FIELD OF THE INVENTION The invention is related to the field of compounds for the use in the preventive treatment of esophageal adenocarcinoma. More specifically, it relates to inhibitors of BMP2 and BMP4. The invention further relates to pharmaceutical compositions for use in the treatment of Barrett's Esophagus or for the prevention of esophageal adenocarcinoma.

BACKGROUND

Barrett's esophagus (BE) is a condition in which the normal multi-layered squamous epithelium is substituted by a (specialized) columnar epithelium (i.e. intestinal or other columnar type of metaplasia). This process is assumed to be the result of longstanding gastro-esophageal reflux disease and is most prevalent in middle aged, Caucasian males. The specialized intestinal type of columnar metaplasia in particular, confers a significantly increased risk for the development of esophageal adenocarcinoma (EAC). EAC is a highly malignant disease with very poor prognosis. The incidence of Barrett's esophagus and EAC is increasing rapidly and developing novel preventive and treatment strategies is of pivotal importance.

Treatment of Barrett's oesophagus with no malignant features, aim on reducing inflammation and includes treatment with compounds to relieve reflux or anti-reflux surgery. In case of malignant degeneration treatment is by endoscopic ablative therapies or surgically removing the affected part of the esophagus. The remains therefore a need in the art for an alternative treatment.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that following treatment with an BMP2/4 inhibitor, implantations in mice of Barrett tissue biopsies from a patient suffering from Barrett's esophagus, the typical phenotypical histological symptoms disappeared. In these in vivo organoids, the cells formed a multi-layered epithelium that expressed the squamous marker p63 and CK5 (Figure 3K-L), indicating the regeneration of normal multi-layered squamous epithelium of the esophagus and thus the disappearance of Barrett's esophagus. The epithelial layer of the treated organoids also lack the presence of columnar and goblet cells (Figure 3J). This shows the effectivity of a BMP2/4 inhibitor in a treatment of Barrett's esophagus and is therefore effective in the prevention of esophageal adenocarcinoma.

The invention provides a kit of parts comprising an inhibitor of BMP2 and an inhibitor of BMP4 or an inhibitor of BMP2 and BMP4 for use in the treatment of Barrett's Esophagus or for the prevention of esophageal adenocarcinoma.

Preferably, said kit of parts comprises an inhibitor of BMP2 and a n inhibitor of BMP4 or an inhibitor of BMP2 and BMP4 is selected from the group consisting of: chordin, noggin, follistatin, BMP3, Inhibin, an anti BMP2 and an anti BMP4 antibody, an antibody binding to BMP2 and BMP4.

In a preferred embodiment, said inhibitor of BMP2 and BMP4 is an isolated, synthetic or recombinant antibody, which binds within residues 24-31, 57-68, 70-72, 89, 91, 101, 103, 104 and 106 of SEQ ID NO:l.

Preferably, the isolated, synthetic or recombinant antibody according to the invention, binds to: a. at least one residue selected from the group consisting of Ser24, Asp25, Val26, Gly27, Trp28, Asn29,Asp30,Trp31, b. at least one residue selected from the group consisting of Ser57, Thr58, Asn59, His60, Ala61,

Ile62, Val63, Gln64, Thr65, Leu66, Val67, and Asn68, c. at least one residue selected from the group consisting of Val70, Asn71 and Ser72, d. at least one residue selected from the group consisting of Tyrl03 and Glnl04, and e. at least one residue selected from the group consisting of Met89, Tyr91, LyslOl, and Metl06 of SEQ ID NO:l.

Preferably, the isolated, synthetic or recombinant antibody according to the invention, binds to Asp30, Trp31, Leu66 and LyslOl.

Preferably, said isolated, synthetic or recombinant antibody is a single chain antibody.

Preferably said single chain antibody comprises: a. a heavy chain CD 1 consisting of the amino acid sequence of SEQ ID NO: 5 or a sequence not differing more than 1 amino acid thereof, b. a heavy chain CD 2 consisting of the amino acid sequence of SEQ ID NO:6, or a sequence not differing more than 1 amino acid thereof, and c. a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 7 or a sequence not differing more than 1 amino acid thereof. In a preferred embodiment, the isolated, synthetic or recombinant antibody according to the invention comprises the amino acid sequence of SEQ I D NO: 12 or a sequence at least 70%, more preferably 71, 72, 73, 74, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical thereto.

