to the myeloma lgA1 Bur Numbering scheme, see Science. 1976 Sep 10; 193(4257): 1017-20 by Liu et al) to remove the tailpiece; and wherein both light chains are genetically fused to Dill QUAD via a linker represented by SEQ I D NO:4. Dil l QUAD is a HSA fragment represented by SEQ ID NO: 8.

In the Figures and Examples herein, lgA1-HC(DIII-QUAD)₂ or lgA2-HC(DIII-QUAD)₂ represents an lgA1 or lgA2 wherein both heavy chains are truncated at position 453 (According to the myeloma lgA1 Bur Numbering scheme, see Science. 1976 Sep 10; 193(4257): 1017-20 by Liu et al) to remove the tailpiece and genetically fused to Dil l QUAD via a linker represented by SEQ ID NO:4. Dill QUAD is a HSA fragment represented by SEQ ID NO: 8.

Figure 1 : Schematic depiction of wild type IgA (IgA-WT), lgA-HC(DIII-WT)₂, IgA- LC(DIII-WT)₂, lgA-HC(DIII-QMP)₂ (previously referred to as lgA-HC(DIII)₂) and IgA- LC(DI I l-QMP)₂ (previously referred to as IgA- LC(DI 11)₂.

Figure 2A: Graphs showing ADCC activity of wild type lgA1 (lgA1-WT), lgA1- HC(DIII-QMP)₂ (previously referred to as lgA1-HC(DIII)₂) and lgA1-LC(DIII-QMP)₂ (previously referred to as lgA1 -LC(DII l)₂). Of course, the wild type lgA1 does not comprise any HSA fragment.

Figure 2B: Graphs showing ADCC activity of wild type lgA2 (lgA2-WT), lgA2- HC(DIII-QMP)₂ (previously referred to as lgA2-HC(DI ll)₂) and lgA2-LC(DIII-QMP)₂ (previously referred to as I g A2- LC( D 111 )₂) . Of course, the wild type lgA2 does not comprise any HSA fragment.

Figure 3A: ELISA data showing that lgA1-HC(DIII-QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂ (previously referred to as lgA1- HC(DIII)₂, lgA1-LC(DII l)₂, lgA2-HC(DI 11)₂ and lgA2-LC(DI 11)₂, respectively) all bind to recombinant human FcRn at pH 5.5. Neither lgA1-WT nor lgA2-WT display binding at pH 5.5. Figure 3B: ELISA data showing that none of lgA1-HC(DIII-QMP)₂, lgA1-LC(DI 11- QMP)₂, lgA2-HC(DIII-QMP)₂ or lgA2-LC(DIII-QMP)₂, (previously referred to as lgA1- HC(DIII)₂, lgA1-LC(DIII)₂, lgA2-HC(DI 11)₂ or lgA2-LC(DI 11)₂, respectively) bind to recombinant human FcRn at pH 7.4. Neither lgA1-WT nor lgA2-WT display binding at pH 7.4.

Figure 4: HERA (human endothelial cell-based recycling assay). The recycling of lgA1-HC(DIII-QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DI 11- QMP)₂ (previously referred to as lgA1-HC(DIII)₂, lgA1-LC(DIII)₂, lgA2-HC(DI 11)₂ and lgA2-LC(DI ll)₂, respectively) is improved over lgA1-WT and lgA2-WT.

Figure 5A: Shows the in vivo half-life of wild type lgA1 (lgA1-WT), lgA1-HC(DIII- QMP)₂ (previously referred to as lgA1-HC(DI ll)₂) and lgA1-LC(DIII-QMP)₂

(previously referred to as lgA1-LC(DIII)₂) in a Tg32 mouse model.

Figure 5B: Shows the in vivo half-life of wild type lgA2 (lgA2-WT), lgA2-HC(DI II- QMP)₂ (previously referred to as lgA2-HC(DI ll)₂) and lgA2-LC(DIII-QMP)₂

(previously referred to as lgA2-LC(DIII)₂) in a Tg32 mouse model.

