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Title:
ANTI-BSEP-SPECIFIC ANTIBODIES
Document Type and Number:
WIPO Patent Application WO/2017/125601
Kind Code:
A1
Abstract:
The present disclosure provides monoclonal antibodies and antibody fragments that specifically bind to extracellular epitopes of BSEP and modulate BSEP function. Numerous compositions and methods are claimed that make use of said invention.

Inventors:
TILLER THOMAS (DE)
BRACKERTZ BETTINA (DE)
KLATTIG JÜRGEN (DE)
HÄUSSINGER DIETER (DE)
KEITEL-ANSELMINO VERENA (DE)
KUBITZ RALF (DE)
STINDT JAN (DE)
Application Number:
PCT/EP2017/051305
Publication Date:
July 27, 2017
Filing Date:
January 23, 2017
Export Citation:
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Assignee:
MORPHOSYS AG (DE)
HEINRICH-HEINE-UNIVERSITÄT (DE)
International Classes:
C07K16/28
Other References:
STINDT JAN ET AL: "Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency.", HEPATOLOGY (BALTIMORE, MD.) FEB 2016, vol. 63, no. 2, 15 December 2015 (2015-12-15), pages 524 - 537, XP055276888, ISSN: 1527-3350, DOI: 10.1002/hep.28311
SANTA CRUZ BIOTECHNOLOGY: "anti bsep antibody, H-180: sc-25571", 1 January 1993 (1993-01-01), pages 1 - 1, XP055276751, ISBN: 978-0-12-198390-1, Retrieved from the Internet [retrieved on 20160531]
Attorney, Agent or Firm:
HUTTER, Bernd (DE)
Download PDF:
Claims:
Claims

1. An isolated monoclonal antibody or antibody fragment which specifically binds to the extracellular region of the polypeptide of SEQ ID No.: 1.

2. The antibody or antibody fragment of claim 1 , wherein said antibody or antibody fragment binds to an epitope on amino acids 84-147, 237-240, 341 -353, 777-794 or 1001 -101 1 of the polypeptide of SEQ ID No.:1.

3. The antibody or antibody fragment of claim 2, wherein said antibody or antibody fragment binds to an epitope on amino acids 84-147, 237-240, 341 -361 of the polypeptide of SEQ ID No. 1.

4. The antibody or antibody fragment of claim 2, wherein said antibody or antibody fragment binds to an epitope on amino acids 777-794 or 1001 -101 1 of the polypeptide of SEQ ID No.:1.

5 The antibody or antibody fragment of any one of the preceding claims, wherein said antibody or antibody fragment is cross-reactive with rat BSEP.

6. The antibody or antibody fragment of any one of the preceding claims, wherein said antibody or antibody fragment stimulates the function of the polypeptide of SEQ ID No. l as measured via the transport of [3H]-taurocholic acid by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90% or by at least 100%.

7. The antibody or antibody fragment of any one of the preceding claims, wherein said antibody or antibody fragment comprises

(a) the HCDR1 region of SEQ ID No.: 6, the HCDR2 region of SEQ ID No.: 7, the HCDR3 region of SEQ ID No.: 8, the LCDR1 region of SEQ ID No.: 9, the LCDR2 region of SEQ ID No.: 10 and the LCDR3 region of SEQ ID No.: 1 1 ;

(b) the HCDR1 region of SEQ ID No.: 16, the HCDR2 region of SEQ ID No.: 17, the HCDR3 region of SEQ ID No.: 18, the LCDR1 region of SEQ ID No.: 19, the LCDR2 region of SEQ ID No.: 20 and the LCDR3 region of SEQ ID No.: 21 ;

(c) the HCDR1 region of SEQ ID No.: 26, the HCDR2 region of SEQ ID No.: 27, the HCDR3 region of SEQ ID No.: 28, the LCDR1 region of SEQ ID No.: 29, the LCDR2 region of SEQ ID No.: 30 and the LCDR3 region of SEQ ID No.: 31 ; (d) the HCDR1 region of SEQ ID No.: 36, the HCDR2 region of SEQ ID No.: 37, the HCDR3 region of SEQ ID No.: 38, the LCDR1 region of SEQ ID No.: 39, the LCDR2 region of SEQ ID No.: 40 and the LCDR3 region of SEQ ID No.: 41 ; or

(e) the HCDR1 region of SEQ ID No.: 46, the HCDR2 region of SEQ ID No.: 47, the HCDR3 region of SEQ ID No.: 48, the LCDR1 region of SEQ ID No.: 49, the LCDR2 region of SEQ ID No.: 50 and the LCDR3 region of SEQ ID No.: 51.

8. The antibody or antibody fragment of any one of the preceding claims, wherein said antibody or antibody fragment comprises

(a) a variable heavy chain of SEQ ID No.: 4 and a variable light chain of SEQ ID No.: 5;

(b) a variable heavy chain of SEQ ID No.: 14 and a variable light chain of SEQ ID No.: 15;

(c) a variable heavy chain of SEQ ID No.: 24 and a variable light chain of SEQ ID No.: 25;

(d) a variable heavy chain of SEQ ID No.: 34 and a variable light chain of SEQ ID No.: 35; or

(e) a variable heavy chain of SEQ ID No.: 44 and a variable light chain of SEQ ID No.: 45.

9. An antibody or antibody fragment which cross-competes with an antibody or antibody fragment of any of the preceding claims.

10. An antibody or antibody fragment according to one of the preceding claims for use in medicine.

1 1. An antibody or antibody fragment according to any one of claim 1 to 9 for use in the detection or diagnosis of cancer.

12. A nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the antibody or antibody fragment according to any one of claims 1 to 9.

13. A vector composition comprising a vector or a plurality of vectors comprising the nucleic acid sequence or plurality of nucleic acid sequences of claim 12.

14. A cell comprising the nucleic acid composition of claim 12 or the vector composition of claim 13.

15. A pharmaceutical composition comprising the antibody or antibody fragment according to one of the claims 1 to 9 and a pharmaceutically acceptable carrier or excipient.

Description:
Anti-BSEP-specific antibodies

Field of the invention

The present application relates to antibodies or antibody fragments which specifically bind to the bile salt export pump, BSEP, and modulate its activity. The invention also relates to nucleic acids, vectors and host cells capable of expressing said antibodies or fragments thereof, pharmaceutical compositions comprising said antibodies or fragments thereof and uses of said antibodies or fragments thereof for treatment and diagnosis of specific diseases.

Background of the invention

BSEP, also known as ABCB1 1 , is a membrane-associated protein of the superfamily of ATP-binding cassette (ABC) transporters. It transports various molecules across extra- and intra-cellular membranes. More specifically, BSEP is a member of the MDR TAP subfamily. It is involved in drug resistance. In particular it is responsible for the transport of taurocholate and other cholate conjugates from hepatocytes (liver cells) to the bile. In humans, the activity of this transporter is the major determinant of bile formation and bile flow.

BSEP is associated with progressive familial intrahepatic cholestasis type 2 (PFIC2). PFIC2 is caused by mutations in the ABCB1 1 gene, causing an increase of the risk of hepatocellular carcinoma in early life.

Certain antibodies which specifically bind to BSEP are known. For example, the polyclonal mouse antibody H-180 (SantaCruz Biotechnology), or the mouse antibody F6 (SantaCruz Biotechnology), both raised against amino acids 1 -180 of BSEP. However, there are no monoclonal antibodies which specifically recognize extracellular epitopes of BSEP and which modulate BSEP functionality. Such antibodies and antibody fragments are valuable for numerous applications, including diagnostic and therapeutic applications. Summary of the invention

The applicant for the first time discloses monoclonal antibodies and antibody fragments that specifically bind an extracellular epitope of BSEP. Applicant also describes how such antibodies can be generated.