The invention further provides a pharmaceutical composition comprising a kit of parts comprising an inhibitor of BMP2 and an inhibitor of BMP4 or an inhibitor of BMP2 and BMP4 as defined above and a pharmaceutically acceptable carrier or diluent for use in the treatment of Barrett's Esophagus or for the prevention of esophageal adenocarcinoma.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the generation of in vivo BE organoids. Figure 2 shows that in vivo organoid recapitulates the original patient biopsy. A) H&E staining of the original biopsy and D) in vivo organoid. I HC using an anti-human mitochondria antibody of the B) original biopsy and E) in vivo organoid. Alcian blue staining of the C) original biopsy and D) in vivo organoid.

Figure 3 shows tissue after periodic treatment with C8C8, an antiBMP2 and BMP antibody, show expression of the squamous markers p63 and CK5. A) to C) original biopsy ; D) to F) control group and G) to I) C4C4 treatment group J) to L) C8C8 treatment group.

Figure 4 A-E show the Inhibition of BMP2/4 drives the development of squamous epithelium in a conditional Noggin knockout model.

Figure 4(A) shows that Rosa26-cre mice were crossed with loxp [noggin] loxp mice and injected with tamoxifen for 3 days (Img, i.p.). Noggin-/- mice were sacrificed after 4, 8, 12, 16 or 20 weeks.

Figure 4 (B) shows a H&E staining of multilayered glands (MLGs) developed at the squamo-columnar junction (SCJ) in noggin-/- mice upon tamoxifen injection (3 days, Img i.p.) at different time points. Figure 4 (C) shows that Rosa26-cre mice were crossed with loxp [noggin] loxp mice and injected with tamoxifen for 3 days (Img, i.p.). Noggin-/- mice were injected with tamoxifen for 3 days (Img, i.p.) and treated with saline or VHH BMP2/4 antibodies for 8 weeks. All mice were treated with proton pump inhibitors (PPIs) during the experiment.

Figure 4 (D) shows SCJ in wild type or noggin-/- mice after treatment with saline or VHH BMP2/4 antibodies. The SCJ with affected areas (dotted line) was stained for columnar marker K19 and squamous markers p63, K14 and K5.

Figure 4€ shows MLGs at the SCJ in noggin-/- mice treated with saline or VHH BMP2/4 antibodies. Affected areas were IHC stained for BMP4, downstream target pSMADl,5,8, squamous markers K5 and K14 (blue) and K7 (brown, 3 ^rd panel)) and ki67 (brown, 4 ^th panel).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Barrett's esophagus is a serious complication of gastroesophageal reflux disease (GE D). In Barrett's esophagus, normal tissue lining the esophagus is replaced by tissue that resembles the lining of the intestine. Barrett's esophagus does not have any specific symptoms, although patients with Barrett's esophagus may have symptoms related to GERD. It does, though, increase the risk of developing esophageal adenocarcinoma, which is a serious, potentially fatal cancer of the esophagus. Diagnosis of Barrett's oesophagus may be done using endoscopy, histology, and/or using biomarkers, for instance as described in US 20120009597 Al.

As used herein, the term "BMP2" is used to refer to mature bone morphogenic protein 2, preferably of human origin. The nucleotide sequence of human pro-BMP2 is publicly available by reference to GenBank Accession No. NM_001200.

As used herein, the term "BMP4" is used to refer to human mature bone morphogenic protein 4. The nucleotide sequence of human pro-BMP4 is publicly available by reference to GenBank Accession No. NM_130851. As used herein, the term "inhibitor" of BMP2 or 4, refers to any compound which interferes with, blocks or decreases the signalling of said molecule. Said compound may be a small molecule or antibody.

The term "BMP4 signalling" as used herein refers to the ability of BMP4 to activate the canonical (the phosphorylation of SMAD 1/5/8) Assays to test BMP4 signalling are described for instance in Shaifur Rahman et al., "TGF-β/ΒΜΡ signaling and other molecular events: regulation of osteoblastogenesis and bone formation" in Bone Research 3, Article number: 15005 (2015). The term "BMP2 signalling" as used herein refers to the ability of BMP2 to activate the canonical (the phosphorylation of SMAD 1/5/8).