Figure 6: Shows the in vivo half-life of wild type lgA1 (lgA1-WT), lgA1-HC(DIII- WT)₂,lgA1-LC(DIII-WT)₂, lgA1-HC(DIII-QMP)₂ and lgA1-LC(DIII-QMP)₂ in a Tg32 mouse model. DIII-QMP but not DIII-WT extend the half-life of IgA.

Figure 7: ELISA data showing that lgA1-LC(DIII-QUAD)₂ and lgA1-LC(DIII-QMP)₂, bind to recombinant human FcRn at pH 5.5 but not (or only weakly or at low affinity, DII I-QUAD) at pH 7.4, whereas lgA1-LC(DIII-WT)₂ does not display binding at pH 5.5 or pH 7.4.

Figure 8A: Shows the in vivo transmucosal delivery of wild type lgA2 (lgA2-WT) and lgA2-HC(DIII-QMP)₂ in a Tg32 mouse model. lgA2-HC(DIII-QMP)₂ reaches the blood in 3-times higher levels than lgA2-WT. Figure 8B: Shows the administration regimen.

Examples:

Example 1 - Protein Production and Preparation

cDNA encoding the variable regions of the HC (heavy chain) and LC (light chain) derived from trastuzumab in frame of human lgA1 and lgA2 were obtained in expression vectors with the following backbones: pEE14.4-kappaLC, pEE14.4-lgA1 and pEE14.4-lgA2(m1), as previously reported (Meyer et al., 2016, MAbs, 8(1), pages 87-98). The encoded antibodies are IgA antibodies having the heavy chain variable region and the light chain variable region of Trastuzumab. Trastuzumab is a monoclonal antibody that binds to HER2 (Her2). cDNA encoding an engineered human albumin fragment represented by SEQ ID NO:2 was sub-cloned in frame with the HC or LC via a segment encoding a glycine-serine linker ((GGS)4GG). The segments encoding the tailpiece were removed by truncating the heavy chain at position 453 (According to the myeloma lgA1 Bur Numbering scheme, see Science. 1976 Sep 10; 193(4257): 1017-20 by Liu et al). Vectors encoding lgA1-HC(DI II- QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂

(previously and also referred to as lgA1 -HC(DI 11)₂, lgA1-LC(DIII)₂, lgA2-HC(DI 11)₂ and lgA2-LC(DI 11)₂, respectively) were transiently co-transfected into adherent HEK293E cells using Lipofectamine2000™. Growth medium was harvested and replaced every second day for 2 weeks prior to purification using a

CaptureSelectTM IgA affinity matrix (Life Technologies) packed column (Atoll), as described by the manufacturer. The collected fusion proteins were up-concentrated and buffer-changed to phosphate-buffered saline (PBS) (Sigma-Aldrich) using Amicon Ultra-15 ml 50K columns (Millipore) prior to size exclusion chromatography using a Superdex 200 increase 10/300GL column (GE Healthcare) coupled to an AKTA Avant instrument (GE Healthcare). Monomeric fractions were collected and up-concentrated by Amicon Ultra-0.5 ml 100K columns (Millipore) and analyzed on a Superdex 200 increase 3.2/300 column (GE Healthcare) coupled to an AKTA FPLC instrument (GE Healthcare).