The present disclosure provides isolated monoclonal antibodies and antibody fragments that specifically bind to the extracellular region of the polypeptide of SEQ ID No.: 1 , more precisely on amino acids 84-147, 237-240, 341-353, 777-794 or 1001-1011 of BSEP.

Further, the present disclosure provides monoclonal antibodies and antibody fragments which specifically bind to an epitope on amino acids 84-1 7, 237-240, 341-361 of the polypeptide of SEQ ID No.:1.

Further, the present disclosure provides human monoclonal antibodies and antibody fragments which specifically bind to an epitope on amino acids 777-794 or 1001-1011 of the polypeptide of SEQ ID No.:1.

Further, the present disclosure provides recombinant and non-natural antibodies and antibody fragments that specifically bind to the extracellular region of the polypeptide of SEQ ID No.: 1.

Further, the present disclosure provides monoclonal antibodies and antibody fragments which specifically bind to the extracellular region of the polypeptide of SEQ ID No.: 1 , wherein said antibody or antibody fragment is cross-reactive with rat BSEP.

Further, the present disclosure provides monoclonal antibodies and antibody fragments which specifically bind to the extracellular region of the polypeptide of SEQ ID No.: 1 , wherein said antibody or antibody fragment is an agonistic or antagonistic antibody, preferably an agonistic antibody.

Further, the present disclosure provides monoclonal antibodies and antibody fragments which specifically bind to the extracellular region of the polypeptide of SEQ ID No.: 1 , wherein said antibody or antibody fragment is a stimulatory or inhibitory antibody, preferably a stimulatory antibody. Further, the present disclosure provides human monoclonal antibodies and antibody fragments which specifically binds to the extracellular region of the polypeptide of SEQ ID No.: 1 , wherein said antibody or antibody fragment has no functional BSEP modulating activity.

Further, the present disclosure provides monoclonal antibodies and antibody fragments which specifically binds to the extracellular region of the polypeptide of SEQ ID No.: 1 and wherein said antibody or antibody fragment has an EC50 to the polypeptide of SEQ ID No.: 1 of less than 10 nM.

Further, the present disclosure provides monoclonal antibodies and antibody fragments which specifically bind to the extracellular region of the polypeptide of SEQ ID No.: 1 , wherein said antibody or antibody fragment has an EC50 to the polypeptide of SEQ ID No.: 1 of less than 10 nM and wherein the polypeptide of SEQ ID No.: 1 is transiently expressed on HKB11 cells.

Further, the present disclosure provides monoclonal antibodies and antibody fragments, wherein said antibody or antibody fragment comprises

(a) the HCDR1 region of SEQ ID No.: 6, the HCDR2 region of SEQ ID No.: 7, the HCDR3 region of SEQ ID No.: 8, the LCDR1 region of SEQ ID No.: 9, the LCDR2 region of SEQ ID No.: 10 and the LCDR3 region of SEQ ID No.: 1 1 ;

(b) the HCDR1 region of SEQ ID No.: 16, the HCDR2 region of SEQ ID No.: 17, the HCDR3 region of SEQ ID No.: 18, the LCDR1 region of SEQ ID No.: 19, the LCDR2 region of SEQ ID No.: 20 and the LCDR3 region of SEQ ID No.: 21 ;

(c) the HCDR1 region of SEQ ID No.: 26, the HCDR2 region of SEQ ID No.: 27, the HCDR3 region of SEQ ID No.: 28, the LCDR1 region of SEQ ID No.: 29, the LCDR2 region of SEQ ID No.: 30 and the LCDR3 region of SEQ ID No.: 31 ;

(d) the HCDR1 region of SEQ ID No.: 36, the HCDR2 region of SEQ ID No.: 37, the HCDR3 region of SEQ ID No.: 38, the LCDR1 region of SEQ ID No.: 39, the LCDR2 region of SEQ ID No.: 40 and the LCDR3 region of SEQ ID No.: 41 ;

or

(e) the HCDR1 region of SEQ ID No.: 46, the HCDR2 region of SEQ ID No.: 47, the HCDR3 region of SEQ ID No.: 48, the LCDR1 region of SEQ ID No.: 49, the LCDR2 region of SEQ ID No.: 50 and the LCDR3 region of SEQ ID No.: 51.

Further, the present disclosure provides monoclonal antibodies and antibody fragments, wherein said antibody or antibody fragment comprises

(a) a variable heavy chain of SEQ ID No.: 4 and a variable light chain of SEQ ID No.: 5; (b) a variable heavy chain of SEQ ID No.: 14 and a variable light chain of SEQ ID No.: 15;

(c) a variable heavy chain of SEQ ID No.: 24 and a variable light chain of SEQ ID No.: 25;

(d) a variable heavy chain of SEQ ID No.: 34 and a variable light chain of SEQ ID No.: 35; or

(e) a variable heavy chain of SEQ ID No.: 44 and a variable light chain of SEQ ID No.: 45.

Further, the present disclosure provides monoclonal antibodies and antibody fragments with germline sequence deviations in antibody heavy and light chain framework regions FR1 , FR2, FR3 and FR4, that likely contribute to BSEP binding specificity.

Further, the present disclosure provides human monoclonal antibodies and antibody fragments which cross-compete with an antibody or antibody fragment of any of the foregoing antibodies or antibody fragments.

Further, the invention provides methods of using aforementioned antibodies for detection of BSEP and of cells or tissues that express BSEP; for diagnosis, prognosis, imaging, or staging of a BSEP-mediated disease; for modulating an activity or function of a BSEP protein and for treatment of a BSEP-mediated disease.

Further, the present disclosure provides aforementioned antibodies and antibody fragments for use in medicine.

Further, the present disclosure provides aforementioned antibodies and antibody fragments for use in the detection or diagnosis of cancer.

Further, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding aforementioned antibodies or antibody fragments.

Further, the present disclosure provides a composition comprising a vector or a plurality of vectors comprising the aforementioned nucleic acid sequence or a plurality of aforementioned nucleic acid sequences.

Further, the present disclosure provides a cell comprising aforementioned nucleic acid compositions or aforementioned vector. Further, the present disclosure provides a pharmaceutical composition comprising aforementioned antibodies or antibody fragments and a pharmaceutically acceptable carrier or excipient.

Detailed description of the invention

The disclosure pertains to a number of monoclonal antibodies or antibody fragments that recognize BSEP.

The term "BSEP" refers to a protein known as Bile sort export pump. Certain synonyms of BSEP do exits, including ABCB1 1 , ABC16, ATP-binding cassette sub-family B member 1 1 , BRIC2, BSEP, PFIC2, PFIC-2, PGY4, SPGP.

Human BSEP has the following amino acid sequence (UniProt 095342):

Homologues of BSEP are also known from other species, including chimpanzee (NCBI gene ID: 470717), cattle (NCBI gene ID: 531 150), dog (NCBI gene ID: 488390), horse (NCBI gene ID: 100052539), mouse (UniProt Q9QY30), rat (UniProt 070127), opossum (NCBI gene ID: 100136688) and rabbit (UniProt Q9N0V3).

In most preferred embodiments BSEP is human BSEP. In other preferred embodiments BSEP is the protein of SEQ ID No.: 1.

Rat BSEP has the following amino acid sequence (UniProt 070127):

In certain embodiments the present disclosure provides monoclonal antibodies and antibody fragments that are cross-reactive with rat BSEP.

In other embodiments the present disclosure provides monoclonal antibodies and antibody fragments that are cross-reactive with a polypeptide of SEQ ID No.:52.