The term "C4" as used herein refers to single chain antibody which binds to the hydrophobic groove of the wrist of BMP4. The term "C4C4" as used herein refers to a dimer of said C4 single chain antibody.

The term "C8" as used herein refers to an antibody according to the invention binds within residues 24-31, 57-68,70-72, 89, 91, 101, 103, 104 and 106 of BMP4 (as depicted in SEQ ID NO:l of

WO2016/042050). This region represents a "hydrophobic pocket" within the wrist epitope of BMP4. An advantage of antibodies which bind to this region is that these antibodies have a very high affinity for BMP4 and BMP2 and are capable of efficiently inhibiting BMP4 and BMP2 signalling.

The term "C8C8" as used herein refers to a dimer of said C8 antibody.

As used herein, esophageal cancer refers to cancer that starts in the esophagus, including but not limited to squamous cell carcinoma and adenocarcinoma.

Embodiments The invention is based on the finding that inhibition of both BMP2 and BMP4 signalling effectively restores the normal tissue lining the esophagus, and is therefore effective in the preventive treatment of esophageal adenocarcinoma. The invention therefore provides a kit of parts comprising an inhibitor of BMP2 and an inhibitor of BMP4. Alternatively, a compound which inhibits BMP2 and BMP4 signalling is also suitable for use in the treatment of Barrett's Esophagus or for the prevention of esophageal adenocarcinoma.

Any combination of compounds which inhibit BMP2 and BMP4 signalling may be used in the treatment of the invention. For instance, any compound which inhibits BMP2 signalling and another compound which inhibits BMP4 signalling may be used. Such compounds may be provided simultaneously or sequentially in a treatment. In another embodiment, an inhibitor which simultaneously inhibits of BMP2 and BMP4 signalling is provided for use in the treatment of the invention. Preferred compounds include, but are not limited to chordin, noggin, follistatin, BMP3, Inhibin (see Ezra Wiater and Wylie Vale, March 7, 2003, The Journal of Biological Chemistry, 278, 7934-7941). Antibodies capable of inhibiting BMP2 signalling and/or BMP4 signalling are known in the art and described for instance in WO2016/042050.

In a preferred embodiment said epitope is located in the wrist within residues 10-17, 24-31, 45-72, 89, 91, 101, 103, 104, and 106 of BMP4. Preferably, an antibody according to the invention binds within residues 10-17, 45-56, and 69 of BMP4. This epitope contains a hydrophobic groove, which the inventors believe is important for BMP4 specific binding. An advantage of the antibodies according to this embodiment is that said antibodies have a low affinity for other members of the BMP family and are highly effective in specifically inhibiting BMP4 signaling. Preferably, said antibody does not substantially bind to BMP2, BMP5, BMP6 or BMP7. A further advantage thereof is that said antibody does not inhibit BMP2 mediated signaling, thereby diminishing or even avoiding adverse side effects when used in vivo. More preferably, said antibody specifically binds to at least one residue selected from the group consisting of LyslO, Asnll, Lysl2, Asnl3, Cysl4, Argl5, Argl6, and Hisl7, at least one residue selected from the group consisting of Gly45, Asp46, Cys47, Pro48, Phe49, Pro50, Leu51, Ala52, Asp53, His54, Leu55 and Asn56, and to Ser69 of BMP4. Preferably, said antibody binds to more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 preferably 16 residues thereof. In a highly preferred

embodiment, said antibody specifically binds to at least Lysl2, Argl5, Asp46, and Pro50 of BMP4.