Example 2

ADCC (antibody-dependent cellular cytotoxicity) assay

Materials and Methods

ADCC was measured using a chromium-release assay, as previously described (Dechant et al., 2007, J. Immunol., 179(5), pages 2936-2943; Meyer et al., 2016, MAbs, 8(1), pages 87-98). Briefly, SKBR3 cells (a human breast cancer cell line that expresses Her2) labeled with 100 pCi 51 chromium (per 10⁶ cells) were added to titrated amounts of IgA (hlgA) variants (i.e. lgA1-HC(DIII)₂, lgA1-LC(DII l)₂, lgA2- HC(DIII)₂ and lgA2-LC(DI ll)₂, as described in Example 1) and lgA1-WT and lgA2- WT in round-bottom microtiter plates (Corning Inc.). PMNs (polymorphonuclear leukocytes) were isolated from blood from healthy donors (MiniDonorDienst UMC Utrecht) by ficoll-histopaque density gradient and added with an effector-to-target (E:T) ratio of 40: 1 in a final assay volume of 200 mI/well. The cells were incubated for 4 hours at 37°C/5% C0₂. Subsequently, the supernatant was transferred to a LumaPlate (Perkin Elmer) and counted in a liquid scintillation counter (MicroBeta; Perkin Elmer). Lysis was calculated using the following formula: % lysis = ((counts of sample - minimal release)/(maximum release - minimum release))*100. Culture medium was used to determine minimal release and 3% TritonX-100 (Roche Diagnostics) to determine maximum release.

Results

The data in Figure 2A and Figure 2B shows no or only minor differences were observed in ADCC activity when titrated amounts of lgA1-WT, lgA2-WT, and lgA1- HC(DMI-QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂ (previously referred to as lgA1-HC(DIII)₂, lgA1-LC(DIII)₂, lgA2-HC(DII l)₂ and lgA2- LC(DIII)₂, respectively) were added to freshly isolated PMNs and Her2-expressing SKBR3 cells.

ELISA Assays

Materials and Methods

Antigen (Her2) binding

Ninety-six-well ELISA plates (Costar) were coated with 1.0 mg/ml recombinant human Her2 (Nordic BioSite) diluted in PBS, and incubated ON at 4°C. Plates were blocked with PBS containing 4% skimmed milk (S) (Sigma-Aldrich) for 1 hour at room temperature (RT) and washed 4 times with PBS containing 0.05% Tween 20 (T) (Sigma-Aldrich). 100 mI of titrated amounts of purified anti-Her2 IgA (hlgA) variants, diluted in PBS/S/T, were added to the plates and incubated for 1 hour on a shaker at RT. The plates were washed as before, and 100 mI of an alkaline phosphatase (ALP) conjugated anti-human IgA (a-chain specific) antibody produced in goat (Sigma-Aldrich, USA) was added (1 :2000) and incubated for 1 hour at RT. The plates were washed as above, and bound proteins were visualized by adding 100 mI of ALP substrate (1 mg/ml phosphate in diethanolamine buffer) (Sigma- Aldrich). Absorbance was measured at 405 nm with a Sunrise spectrophotometer (TECAN).

hFcaR binding

Ninety-six-well ELISA plates (Costar) were coated and incubated with IgA (hlgA) variants -Dill as above followed by adding 100 pi of His-tagged human FcaR (Nordic BioSite), at a concentration of 2 pg/ml and incubated 1 hour at RT. The plates were washed before 100 pi of an ALP conjugated anti-His-tag antibody (Abeam) was added (1 :5000) and incubated for 1 hour at RT. The plates were washed and bound proteins were visualized by adding 100 mI of ALP substrate. hFcRn binding

Ninety-six-well ELISA plates (Costar) were coated as above followed by adding of IgA (hlgA) variants. Wells were washed either with PBS/T pH 5.5 or PBS/T pH 7.4 before 2 pg/ml of GST-fused soluble recombinant human FcRn, diluted in PBS/S/T pH 5.5 or in pH 7.4, was added and incubated for 1 hour at RT. The plates were washed at either pH 5.5 or pH 7.4, and bound receptors were detected using horseradish peroxidase (HRP) conjugated goat anti-GST antibody (GE Healthcare), diluted (1 :8000) in PBS/S/T with pH 5.5 or 7.4, and incubated 1 hour at RT. The plates were washed as before and 100 pi of 3,3',5,5'-tetramethybenzidine (TMB) substrate solution (Merck Millipore) was then added to the wells. Absorbance was measured at 620 nm with a Sunrise spectrophotometer (TECAN). The reaction was stopped by adding 50 pi 1M HCI, and then measured at 450 nm.