In other embodiments the present disclosure provides monoclonal antibodies and antibody fragments that specifically bind to a polypeptide of SEQ ID No.: 1 and a polypeptide of SEQ ID No. :52.

The human BSEP protein is a multi-pass transmembrane protein. The membrane topology of human BSEP is known (see also JHumGenet (2010) 55:308-13). BSEP consists of 1321 amino acids with a molecular mass of -160 kDa. Like other full-length transporters of the ABC superfamily, the topology is predicted to be a tandemly duplicated structure with each half of the molecule consisting of six predicted transmembrane domains (TMD) and a large cytoplasmic nucleotide binding domain (NBD) in a TMD-NBD-TMD-NBD organization (Lam et al. Semin Liver Dis. 2010 May; 30(2): 125-133). See Figure 1 . The following table indicates the location of the amino acids of human BSEP:

Table 1 : Topology of human BSEP

In preferred embodiments, the antibody and antibody fragments of the present disclosure bind to an extracellular epitope of BSEP.

In other preferred embodiments, the antibody and antibody fragments of the present disclosure bind to an epitope on amino acids 84-147, 237-240, 341-353, 777-794 or 1001- 1011 of BSEP.

In other preferred embodiments, the antibody and antibody fragments of the present disclosure exclusively bind to an epitope of the N-terminal part (amino acids 1 - 659) of human BSEP, more specifically to an epitope on amino acids 84-147, 237-240 or 341-353. In other preferred embodiments, the antibody and antibody fragments of the present disclosure exclusively bind to an epitope of the C-terminal part (amino acids 659 - 1321 ) of human BSEP, more specifically to an epitope on amino acids 777-794 or 1001 -101 1.

In other preferred embodiments, the antibody or antibody fragment of the present disclosure binds to an epitope on amino acids 84-147, 237-240, 341-361 of the polypeptide of SEQ ID No.: 1.

In other preferred embodiments, the antibody or antibody fragment of the present disclosure binds to an epitope on amino acids 777-794 or 1001 -101 1 of the polypeptide of SEQ ID No.: 1.

In other preferred embodiments, the antibody and antibody fragments of the present disclosure bind to an epitope on amino acids 84-147 of human BSEP.

In other preferred embodiments, the antibody and antibody fragments of the present disclosure bind to an epitope on amino acids 237-240 of human BSEP.

In other preferred embodiments, the antibody and antibody fragments of the present disclosure bind to an epitope on amino acids 341 -353 of human BSEP.

In other preferred embodiments, the antibody and antibody fragments of the present disclosure bind to an epitope on amino acids 777-794 of human BSEP.

In other preferred embodiments, the antibody and antibody fragments of the present disclosure bind to an epitope on amino acids 1001-101 1 of human BSEP.

The term "antibody" as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds which interacts (e.g., by binding, steric hindrance, stabilizing spatial distribution) with an antigen. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FR's arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies. The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, SgG3, lgG4, lgA1 and lgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology. The term "immunoglobulin format" refers to a full length antibody as defined herein above.

The phrase "antibody fragment", as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen. Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23: 1 126-1 136). Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1 -VH-CH1 ) which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641 ,870). The term "Fab format" refers to a Fab fragment of an antibody.

A "human antibody" or "human antibody fragment", as used herein, includes antibodies and antibody fragments having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86). The structures and locations of immunoglobulin variable domains, e.g., CDRs, may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991 ), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabat et al., (1991 ) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948. "Human" antibodies can also be produced in transgenic mice, e.g. the XenoMouse. In preferred embodiments of the present disclosure the antibodies or antibody fragments are human antibodies or antibody fragments. In other preferred embodiments of the present disclosure the antibodies or antibody fragments are monoclonal human antibodies or antibody fragments.

A "humanized antibody" or "humanized antibody fragment" is defined herein as one that is (i) derived from a non-human source, or ii) CDR-g rafted, wherein in (i) and (ii) the CDRs of the variable domain are from a non-human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.

The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule which has constant antibody regions derived from, or corresponding to, sequences found in one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from, or corresponding to, sequences found in humans, e.g. in the human germ line or somatic cells, and the variable antibody regions (e.g. VH , VL , CDR or FR regions) are derived from sequences found in a non-human animal, e.g. a mouse, rat, rabbit or hamster. In another embodiment of the present disclosure the antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment. In another embodiment of the present disclosure the antibody or antibody fragment is of the IgG isotype.

BSEP mediates the excretion of monovalent conjugated bile acids and has low affinity for certain drugs that are also substrates for MDR1. Human and rodent BSEP exhibit high affinity and selectivity toward conjugated bile salts in the order of taurochenodeoxycholate (TCDCA), taurocholate (TCA) >taurodeoxycholate (TDCA) >tauroursodeoxycholate (TUDCA) -glycocholate (GCA) (Lam et al Semin Liver Dis. 2010 May; 30(2): 125-133). BSEP also has a low affinity for a limited number of drugs that are substrates for MDR1 e.g. pravastatin (Kivisto, K.T. and M. Niemi, Pharm Res, 2007. 24(2): p. 239-47). Inhibition of BSEP is related to a decrease in bile salt secretion and in some cases, development of cholestasis.

In-vitro evaluations of polymorphisms in the ABCB1 1 gene revealed 616A>G, 1674G>C, 1772A>G, and 3556G>A associated with significantly impaired taurocholate transport activity; the 890A>G variant had mildly impaired function and 3556G>A associated with reduced cell surface total protein expression compared with wild-type BSEP (Ho, R.H., et al. Pharmacogenet Genomics, 2010. 20(1 ): p. 45-57).

Mutations in the BSEP transporter gene lead to an inheritable cholestatic disorder, Progressive Familial Intrahepatic Cholestasis type 2 (PFIC-2), characterized by severe jaundice, hepatomegaly and high plasma levels of bile acids and aminotransferases.

A number of mutations are present and amongst those functionally analyzed, a majority correlate with decreased protein expression and function, altered membrane targeting and increased degradation. In humans, unlike in rats, there is no compensatory mechanism for the loss of this transporter. Due to this, mutations or chemical inhibitors can result in decreased biliary bile salt secretion, leading to decreased bile flow and accumulation of bile salts inside the hepatocyte, resulting in hepatotoxicities.

Drugs such as bosentan, troglitazone and CI-1034 cause clinical hepatotoxicity that is related to inhibition of BSEP (Sahi, J., et al. Chem Biol Interact, 2006. 159(2): p. 156-68) Drug- induced liver injury is a relevant clinical issue, in severe cases ending in liver transplantation. Therefore, inversion of drug-induced or inherent BSEP inhibition, by stimulating, agonistic anti- BSEP antibodies might be relevant for the treatment of drug-induced liver toxicities. The terms "antagonist" and "antagonistic" as used herein refer to or describe an agent that is capable of, directly or indirectly, partially or fully blocking, inhibiting, reducing, or neutralizing the biological activity of a molecule, such as BSEP.

The terms "inhibit" and "inhibitory" as used herein refer to or describe an agent that is capable of, directly or indirectly, partially or fully blocking, reducing, or neutralizing the biological activity of a molecule, such as BSEP.

In certain embodiments the present disclosure provides monoclonal antibodies and antibody fragments which inhibit the function of the BSEP by at least 10%. In other embodiments said antibodies and antibody fragments inhibit the function of the BSEP by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80% or by at least 90%. In preferred embodiments the function of BSEP is measured via the transport of [3H]-taurocholic acid in a rapid filtration assay. In other preferred embodiments the function of BSEP is measured as described in Example 6. In other embodiments of the present disclosure the antibody or antibody fragments stimulate the function of the polypeptide of SEQ ID No.:1 as measured via the transport of [3H]-taurocholic acid by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90% or by at least 100%.