In a preferred embodiment, an antibody according to the invention is a single chain antibody. In a preferred embodiment, said antibody comprises a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 or a sequence not differing more than 2 amino acid thereof, a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:4, or a sequence not differing more than 1 amino acid thereof, and preferably further a heavy chain CDRl consisting of the amino acid sequence of SEQ ID NO: 2 or a sequence not differing more than 1 amino acid thereof. In a highly preferred embodiment, said antibody comprises the amino acid sequence of SEQ ID NO: 11. In another embodiment, an antibody according to the invention binds within residues 24-31, 57- 68,70-72, 89, 91, 101, 103, 104 and 106 of BMP4 (of SEQ ID NO:l). This region represents a

"hydrophobic pocket" within the wrist epitope of BMP4. An advantage of antibodies which bind to this region is that these antibodies have a very high affinity for BMP4 and BMP2 and are also capable of efficiently inhibiting BMP4 and BMP2 signaling. Preferably, said antibody specifically binds to at least one residue selected from the group consisting of Ser24, Asp25, Val26, Gly27, Trp28,

Asn29,Asp30,Trp31; at least one residue selected from the group consisting of Ser57, Thr58, Asn59, His60, Ala61, Ile62, Val63, Gln64, Thr65, Leu66, Val67, and Asn68; at least one residue selected from the group consisting of Val70, Asn71 and Ser72; at least one residue selected from the group consisting of Tyrl03 and Glnl04; and Met89, Tyr91, LyslOl, and to Metl06 of BMP4. Preferably, said antibody binds to more than 9, 10, 11, 12, 13 preferably 14 residues thereof. In a highly preferred embodiment, said antibody specifically binds to Asp30, Trp31, Leu66 and LyslOl. In a preferred embodiment, said antibody is a single chain antibody. In a preferred embodiment, said single chain antibody is capable of binding to a "hydrophobic pocket" region within the wrist epitope of BMP4 as described above. In a preferred embodiment, an antibody according to the invention comprises a heavy chain CD 3 consisting of the amino acid sequence of SEQ ID NO: 7 or a sequence not differing more than 1 amino acid thereof. Without wishing to be bound by theory, the inventors believe that this CDR3 is important for the binding interaction with the hydrophobic pocket of the wrist of BMP4. Preferably, said antibody further comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 5 or a sequence not differing more than 1 amino acid thereof, and a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:6, or a sequence not differing more than 1 amino acid thereof. In a highly preferred embodiment, said antibody comprises the amino acid sequence of SEQ ID NO: 12.

In yet another embodiment of the antibody of the invention, said antibody binds specifically within residues 34, 35, 39, 86-88, 90, 97, 98, 100, 102 and 109 of BMP4. This region represents the so called "knuckle" epitope of BMP4. Antibodies specifically binding to residues in this region also have a high affinity for BMP4, but in addition also a high affinity for BMP2 and slightly less for BMP5, and BMP6. Preferably, said antibody binds specifically binding to Ala34, Gln39, Ser88, Leu90 and LeulOO.

In a preferred embodiment, said antibody is a single chain antibody. In a preferred embodiment, an antibody capable of binding to said knuckle as described above comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 8 or a sequence not differing more than 1 amino acid thereof and a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:9, or a sequence not differing more than 1 amino acid thereof. Without wishing to be bound by theory, the inventors believe that these CDRs are important for the binding interaction with the knuckle of BMP4. Said antibody preferably further comprises a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 10 or a sequence not differing more than 1 amino acid thereof. In a preferred embodiment, said antibody comprises the amino acid sequence of SEQ ID NO: 13. BMP4 inhibitors are known in the art and described for instance in Calpe et al. , MABS 2016, VOL. 8, NO. 4, 678-688. BMP2 inhibitors are known in the art and described for instance in Khattab et al. Bone.

2014 Dec;69:165-73 and Hirata-Tsuchiya et al. Mol Endocrinol. 2014 Sep;28(9):1460-70.

Also preferred are antibodies described on p. 20-22, which bind to the hydrophobic groove of the wrist of BMP4 and inhibit BMP4 signalling. In a preferred embodiment antibodies which bind to the hydrophobic pocket of the wrist of BMP4 are used. These antibodies inhibit the signalling of both BMP2 and BMP4. Such antibodies are disclosed in WO2016/042050 on p. 22-24. Preferably, said antibody specifically binds to at least one residue selected from the group consisting of Ser24, Asp25, Val26, Gly27, Trp28, Asn29,Asp30,Trp31; at least one residue selected from the group consisting of Ser57, Thr58, Asn59, His60, Ala61, Ile62, Val63, Gln64, Thr65, Leu66, Val67, and Asn68; at least one residue selected from the group consisting of Val70, Asn71 and Ser72; at least one residue selected from the group consisting of Tyrl03 and Glnl04; and Met89, Tyr91, LyslOl, and to Metl06 of BMP4. Preferably, said antibody binds to more than 9, 10, 11, 12, 13 preferably 14 residues thereof. In a highly preferred