The recombinant human FcRn was produced and purified as follows: A vector encoding soluble recombinant GST-tagged human FcRn (pcDNA3-GST-h^2- microglobulin) was transiently transfected into HEK293E cells, and secreted receptor was purified using a GST rap FF column, all as described previously (Berntzen et ai, J. Immunol. Methods, 2005, 298(1-2), pages 93-104). Production of recombinant His-tagged mouse and human FcRn was done by using a Baculovirus expression vector system (Firan et ai, Int. Immunol., 2001 , 13(8), pages 993-1002). The receptors were purified using a HisTrap HP column supplied with Ni²⁺ ions (GE Healthcare), as previously reported (Sand et ai, J. Biol. Chem., 2014, 289(24), pages 17228-17239).

Results

To address whether the presence of Dill QMP affects binding to cognate antigen of IgAs, titrated amounts of the lgA1-WT, lgA2-WT, and lgA1-HC(DIII-QMP)₂, lgA1- LC(DIM-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂ (previously referred to as lgA1-HC(DII l)₂, lgA1-LC(DIII)₂, lgA2-HC(DII l)₂ and lgA2-LC(DIII)₂ respectively) were compared for binding to recombinant Her2 coated in ELISA plates. Equal binding responses were detected (data not shown). An analogous set-up was used to measure binding to recombinant human FcaRI, and we found that lgA1-WT, lgA2-WT, and lgA1-HC(DIII-QMP)₂, lgA1-LC(DI II- QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂ (previously referred to as lgA1 -HC(DII l)₂, lgA1-LC(DIII)₂, lgA2-HC(DI 11)₂ and lgA2-LC(DIII)₂ respectively) bound to the receptor (data not shown).

Binding to recombinant human FcRn was tested, and we confirm that lgA1-HC(DIII- QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂

(previously referred to as lgA1 -HC(DI 11)₂, lgA1-LC(DI ll)₂, lgA2-HC(DI 11)₂ and lgA2- LC(DIII)₂ respectively) bind FcRn in a strictly pH-dependent fashion, with strong binding at acidic pH and no binding at neutral pH. Neither lgA1-WT nor lgA2-WT displayed significant binding (Figure 3A and 3B).

Example 3 - HERA assay (human endothelial cell-based recycling assay) Materials and Methods

A human endothelial recycling assay were performed, as previously reported (Grevys et al., 2018, Nat. Commun., 9(1): 621). Briefly, 7.5 10⁴ HMEC1 cells stably expressing HA-hFcRn-EGFP were seeded into 24-well plates per well (Costar) and cultured for one day in growth medium. The cells were washed and starved for 1 h in Hank’s balanced salt solution (HBSS) (Life Technologies). 400 nM of lgA1-WT, lgA2-WT, and lgA1-HC(DIII-QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DI 11- QMP)₂ and lgA2-LC(DIII-QMP)₂ (previously referred to as lgA1-HC(DI ll)₂, lgA1- LC(DIII)₂, lgA2-HC(DII l)₂ and lgA2-LC(DIII)₂ respectively) were diluted in 250 mI HBSS (pH 7.4) and added to the cells followed by 4 h incubation. The medium was removed and the cells were washed with ice cold HBSS (pH 7.4) before fresh warm HBSS (pH 7.4) or growth medium without FCS and supplemented with MEM non- essential amino acids (ThermoFisher) was added. Samples of the growth medium were collected the next day. The amounts of lgA1-WT, lgA2-WT, and lgA1-HC(DIII- QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂

(previously referred to as lgA1 -HC(DI 11)₂, lgA1-LC(DI ll)₂, lgA2-HC(DI 11)₂ and lgA2- LC(DIII)₂ respectively) variants in the growth medium were quantified using the antigen (Her2) ELISA as described above.