The terms "agonist" and "agonistic" as used herein refer to or describe an agent that is capable of, directly or indirectly, substantially inducing, activating, promoting, stimulating, increasing, or enhancing the biological activity of a molecule, such as BSEP.

The terms "stimulate" and "stimulatory" as used herein refer to or describe an agent that is capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a molecule, such as BSEP.

In certain embodiments the present disclosure provides monoclonal antibodies and antibody fragments which stimulate the function of the BSEP by at least 10%. In other embodiments said antibodies and antibody fragments stimulate the function of the BSEP by at least 20%, by at least 30%, by at least 40% or by at least 50%. In preferred embodiments the function of BSEP is measured via the transport of [3H]-taurocholic acid in a rapid filtration assay. In other preferred embodiments the function of BSEP is measured as described in Example 6.

The term "isolated" refers to a compound, which can be e.g. an antibody or antibody fragment, that is substantially free of other antibodies or antibody fragments having different antigenic specificities. Moreover, an isolated antibody or antibody fragment may be substantially free of other cellular material and/or chemicals. Thus, in some aspects, antibodies provided are isolated antibodies which have been separated from antibodies with a different specificity. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. An isolated antibody that specifically binds to an epitope, isoform or variant of a target may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., species homologs). In preferred embodiments of the present invention the antibody or antibody fragment is an isolated antibody or antibody fragment.

The term "recombinant antibody", as used herein, includes all antibodies that are prepared, expressed, created or segregated by recombinant means, such as antibodies isolated from an animal (e.g. , a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, antibodies selected and isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences. Preferably, such recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis. A recombinant antibody may be a monoclonal antibody. In preferred embodiments of the present invention the antibody or antibody fragment is a recombinant antibody or antibody fragment.

The term "non-natural antibody", as used herein, refers to an antibody or antibody fragment which does not occur with an identical amino acid sequence in nature. Non-natural antibodies or antibody fragment are typically engineered, modified or recombinantly expressed or manipulated. In preferred embodiments of the present invention the antibody or antibody fragment is a non-natural antibody or antibody fragment.

The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a unique binding site having a unique binding specificity and affinity for particular epitopes. In certain embodiments of the present disclosure the antibody or antibody fragment is a monoclonal antibody or antibody fragment.

As used herein, an antibody "binds specifically to", "specifically binds to", is "specific to/for" or "specifically recognizes" an antigen if such antibody is able to discriminate between such antigen and one or more reference antigen(s), since binding specificity is not an absolute, but a relative property. The reference antigen(s) may be one or more closely related antigen(s), which are used as reference points, e.g. a mutated of scrambled version of BSEP. In its most general form (and when no defined reference is mentioned), "specific binding" is referring to the ability of the antibody to discriminate between the antigen of interest and an unrelated antigen, as determined, for example, in accordance with one of the following methods. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. For example, a standard ELISA assay can be carried out. The scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxidase and tetramethyl benzidine with hydrogen peroxide). The reaction in certain wells is scored by the optical density, for example, at 450 nm. Typical background (^negative reaction) may be 0.1 OD; typical positive reaction may be 1 OD. This means the difference positive/negative can be more than 10-fold. Typically, determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like. The term "binds specifically" in particular also means that no detectable binding to any negative control peptide, such as YFP, is observed.

As used herein, the term "affinity" refers to the strength of interaction between the polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts through weak non-covalent forces with its target at numerous sites; the more interactions, the stronger the affinity.

The term "EC 5 o" or "EC50", as used herein refers to the half maximal effective concentration, which includes the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time. The EC50 essentially represents the concentration of an antibody where 50% of its maximal effect is observed. Thus, reduced binding is observed with an increased EC50, or half maximal effective concentration value.

In certain embodiments the present disclosure provides antibodies and antibody fragments with an EC50 to BSEP of less than 10 nM. In other embodiments the present disclosure provides antibodies and antibody fragments with an EC50 to BSEP of less than 5 nM.

In certain embodiments the present disclosure provides antibodies and antibody fragments with an EC50 to BSEP of less than 10 nM, wherein BSEP is transiently expressed on H B1 1 cells. In other embodiments the present disclosure provides antibodies and antibody fragments with an EC50 to BSEP of less than 5 nM.

"Cross competes" means the ability of an antibody, antibody fragment or other antigen- binding moieties to interfere with the binding of other antibodies, antibody fragments or antigen- binding moieties to a specific antigen in a standard competitive binding assay. The ability or extent to which an antibody, antibody fragment or other antigen-binding moieties is able to interfere with the binding of another antibody, antibody fragment or antigen-binding moieties to a specific antigen, and, therefore whether it can be said to cross-compete according to the invention, can be determined using standard competition binding assays. One suitable assay involves the use of the Biacore technology (e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-competing uses an ELISA-based approach. A high throughput process for "epitope binning" antibodies based upon their cross-competition is described in International Patent Application No. WO 2003/48731 . Cross-competition is present if the antibody or antibody fragment under investigation reduces the binding of one of the antibodies described in Table 2 to BSEP by 60% or more, specifically by 70% or more and more specifically by 80% or more and if one of the antibodies described in Table 2 reduces the binding of said antibody or antibody fragment to BSEP by 60% or more, specifically by 70% or more and more specifically by 80% or more.

The term "epitope" includes any proteinacious region which is specifically recognized by an antibody or fragment thereof or a T-cell receptor or otherwise interacts with a molecule. Generally epitopes are of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by one of skill in the art, practically anything to which an antibody can specifically bind could be an epitope. An epitope can comprise those residues to which the antibody binds and may be "linear" or "conformational." The term "linear epitope" refers to an epitope wherein all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein (continuous). The term "conformational epitope" refers to an epitope in which discontinuous amino acids that come together in three dimensional conformations. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another. "Binds the same epitope as" means the ability of an antibody, antibody fragment or other antigen-binding moiety to bind to a specific antigen and having the same epitope as the exemplified antibody. The epitopes of the exemplified antibody and other antibodies can be determined using epitope mapping techniques. Epitope mapping techniques are well known in the art. For example, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., hydrogen/deuterium exchange, x-ray crystallography and two-dimensional nuclear magnetic resonance.

Compositions of the disclosure may be used for numerous applications. For example, the antibodies and antibody fragments of the present disclosure may be used for therapeutic applications. Merely as an example, numerous medicaments block the BSEP protein. As a consequence, salt of the bile are not transported out of the liver thereby leading to a damage of the liver. Parallel administration of an agonistic or stimulatory anti-BSEP antibody or an antibody fragment may prevent such liver damage. The disclosure, therefore, includes a pharmaceutical composition containing an antibody (or functional antibody fragment) of the present disclosure and a pharmaceutically acceptable carrier or excipient therefore. Therefore, in certain embodiments the present disclosure provides an antibody of antibody fragment of the present disclosure for use in medicine. In a related aspect, the disclosure provides a method for treating a disease or disorder. Such method contains the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains an antibody or antibody fragment of the present disclosure.

Such carriers, diluents and excipients are well known in the art, and the skilled artisan will find a formulation and a route of administration best suited to treat a subject with the anti- BSEP antibodies or antibody fragments of the present disclosure.

A "therapeutically effective amount" or "effective amount", as used herein, refers to the amount of the anti-BSEP antibody necessary to elicit the desired biological response. In accordance with the subject invention, the therapeutic effective amount is the amount of the anti-BSEP antibody necessary to treat and/or prevent a disease.