Results Using the human endothelial cell-based recycling assay, it was found that lgA1- HC(DIM-QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂ (previously referred to as lgA1 -HC(DI 11)₂, lgA1-LC(DI ll)₂, lgA2-HC(DI 11)₂ and lgA2- LC(DIII)₂ respectively) were recycled and rescued from intracellular degradation, whereas the unfused IgA (WT) was degraded (Figure 4). lgA1-LC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂ were rescued most efficiently.

Example 4 - In vivo half-life studies

Materials and Methods

Homozygote Tg32 mice (B6.Cg-Fcg/ftm1 Dcr Tg(FCGR7)32Dcr/DcrJ; The Jackson Laboratory), which express human FcRn, but not mouse albumin and not mouse FcRn, were used to determine the half-life of IgA variants. Male mice, age 7-9 weeks, weighing between 17 and 27 g (5 mice per group), received equimolar amounts (1.3 mg/kg for the constructs containing IgA-DIII and 1 mg/kg for the IgA- WT constructs) of IgA variants diluted in PBS by intravenous injections. Blood samples (25 mI) were drawn from the retro-orbital sinus at days 1 , 2, 3, 4, 5, 7, 10,

12, 16, 19, 23, 30 and 37 after injection. The blood samples were immediately mixed with 1 mI 1% K3-EDTA to prevent coagulation and then centrifuged at 17,000 x g for 5 min at 4°C. Plasma was isolated and diluted 1 :10 in 50%

glycerol/PBS solution and then stored at -20°C until analysis by ELISA. Plasma samples were diluted 1 :400 in PBS/S/T and 100 mI was added per ELISA well. The study was carried out at the Jackson Laboratory (JAX Services, Bar Harbor, ME). The experiments and procedures used were approved by the Animal Care and Use Committee at The Jackson Laboratory and performed in accordance with the approved guidelines and regulations.

Measurements of half-life of HSA variants are critically dependent on the right animal model for testing as cross-species differences in binding of albumin to FcRn limit the use of conventional animal models. The Tg32 mouse model is genetically modified to lack the mouse (murine) FcRn and instead expresses human FcRn under the control of the native human FcRn promoter. Murine FcRn is known to bind murine serum albumin (MSA) stronger than HSA. As a consequence, the binding of HSA is hard to assess due to competition with native MSA binding to mFcRn.

Accordingly, the Tg32 mouse model is much more reliable for predicting half-life of HSA variants in human circulation than other models (see Nilsen et al, Current Opinion in Chemical Engineering 2018, 19:68-76). Half-life calculation

The plasma concentration of IgA variants is presented as percentage remaining in the circulation at time points post injection compared to the concentration on day 1. Nonlinear regression analysis was performed to fit a straight line through the data using the Prism 7 software, and the b-phase half-life was calculated using the formula: t1/2 = log 0.5/(log Ae/AO) ^c t, where t1/2 is the half-life of the IgA variants, Ae is the amount of IgA remaining, A0 is the amount of IgA on day 1 and t is the elapsed time.

Results

The half-life of IgM-WT, lgA2-WT, and lgA1-HC(DIII-QMP)₂, lgA1-LC(DIII-QMP)₂, lgA2-HC(DIII-QMP)₂ and lgA2-LC(DIII-QMP)₂ (previously and also referred to as lgA1-HC(DIII)₂, lgA1-LC(DIII)₂, lgA2-HC(DI 11)₂ and lgA2-LC(DIII)₂ respectively) was determined in human FcRn transgenic mice (Roopenian et al., 2015, MAbs, 7(2) pages 344-351) and it was found that fusion of Dill QMP (SEQ ID NO: 2) to the light or to the heavy chains of lgA1 extends the half-life by more than 3-fold (~3-4 -fold) compared to lgA1-WT (Figure 5A). The lgA1-WT was rapidly cleared from the bloodstream and had a half-life of ~1 day in the mice. It was also found that fusion of Dill QMP (SEQ ID NO:2) to the light or to the heavy chains of lgA2 extends the half-life by more than 2-fold compared to lgA2-WT (Figure 5B). The lgA2-WT was rapidly cleared from the bloodstream and had a half-life of ~1 day in the mice. For the lgA2 molecules, the effect was more pronounced for lgA2-HC(DI ll)₂ than for lgA2-LC(DI ll)₂. These results are represented in the following table:

Tl /2p SD

IgAl -WT 1 0 ± 0.1

!gA1 -LC(DIH)₂ 3.9 ± 0.3

lgA2-WT 0.9 ± 0.1

IgA2-HC(D!II)₂ 3.3 ± 0.2

lgA2-LC(DIII), 2.1 ± 0.1 These data show that Dill QMP can be used to increase the serum persistence of IgA. Thus, these data demonstrate that fusion of HSA fragments in accordance with the invention are useful for extending the in vivo half-life of IgA antibodies.

Example 5 - In vivo half-life studies

Materials and Methods

The in vivo half-life studies and half-life calculation were carried out as described in Example 4.

Results

The half-life of wild type lgA1 (lgA1-WT), lgA1-HC(DIII-WT)₂,lgA1-LC(DIII-WT)₂, lgA1-HC(DIII-QMP)₂ and lgA1-LC(DIII-QMP)₂ was determined and it was found that fusion of Dill QMP (SEQ ID NO: 2) to the light or to the heavy chains of lgA1 extends the half-life by more than 3-fold (~3.5-5 -fold) compared to lgA1-WT, lgA1- HC(DIII-WT)2 and lgA1-LC(DIII-WT)₂ (Figure 6). The effect was more pronounced for lgA1-LC(DIII-QMP)₂ than for lgA1-HC(DIII-QMP)₂. The lgA1-WT was rapidly cleared from the bloodstream and had a half-life of ~1 day in the mice. The same clearance was observed for lgA1 fused the non-mutated Dill fragments lgA1- HC(DIII-WT)₂ and lgA1-LC(DIII-WT)_2.

Thus, this data shows an in vivo half-life comparison of lgA1 fused to DIII-WT and DIII-QMP in hFcRn Tg AlbKO mice. Fusion of DIII-WT does not extend the half-life and DIII-WT fused lgA1 shows similar short half-life as that of non-fused parental lgA1-WT. However, DIII-QMP fused to lgA1 extends the half-life by almost 3.5-5 fold compared to the parental lgA1-WT. This data shows that DIII-QMP but not DIII- WT extends the half-life of IgA.

Example 6 - FcRn binding studies

Materials and Methods

The hFcRn binding studies were carried out as described in Example 2 (ELISA assays).

The constructs tested were lgA1-LC(DIII-QUAD)2, lgA1-LC(DIII-QMP)2, and lgA1- LC(DIII-WT)2 The lgA1 light chain fused to DIN-QUAD is the same construct as described for the lgA1 light chain fused to DIII-QMP but with an additional Alanine (A) mutation at the position corresponding to the amino acid position 547 of SEQ ID NO:1 (in this experiment the HSA fragment used is shown in SEQ ID NO:8). Results

The results are shown in Figure 7 where pH-dependent FcRn binding in ELISA (pH 5.5 (left) and 7.4 (right)) comparing IgM fused to DIII-WT, DIII-QMP and DIN-QUAD (E505Q/T527M/V547A/K573P) is shown. lgA1 harboring the DIII-WT does not show any binding to FcRn at pH 5.5 or pH 7.4, whereas lgA1 fused to DIII-QMP bind strongly to FcRn at pH 5.5 but not at pH 7.4. lgA1 fused to DIII-QUAD binds strongly to FcRn at pH 5.5 and with some affinity (weak binding or low affinity) at pH 7.4. lgA1-fused to DIII-QUAD (SEQ ID NO:8) binds stronger to human hFcRn at pH 5.5 than the DIII-QMP version.