"Subject" or "species", as used in this context refers to any mammal, including rodents, such as mouse or rat, and primates, such as cynomolgus monkey (Macaca fascicularis), rhesus monkey (Macaca mulatta) or humans (Homo sapiens). Preferably the subject is a primate, most preferably a human. The antibodies and antibody fragments of the present disclosure may also be used in diagnostic applications.. BSEP is specifically expressed in the liver. The entire liver tissue may be visualized with a labelled e.g. radio-labelled) anti-BSEP antibody. BSEP-negative metastases, e.g. derived from gastric carcinoma, which settle in the liver, will not be labelled and will therefore be detected. Such a method will be very sensitive and will have a high resolution. Therefore, in certain embodiments the present disclosure provides a method for the detection of cancer metastases in the liver utilizing antibody of antibody fragment of the present disclosure.

BSEP is a specific marker for hepatocellular carcinoma. Antibodies and antibody fragments of the present disclosure may also be used in diagnostic applications for imaging extrahepatic metastases of hepatocellular carcinoma. In preferred embodiments said cancer is hepatocellular carcinoma. In other preferred embodiments said cancer is hepatoblastoma.

In one embodiment, the present disclosure refers to an antibody or antibody fragment which specifically binds to BSEP on cells.

In one embodiment, the present disclosure refers to an antibody or antibody fragment which specifically binds to a cell expressing BSEP.

In one embodiment, the present disclosure refers to an antibody or antibody fragment which specifically binds a cancer cell expressing BSEP.

In one embodiment, the present disclosure refers to an antibody or antibody fragment which specifically binds to a cell expressing the N-terminal part (amino acids 1 - 659) of BSEP.

In one embodiment, the present disclosure refers to an antibody or antibody fragment which specifically binds to a cell expressing the C-terminal part (amino acids 659-1321 ) of BSEP.

In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises

the HCDR1 region of SEQ ID No.: 6, the HCDR2 region of SEQ ID No.: 7, the HCDR3 region of SEQ ID No.: 8, the LCDR1 region of SEQ ID No.: 9, the LCDR2 region of SEQ ID No.: 10 and the LCDR3 region of SEQ ID No.: 11. In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises

the HCDR1 region of SEQ ID No.: 16, the HCDR2 region of SEQ ID No. : 17, the HCDR3 region of SEQ ID No.: 18, the LCDR1 region of SEQ ID No.: 19, the LCDR2 region of SEQ ID No.: 20 and the LCDR3 region of SEQ ID No. : 21 .

In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises

the HCDR1 region of SEQ ID No.: 26, the HCDR2 region of SEQ ID No. : 27, the HCDR3 region of SEQ ID No. : 28, the LCDR1 region of SEQ ID No. : 29, the LCDR2 region of SEQ ID No.: 30 and the LCDR3 region of SEQ ID No.: 31

In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises

the HCDR1 region of SEQ ID No.: 36, the HCDR2 region of SEQ ID No. : 37, the HCDR3 region of SEQ ID No. : 38, the LCDR1 region of SEQ ID No.: 39, the LCDR2 region of SEQ ID No. : 40 and the LCDR3 region of SEQ ID No.: 41 .

In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises

the HCDR1 region of SEQ ID No.: 46, the HCDR2 region of SEQ ID No. : 47, the HCDR3 region of SEQ ID No.: 48, the LCDR1 region of SEQ ID No.: 49, the LCDR2 region of SEQ ID No. : 50 and the LCDR3 region of SEQ ID No.: 51 .

In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain of SEQ ID No.: 4 and a light chain of SEQ ID No.: 5. In another embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain and a light chain that has at least 60%, at least 70 %, at least 80%, at least 90% or at least 95% identity to the a heavy chain of SEQ ID No.: 4 and to the light chain of SEQ ID No.: 5.

In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain of SEQ ID No.: 14 and a light chain of SEQ ID No.: 15. In another embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain and a light chain that has at least 60%, at least 70 %, at least 80%, at least 90% or at least 95% identity to the a heavy chain of SEQ ID No : 14 and to the light chain of SEQ ID No.: 15.

In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain of SEQ ID No.: 24 and a light chain of SEQ ID No.: 25. In another embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain and a light chain that has at least 60%, at least 70 %, at least 80%, at least 90% or at least 95% identity to the a heavy chain of SEQ ID No.: 24 and to the light chain of SEQ ID No.: 25. In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain of SEQ ID No.: 34 and a light chain of SEQ ID No.: 35. In another embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain and a light chain that has at least 60%, at least 70 %, at least 80%, at least 90% or at least 95% identity to the a heavy chain of SEQ ID No.: 34 and to the light chain of SEQ ID No.: 35.

In one embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain of SEQ ID No.: 4 and a light chain of SEQ ID No.: 5. In another embodiment, the present disclosure refers to an antibody or antibody fragment specific for BSEP, wherein said antibody or antibody fragment comprises a heavy chain and a light chain that has at least 60%, at least 70 %, at least 80%, at least 90% or at least 95% identity to the a heavy chain of SEQ ID No.: 44 and to the light chain of SEQ ID No.: 45.

In one embodiment, the present disclosure refers to a nucleic acid molecule comprising

(a) the variable heavy chain DNA sequence of SEQ ID No.: 2 and the variable light chain DNA sequence of SEQ ID No.: 3;

(b) the variable heavy chain DNA sequence of SEQ ID No.: 12 and the variable light chain DNA sequence of SEQ ID No.: 13;

(c) the variable heavy chain DNA sequence of SEQ ID No.: 22 and the variable light chain DNA sequence of SEQ ID No.: 23

(d) the variable heavy chain DNA sequence of SEQ ID No.: 32 and the variable light chain DNA sequence of SEQ ID No.: 33;

or

(e) the variable heavy chain DNA sequence of SEQ ID No.: 42 and the variable light chain DNA sequence of SEQ ID No.: 43.

In one embodiment, the present disclosure refers to two nucleic acid molecules, wherein

one nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 2 and the second nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 3;

one nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 12 and the second nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 13;

one nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 22 and the second nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 23; one nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 32 and the second nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 33;

or

one nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 42 and the second nucleic acid molecule comprises the DNA sequence of SEQ ID No.: 43.

In another embodiment, the present disclosure refers to a vector composition comprising a vector or a plurality of vectors comprising the nucleic acid sequence or plurality of nucleic acid sequences encoding an antibody or antibody fragment as disclosed in Table 2.

In one embodiment, the present disclosure refers to a cell comprising a vector composition comprising a vector or a plurality of vectors comprising the nucleic acid sequence or plurality of nucleic acid sequences encoding an antibody or antibody fragment as disclosed in Table 2.

In another embodiment, the present disclosure refers to a pharmaceutical composition comprising an antibody or antibody fragment as disclosed in Table 2 and a pharmaceutically acceptable carrier or excipient.

In one embodiment the disclosed antibody or antibody fragment specific for BSEP is a human, humanized or chimeric antibody. In certain embodiments, said antibody or antibody fragment specific for BSEP is an isolated antibody or antibody fragment. In another embodiment said antibody or antibody fragment is a recombinant antibody or antibody fragment. In a further embodiment said antibody or antibody fragment is a recombinant human antibody or antibody fragment. In a further embodiment said antibody or antibody fragment is a non-natural human antibody or antibody fragment. In a further embodiment said recombinant human antibody or antibody fragment is an isolated recombinant human antibody or antibody fragment. In a further embodiment said recombinant human antibody or antibody fragment or isolated recombinant human antibody or antibody fragment is monoclonal.