Example 7 - In vivo transmucosal delivery studies

Materials and Methods

Pulmonary delivery studies

Homozygous Tg32 albKO mice (B6.Cg-Alb^em12Mvw Fcgrt^{tm1 Dcr}

Tg(FCGRT)32Dcr/MvwJ, The Jackson Laboratory) were used for intranasal (i.n.) delivery studies. The mice were pre-loaded with human albumin (250 mg kg^-1) 48 hours before intranasal delivery. A mix of female and male mice (Tg32 albKO, 6-8 weeks, 5 mice/group) were anesthetized by intraperitonal injection of Zoletil mix. When sedated, 10mI of 2 mg kg ¹ non-fused (naked) lgA2-WT or lgA2-HC(DI 11- QMP)2 diluted in PBS were given to each nostril followed by breathing in while lying on their backs. Blood was collected by puncture of the saphenous vein and collected using heparinized micro capillary pipettes after 4 and 24 hours. Sera from mice was isolated by centrifugations for 5 minutes at 17000 x g at 4°C and stored at -20 after isolation. Quantification of the amount of the variants in sera was done by ELISA.

Results

Figure 8 shows pulmonary delivery of lgA2-WT and lgA2 heavy chains fused to DIII- QMP, where the amounts of the variants in sera were detected after 4 and 24 hours post intranasal administration. It is shown that lgA2 that has DIII-QMP fused is more efficiently transported across the mucosal lung barrier and reaches the blood in 3-times higher levels after 24 hours than non-fused (naked) lgA2-WT.

Claims

1. A protein comprising at least two polypeptide chains that each comprise an IgA antibody constant domain, wherein two or more of said polypeptide chains have attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn at endosomal pH,

wherein said fragment of human serum albumin is characterized by comprising

2. The protein of claim 1 , wherein said fragment of human serum albumin is further characterized by comprising

(iv) an alanine residue at the position corresponding to amino acid position 547 of SEQ ID NO:1.

3. The protein of claim 1 or claim 2, wherein said fragment of human serum albumin that is capable of binding to FcRn consists of domain III of human serum albumin, wherein domain III of human serum albumin is a polypeptide that consists of 180 to 300 amino acid residues and comprises a sequence with at least 95% sequence identity to SEQ ID NO:5.

4. The protein of any one of claims 1 to 3, wherein said fragment of human serum albumin that is capable of binding to FcRn comprises or consists of SEQ ID NO:2.

5. The protein of any one of claims 1 to 3, wherein said wherein said fragment of human serum albumin that is capable of binding to FcRn comprises or consists of SEQ ID NO:3.

6. The protein of any one of claims 1 to 5, wherein said fragment of human serum albumin that is capable of binding to FcRn is directly attached to the C- terminal end of said two or more of said polypeptide chains.

7. The protein of any one of claims 1 to 5, wherein said fragment of human serum albumin that is capable of binding to FcRn is indirectly attached to the C- terminal end of said two or more of said polypeptide chains.

8. The protein of claim 7, wherein said fragment of human serum albumin that is capable of binding to FcRn is indirectly attached to the C-terminal end of said two or more of said polypeptide chains via a linker.

9. The protein of any one of claims 1 to 8, wherein said two or more polypeptide chains having attached to the C-terminal end thereof a fragment of human serum albumin that is capable of binding to FcRn are fusion polypeptides, wherein each fusion polypeptide comprises a polypeptide chain comprising an IgA antibody constant domain and said fragment of human serum albumin that is capable of binding to FcRn, said fragment of human serum albumin that is capable of binding to FcRn being fused to the C-terminal end of said polypeptide chain comprising an IgA antibody constant domain, optionally via a linker.

10. The protein of any one of claims 1 to 9, wherein said protein consists of two polypeptide chains that each comprise an IgA antibody constant domain.

11. The protein of any one of claims 1 to 9, wherein said protein comprises four polypeptide chains that each comprise an IgA antibody constant domain.

12. The protein of any one of claims 1 to 11 , wherein two of said polypeptide chains have attached to the C-terminal end thereof said fragment of human serum albumin that is capable of binding to FcRn.

13. The protein of any one of claims 1 to 9, 11 or 12, wherein four of said polypeptide chains have attached to the C-terminal end thereof said fragment of human serum albumin that is capable of binding to FcRn.

14. The protein of any one of claims 1 to 13, wherein said protein comprises an

Fc fragment of an IgA antibody.

15. The protein of any one of claims 1 to 14, wherein said protein comprises a Fab fragment of an IgA antibody.

16. The protein of any one of claims 1 to 15, wherein said protein is an IgA antibody, or a fragment thereof.

17. The protein of claim 16, wherein said IgA antibody or fragment thereof is an lgA1 antibody or fragment thereof or an lgA2 antibody or a fragment thereof.

18. The protein of claim 16 or claim 17, wherein said protein is a monomeric IgA antibody or fragment thereof.

19. The protein of claim 16 or claim 17, wherein said protein is a secretory form of an IgA antibody or fragment thereof.

20. The protein of any one of claims 1 to 19, wherein said IgA antibody constant domains to which said fragments of HSA are attached are IgA heavy chain constant domains or IgA light chain constant domains.

21. The protein of claim 20 wherein said IgA is lgA1 and said fragments of HSA are attached to light chain constant domains, or wherein said IgA is lgA2 and said fragments of HSA are attached to heavy chain constant domains.

22. A composition, preferably a pharmaceutical composition, comprising a protein of any one of claims 1 to 21 in admixture with a suitable diluent, carrier or excipient.

23. A nucleic acid molecule comprising a nucleotide sequence that encodes a protein of any one of claims 1 to 21.

24. A set of nucleic acid molecules each comprising a nucleotide sequence, wherein said set of nucleic acid molecules encodes a protein of any one of claims 1 to 21.

25. A host cell comprising a protein of any one of claims 1 to 21 or comprising a nucleic acid molecule of claim 23 or comprising a set of nucleic acid molecules of claim 24.

26. A method of producing a protein of any one of claims 1 to 21 , comprising the steps of:

(i) culturing a host cell comprising one or more nucleic acid molecules of claim 23, or a set of nucleic acid molecules of claim 24, or one or more expression vectors comprising one or more of said nucleic acid molecules or said set of nucleic acid molecules, under conditions suitable for the expression of the encoded protein; and

(ii) isolating or obtaining the protein from the host cell or from the growth

medium/supernatant.

27. The method of claim 26, wherein said method further comprises a step of purification of the protein product and/or formulating the protein product into a composition including at least one additional component.

28. A protein of any one of claims 1 to 21 for use in therapy.

29. The protein for use of claim 28 wherein said protein comprises an antigen binding domain and wherein said protein is for use in the treatment of a disease that is characterized by the expression of said antigen, preferably wherein said disease is cancer.

30. A method of treating a disease that is characterized by the expression of a given antigen which method comprises administering to a patient in need thereof a therapeutically effective amount of a protein of any one of claims 1 to 21 that comprises an antigen binding domain that binds to said antigen, preferably wherein said disease is cancer.

31. Use of the protein of any one of claims 1 to 21 , wherein said protein comprises an antigen binding domain, in the manufacture of a medicament or composition for use in the treatment of a disease that is characterized by the expression of said antigen, preferably wherein said disease is cancer.

32. The protein for use of claim 28 or claim 29, the method of claim 30 or the use of claim 31 , wherein said protein for use, method, or use, comprises transmucosal delivery of said protein.

33. A method of transmucosal delivery of the protein of any one of claims 1 to 21 , wherein said method comprises administering to a patient in need thereof a therapeutically effective amount of a protein of any one of claims 1 to 21.