In another embodiment the disclosed antibody or antibody fragment comprises a heavy chain encoded by SEQ ID No.: 2 and a light chain encoded by SEQ ID No.: 3, or a heavy chain encoded by SEQ ID No.: 12 and a light chain encoded by SEQ ID No.: 13, or a heavy chain encoded by SEQ ID No.: 22 and a light chain encoded by SEQ ID No.: 23, or a heavy chain encoded by SEQ ID No.: 32 and a light chain encoded by SEQ ID No.: 33, or a heavy chain encoded by SEQ ID No.: 42 and a light chain encoded by SEQ ID No.: 43, or a heavy chain and a light chain that has at least 60%, at least 70 %, at least 80%, at least 90% or at least 95% homology aforementioned sequences.

In one embodiment the disclosed antibody or antibody fragment comprises a human heavy chain constant region and a human light chain constant region, in a further embodiment said human heavy chain constant region comprises the amino acid sequences of SEQ ID No.: 4 and the human light chain constant region comprises the amino acid sequences of SEQ ID No.: 5, or said human heavy chain constant region comprises the amino acid sequences of SEQ ID No.: 14 and the human light chain constant region comprises the amino acid sequences of SEQ ID No.: 15, or said human heavy chain constant region comprises the amino acid sequences of SEQ ID No.: 24 and the human light chain constant region comprises the amino acid sequences of SEQ ID No.: 25, or said human heavy chain constant region comprises the amino acid sequences of SEQ ID No.: 34 and the human light chain constant region comprises the amino acid sequences of SEQ ID No.: 35, or said human heavy chain constant region comprises the amino acid sequences of SEQ ID No.: 44 and the human light chain constant region comprises the amino acid sequences of SEQ ID No.: 45, or said human heavy chain constant region comprises the amino acid sequences of SEQ ID No.: 54 and the human light chain constant region comprises the amino acid sequences of SEQ ID No.: 55.

In one embodiment the disclosed antibody or antibody fragment is of the IgG isotype.

In another embodiment said antibody is IgG 1.

In one embodiment said antibody fragment is a bivalent antibody fragment.

In another embodiment, the present disclosure refers to an antibody or antibody fragment that cross-competes with an antibody described in Table 2. In one embodiment the present disclosure refers to an antibody or antibody fragment, wherein said antibody or antibody fragment cross-competes with an antibody or antibody fragment comprising 6 CDRs defined by Kabat of one of the antibodies in Table 2.

In a certain embodiment, the disclosure refers to an antibody or antibody fragment that cross-competes with an antibody described in Table 2 and reduces the specific binding of one of the antibodies described in Table 2 by at least 70%, 80% or 90% in an ELISA-based cross- competition assay. In a certain embodiment, the present disclosure refers to an monoclonal antibody or antibody fragment that cross-competes with an antibody described in Table 2 and reduces the specific binding of one of the antibodies described in Table 2 to BSEP by at least 70%, 80% or 90% in an ELISA-based cross-competition assay.

In certain embodiments, the disclosure refers to an antibody or antibody fragment that binds to a conformational epitope on the C-terminal part of BSEP.

In another embodiment, the present disclosure refers to an antibody or antibody fragment that binds to (e.g., by binding, stabilizing, spatial distribution) the same epitope as one of the antibodies in Table 2. In a further embodiment said antibody or antibody fragment bind to (e.g., by binding, stabilizing, spatial distribution) the same epitope as an antibody or antibody fragment comprising 6 CDRs defined by Kabat of one of the antibodies in Table 2.

Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g. , Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E.Morris, Ed. , 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et a!., (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al. , (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al., (1986) Mol. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., hydrogen/deuterium exchange, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g. , the Omiga version 1 .0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al. , (1981 ) Proc. Natl. Acad. Sci USA 78:3824-3828; for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., (1982) J. Mol. Biol. 157: 105-132; for hydropathy plots.

In one embodiment, the present disclosure refers to an antibody or antibody fragment comprising 6 CDRs defined by Kabat of any of the antibodies in Table2. In another aspect, the disclosure pertains to an isolated monoclonal antibody or fragment thereof comprising 6 CDRs defined by Kabat of each of the antibodies in Table 2. In another embodiment the present disclosure refers to the use of said pharmaceutical composition for the treatment of a disorder or condition associated with the undesired presence of BSEP.

In another embodiment the present disclosure refers to the use of said pharmaceutical composition for the treatment of a disorder or condition associated with the presence of a nonfunctional or inhibited form of BSEP.

In another embodiment the present disclosure refers to the use of said pharmaceutical composition for the treatment of a disorder or condition associated with the presence of BSEP.

In another embodiment said condition associated with the presence of BSEP is hepatocellular carcinoma metastasis.

In another embodiment said condition associated with the undesired presence of BSEP is metastasis. In another embodiment said condition associated with the presence of BSEP is hepatocellular carcinoma metastasis.

Figure legend

FIGURE 1 shows the membrane topology of human BSEP.

FIGURE 2 shows the key steps in the generation of monoclonal antibodies specific for extracellular epitopes of BSEP. BSEP-reactive B cells were isolated from peripheral blood of a PFIC-2 patient with a high anti-BSEP titer by fluorescence-activated single cell-couplet sorting (FACS). Each sorted couplet consists of a B cell bound to a HEK293 cell expressing BSEP-EYFP. Single couplets were then sorted into 96 well plates. Corresponding full-length immunoglobulin heavy and light chain variable region gene transcripts were amplified by single cell RT-PCR and cloned into eukaryotic expression vectors to enable the in vitro production of the respective monoclonal antibodies.

FIGURE 3 shows the overall PGR strategy for immunoglobulin isolation amplification.

FIGURE 4 shows the primer used for PGR strategy for immunoglobulin isolation amplification. Figure 4A discloses SEQ ID NOS 53-70, Figure 4B discloses SEQ ID NOS 71 - 76, and Figure 4C discloses SEQ ID NOS 77-92, all respectively, in order of appearance. FIGURE 5 shows that all antibodies identified in the present work specifically bind to human BSEP. Top row: detection of BSEP-expressing HEK293 cells by EYFP fluorescence. Middle row: testing for BSEP-reactive antibodies. All antibodies identified in the present work specifically bind to human BSEP, as does the commercially available antibody F-6. Polyclonal serum from a patient is also anti-BSEP positive, whereas negative control antibody 054 shows no staining. Bottom row: Nuclei were stained with Hoechst 33580. Scale bar = 20μητ

FIGURE 6 shows the staining of MeOH-fixed human (A) and rat (B) liver cryosections. Top row: staining of Mpr2, a canalicular marker. Middle row: the binders of the present disclosure, but not the control antibody 054, bind and stain the canalicular structure. Bottom row: merge of the Mpr2 and the BSEP staining. Scale bar = 50μιη.

FIGURE 7 shows the staining of PFA-fixed, unpermeabilized HEK293 cells transiently expressing BSEP-EYFP. Top row: detection of BSEP-expressing HEK293 cells by EYFP fluorescence. Middle row: staining with specific antibodies. All binders of the present disclosure do bind to unpermeabilized HEK293 cells transiently expressing BSEP-EYFP. Exceptions are control stainings with the non-binder 054 and the secondary antibody only. Polyclonal serum from a patient is also anti-BSEP positive. Bottom row: merge of the pictures of the top row and the middle row. Scale bar = 20μητ

FIGURE 8 shows the binding of all BSEP-reactive clones as measured by FACS analysis. All BSEP-reactive antibody clones except the non-binder 054 and the control containing only a secondary antibody show extracellular staining of BSEP-expressing ceils.

FIGURE 9 shows the functionality of the binders of the present disclosure. Data represent mean and standard error of three independent measurements and were analyzed using Student ' s t-test (unpaired; two-sided p-values). *, p<0.05; **, p<0.005; *** , p<0.0005; n.s., not significant.

FIGURE 10, left panel, shows the inhibition mechanisms by monovalent Fab and bivalent IgG molecules While extracellular binding of a monovalent Fab fragment may inhibit transport either by direct steric occlusion of the translocation pore or by restricting the conformational freedom necessary for transition between one or more steps of the transport cycle, a bivalent IgG molecule may also crosslink two transporter units. The right panel shows the transport inhibition by antibody Tweed, both in Fab and IgG format at 300, 60, 12 and 6 g/mL IgG or equivalent Fab binding sites. IgG was significantly increased in comparison to the same number of binding sites on Fab molecules over a range of increasing concentrations, strongly suggesting a crosslinking component of inhibition..

FIGURE 1 1 shows the results of the Western Blot analysis. Human (H) and rat (R) BSEP expressed in Sf9 membrane vesicles are recognized by equal amounts of IgG with clone-dependent intensity. (C) Empty control vesicles. The monoclonal anti-BSEP mouse antibody F-6 (Santa Cruz) used as a positive control recognizes human, but not rat BSEP.

FIGURE 12 shows the staining of HEK293 cells transiently transfected with the N- terminal portion (A) or the C-terminal portion (B) of human BSEP. Top row: detection of BSEP- expressing HEK293 cells by EYFP fluorescence. Middle row: staining with specific antibodies. Antibody Batist recognizes an epitope present on the N-terminal half of BSEP. Antibodies Duvetine, Fleece, Malimo and Tweed recognize epitopes present on the C-terminal half of BSEP, The monoclonal anti-BSEP mouse antibody F-6 (Santa Cruz) recognizes an epitope present on the N-terminal half of BSEP but not on the C-terminal part of BSEP.

FIGURE 13 summarizes the binding and functional features of the anti-BSEP antibodies of the present invention.

Examples

Example 1 : Generation of antibodies specific for extracellular epitopes of BSEP

An overview of the procedure is shown in Figure 2.

Example 1.1 Isolation of human peripheral blood mononuclear cells (PBMCs)

General methods of isolating and sorting human PBMCs from venous blood and mononuclear cells from bone marrow are described in J Immunol Methods (2008) 329: 1 12- 124. The PBMCs were isolated as follows.

5 ml venous blood was collected from a human PFIC-2 patient (after informed consent) into 3x Na-EDTA blood collection tubes (Sarstedt) (1.5 ml each). The contents of each monovette were combined into a single 50 ml Falcon (5 ml total). The blood was diluted with 25 ml of 1 x PBS (Invitrogen). 7 ml of FicollPaque (GE Healthcare) was added to new 15 ml conical tubes and 5 ml of diluted blood was layered over the FicollPaque, totaling (6 tubes: each with 7 ml FicollPaque + 5 mL blood). The tubes were spun on a centrifuge for 30 min at 400g. After centrifugation the enriched PBMCs formed a band at the interface between the plasma and the FicollPaque. The PBMCs were removed from each tube with a pipette and transferred to a new 50 ml tube. The PBMCs were washed by diluting to 40 ml with PBS and spinning on a centrifuge for 10 min at 1250 rpm at 4°C. The PBMCs were counted with Trypan Blue and frozen at 1 E+07 cells/tube (6 tubes) in FCS/10%DMSO at -150°C until single cell sort.

Example 1.2 Single cell sorting

The PBMCs were used for the isolation of B cells via magnetic MACS separation using human CD19 MicroBeads, (conjugated to monoclonal anti-human CD19 antibodies; isotype: mouse lgG1 ; Miltenyi Biotec; #130-050-301), MidiMACS Separator and LS Columns; Miltenyi Biotec, # 30-042-401 according to standard protocols. To enrich for BSEP-specific B cells, the isolated B cells were incubated with HEK cells transfected with YFP-BSEP in a ratio of 1 :1 (volume 200 μΙ). The mixture was subjected to a dual centrifugation step starting with 10 min at 1250 rpm, then cells were resuspended and centrifuged 30 min at 625 rpm. The mixture was resuspended in 150 μΙ FACS buffer, stained with 15 μΐ_ CD20-APC (Pharmingen #559776) and 15 μΙ IgG-PE (Pharmingen #555785); at 4°C for 40 min in the dark. Additional B cells remained unstained or were single stained with CD20-APC and IgG-PE for calibration. Cells were washed twice with 150 μΙ of FACS buffer, 1200 rpm, 4°C, 5 min, supernatant was discarded. Stained cell pellet was resuspended in 300 μΙ FACS buffer and passed through a cell strainer on FACS tubes (Eppendorf) to avoid clogs in the cytometer. Cells were analyzed and single sorted at FACS Aria on forward versus side scatter (live cell gate with double discrimination) according to cell surface marker expression of the phenotype of interest. lgG+ memory B cells reactive to BSEP appear CD20-APC+lgG-PE+,BSEP-HEKs-FITC+

Example 1.3 Single cell PGR and cloning into IgG expression vectors

Single cells were sorted in 96 well PGR plates containing 8 U RNAsin (Promega) to inhibit ribonucleases. Plates were then frozen at -80°C until further use. Immunoglobulin gene transcripts of the single sorted B cells selected for BSEP binding were then PGR amplified to amplify the respective VH and VL antibody gene sequences. The primer sequences were slightly modified to include restriction sites for compatibility with the pAMex20 vector system (MorphoSys). The overall PGR strategy and the primer used are shown in Figures 3 and 4A, 4B and 4C. First cDNAs of the single sorted B cells were synthesized. Thereafter nested PGR was conducted. Human IgH, Igk and IgL V gene transcripts were PGR amplified independently. Primers for transcripts encoding heavy chains and kappa light chains were annealed at 58°C, primers for transcripts encoding lambda light chains were annealed at 60°C. Nested PGR reactions with gene-specific primers or primer mixes were performed with 3 unpurified first PGR products. Aliquots of the second PCRs (=PCR products) were run agarose gel and DNA bands were visualized. PGR product sizes were approximately 450 bp for Igy, 510 bp for Igx and 405 bp for Igλ. PGR products were then sequenced (Eurofins MWG Operon, Ebersberg, Germany), and the sequencing results were then blasted with IgBLAST (NCBI) to identify the VH, VK, and VL gene families and sequences were cloned into IgG expression vectors pAMex20_h_lgG1f, pAMex20_lgkappa and pAMex20_h_lglambda, respectively. Sequences are shown in Table 2.

Example 2: Generation of antibodies specific for extracellular epitopes of BSEP - alternative

Antibodies against extracellular epitopes may also be generated by other techniques. Although transmembrane proteins are more difficult to produce than other, soluble proteins, certain techniques do exist that overcome these shortcomings. Examples are the StaR® technology of Heptares (see e.g. Neuropharmacology 201 1 , 60: 36-44) or the CHESS technology of G7 Therapeutics (see e.g. Curr Opin Chem Biol. (2013) 427-435). In certain cases also the generation of linear peptides of the proteins which are located extracellularly might work. Raising antibodies against polypeptides or proteins produced in such way will likewise yield antibodies or antibody fragments according to the present disclosure.

Example 3: All antibodies bind to human BSEP

About 50 immunoglobulins produced recombinantly as described in Example 1 were tested for binding to HKB1 1 cells transiently expressing BSEP-EYFP Five recombinant antibodies reactive to BSEP were identified. The binders were then tested for binding to HEK293 cells transiently expressing BSEP-EYFP. This was done via immunofluorescence staining of MeOH-fixed HE 293 cells. All five binders were BSEP-reactive, whereas clone 54 (a non-binder used as negative control did not). Commercial anti-BSEP mouse antibody F-6 (Santa Cruz Biotechnology; Cat No: sc-74500) was also reactive, as was a polyclonal serum sample from a patient. Results are shown in Figure 5. This experiment demonstrates that the binders generated as described herein above bind to human BSEP.

Example 4: Staining of various BSEP preparations

Human and rat liver cryosections were MeOH-fixed and stained with equal amounts of the antibodies of the present disclosure or control antibodies, labeled with AF-546. MRP2 was counterstained as a canalicular marker. Confocal laser scanning microscopy (Zeiss LSM 510 meta) of the stained sections confirmed that all binders of the present disclosure bind and stain the canalicular structures of human and rat BSEP. Results are shown in Figure 6A and Figure 6B.

Example 5: All binders bind to extracellular epitopes of BSEP

Example 5,11 Staining of unpermealized HEK293 cells

Recombinantly expressed monovalent Fab derivatives of the immunoglobulins of the present disclosure were used to test for staining of PFA-fixed, unpermeabilized HEK293 cells transiently expressing BSEP-EYFP. In order to re-assemble the monovalent Fab fragments into a bivalent form used as primary antibody, the Fabs containing a poly-histidine (His) tag were preincubated with a polyclonal, affinity-purified rabbit anti-His tag antibody at stoichiometry of two Fab per anti-His IgG.

As could be demonstrated, the BSEP-specific binders of the present disclosure specifically stain the surface of PFA-fixed, unpermeabilized HEK293 cells transiently expressing BSEP-EYFP. In contrast, the negative control antibody 054 does not stain said cells. Likewise, the commercially available anti-BSEP antibody F-6 (Santa Cruz Biotechnology; Cat No: sc-74500), does not stain the surface of unpermeabilized HEK293cells (data not shown). Results are shown in Figure 7.

Example 5.2: FACS analysis

The extra-cellular binding of all BSEP-reactive binders of the present disclosure was also demonstrated by FACS analysis. Un- or BSEP-EY P-transfected HEK293 cells were gently fixed with PFA and stained overnight at 4°C with (a) equal amounts of each antibody or (b) preformed (Fab-His) 2 -(mouse anti-His tag-lgG), each complexed with a fluorescently labeled secondary mouse Fab-Fragment. All BSEP-reactive antibody clones except the non- binder 054 showed extracellular staining of BSEP-EYFP-expressing HEK293 cells but not of untransfected cells. Results are shown in Figure 8.

This experiment therefore confirms the finding that all BSEP-specific binders of the present disclosure bind to extracellular epitopes of BSEP.

Example 6: Functionality of the binders

To test the functionality of the binders generated in the present disclosure Sf9 membrane vesicles containing rat BSEP were mixed with equal amounts of immunoglobulins and subjected to multiple freeze/thaw cycles to include immunoglobulins into the vesicular lumen. Then the transport of [3H]-taurocholic acid (TC, 2μΜ) was measured for 10 min using a rapid filtration assay with PBS as a control. Results are shown in Figure 9.

Antibodies Batist, Duvetine and Tweed are inhibiting, antagonistic, inhibitory antibodies. An equimolar mix of all antibodies of the present disclosure also was inhibitory. Antibody Malimo is a stimulating, agonistic, stimulatory antibody. Antibody Fleece binds to BSEP but does not show any functionality. The experiment was repeated with antibodies Malimo and Tweed. The functionality could be reproduced utilizing rat BSEP and human BSEP.

Example 7: In situ rat studies

Livers of anesthetized Wistar rats were perfused in situ with covalently AF546-labeled immunoglobulins and or Fab fragment of BSEP-specific binders of the present disclosure. Livers were harvested, snap-frozen and cryosectioned before staining with Mrp2, a canalicular marker, and cell nuclei using Hoechst 33580. As could be demonstrated the BSEP-reactive IgG and Fab reach the canalicular space in the intact organ, where they are retained by binding to BSEP.

Example 8: EC50 on HKB11 cells EC50 values were determined for immunoglobulins of the present disclosure by FACS binding to transiently transfected HKB1 1 cells.

For mammalian BSEP expression the vector pEYFP-N1-BSEP (yellow fluorescent protein - bile salt export pump fusion protein) or pN1 -BSEP, and as control pEYFP-N1 plasmid were used for transient transfections (Hepatology (2009) Hepatology 50:510-517). Cells were transfected with lipofectamin and further prepared using standard procedures. For antibody staining, 100 μΙ diluted immunoglobulin solution (starting with 150 μg/ ml) were added to the cells. The next steps included: incubation for 1 h on ice; washing the cells 2x with FACS buffer (3%FCS / 0.02%NaN3 in PBS, 5min, 250g, 4°C); resuspending in 100 μΙ of buffer of the respective detection antibody (Alexa 647-conjugated goat anti-human F(ab ' )2 Fragment (Jackson ImmunoResearch: 109-606-097) for human; Alexa 647-conjugates donkey F(ab ' )2 Fragment anti-mouse IgG H+L (Jackson ImmunoResearch: 715-606-150) for mouse). Plates were then incubated for 60 min on ice, washed with FACS and then acquired on a FACS array. Table 3 shows the EC50's measured in this way.

Example 9: Specificity of the prior art antibody F-6

In one experiment the goal was to measure the EC50 value of the prior art antibody F- 6 in the same set up as in Example 8. It was however observed that the prior art antibody F-6 (Santa Cruz Biotechnology; Cat No: sc-74500) did not show specificity for BSEP, but also did show binding to HKB1 1 cells transfected with a YFP control polypeptide. The EC50 of F-6 to HKB11 cells expressing YFP was determined to be 20.67 nM, compared to <10 nM for the antibodies and antibody fragments of the present invention. Therefore, F-6 is not BSEP- specific in the sense of the present disclosure.

Example 10: Reactivity against rat and human BSEP determined by Western Blot

As shown herein above (Example 4), the binders of the present disclosure bind to human BSEP and to rat BSEP.

BSEP binding was also tested by denaturing Western Blot. Human (H) and rat (R) BSEP expressed in Sf9 membrane vesicles are recognized by equal amounts of IgG with clone-dependent intensity. The BSEP-specific antibodies Batist, Duvetine, Fleece and Tweed strongly recognized denatured rat BSEP and human BSEP. The BSEP-specific antibody Malimo recognized denatured human BSEP. The monoclonal BSEP antibody F-6 (Santa Cruz) used as a positive control recognizes human, but not rat BSEP (Figure 1 1 ). Therefore, F6 is not rat BSEP-specific.This indicated that binder Malimo binds to a conformational epitope of BSEP.

Example 11 :

Full-length BSEP was subcloned into its two homologous halves (N-terminal part: amino acids 1-659 and C-terminal part amino acids 659-1321 ) and transfected into HEK293 cells. The N-terminal half contains three putative extracellular loops from ECL1 (amino acids 84-144), ECL2 (237-240), and ECL3 (342-361), the C-terminal half contains three putative extracellular loops from ECL4, ECL5 (aa 777-794), and ECL6 (aa 1001-101 1 ). The binders of the present invention were then tested for binding to HEK293 cells transiently expressing the N-terminal or the C-terminal portion of human BSEP. Antibody Batist recognizes an epitope present on the N-terminal half of BSEP. Antibodies Duvetine, Fleece, Malimo and Tweed recognize epitopes present on the C-terminal part of BSEP, The monoclonal BSEP antibody F-6 (Santa Cruz) recognizes as expected an epitope present on the N-terminal half of BSEP. Results are shown in Figure 12 and summarized in Figure 13.