embodiment, said antibody specifically binds to Asp30, Trp31, Leu66 and LyslOl. In a preferred embodiment, said antibody is a single chain antibody. In a preferred embodiment, an antibody according to the invention comprises a heavy chain CD 3 consisting of the amino acid sequence of SEQ ID NO: 7 or a sequence not differing more than 1 amino acid thereof. Without wishing to be bound by theory, the inventors believe that this CDR3 is important for the binding interaction with the hydrophobic pocket of the wrist of BMP4. Preferably, said antibody further comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 5 or a sequence not differing more than 1 amino acid thereof, and a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:6, or a sequence not differing more than 1 amino acid thereof. In a highly preferred embodiment, said antibody comprises the amino acid sequence of SEQ ID NO: 12.

In a preferred embodiment, said an inhibitor of BMP2 and BMP4 comprises a hetero or homo multimeric molecule with increased antigen affinity for the antigens and/or an increased inhibitory effect on BMP signalling. In a further aspect, the invention therefore provides a multimeric antibody comprising at least one, more preferably at least two antibodies which bind to the hydrophobic pocket of the wrist of BMP4 as described above.

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing an inhibitor of BMP2 and an inhibitor of BMP4 or an inhibitor of BMP2 and BMP4, formulated together with a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising antibodies binding to BMP4 or antigen binding portions thereof are well known in the art and are described in more detail for example in WO2008030611. Such compositions may include one or a combination of (e.g., two or more different) antibodies or a multimeric antibody. Suitably, an antibody or multimeric antibody as defined herein is used. Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e. combined with other agents. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible. Typically, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

Depending on the route of administration, the active compound, i.e. antibody, or antigen binding fragment thereof or multimeric antibody of the invention, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge et al, J. Pharm. ScL 66:1- 19 (1977)). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti- oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble

antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils, such as olive oil and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by

incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, typically from about 0.1 per cent to about 70 per cent, most typically from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.

EXAMPLE

Mice

NOD-scid interleukin-2 (IL2) receptor gamma chain knockout (NSG) were purchased from The Jackson Laboratory and then bred in-house at the Animal Research Institute of the Academic Medical Center of Amsterdam (AMC). All mice were maintained in specific pathogen-free conditions, given autoclaved standard pellet feed and water, and housed in ventilated racks in the Animal Research Institute of the AMC. All animal experiments were approved by the Animal Experimental Committee (DEC) of the AMC.

Human Tissue Collection and Preparation

Human tissue biopsies were obtained from Barrett Esophagus (BE) patients during the routine surveillance program at the Gastroenterology and Hepatology Department in the AMC. All patients were on long-term proton pump inhibition therapy and after a confirmed diagnosis by endoscopy and histology. The collection and use of human tissue for this project was in accordance with the legislation in the Netherlands and approved by the medical ethical committee in the AMC. After obtaining informed consent, fresh samples were collected endoscopically from BE patients. Samples were placed in chilled phosphate-buffered saline (PBS) without MgCI ₂ and CaCI ₂ and supplemented with 200 U/ml of penicillin and 200μg/ml of streptomycin, and kept on ice. Biopsies were divided into approximately l-2mm ³ -sized pieces discarding any necrotic areas and blood clots. Representative samples were fixed in 10% buffered formalin for histological evaluation and the remaining pieces for implantation were placed in Matrigel (354234, Corning) with or without anti-BMP2/4 llama-derived antibody (C8C8; 500μg/ml) or anti-BMP4 llama-derived antibody (C4C4; 500μg/ml) (Calpe et al., 2015) and kept on ice until implantation. All biopsies were implanted within 2h of collection.

Intramuscular Implantation and Monitoring

(Intramuscular procedure previously described in (Read et al., 2016))

Mice were anesthetized via an intraperitoneal injection (ip) of ketamine (lOOmg/ml) and xylazine (20mg/ml) solution. After weighing, mice are injected with 10μΙ of the anaesthetic solution per gram of body weight. The dorsum of the mouse was shaved and prepared with a 2 % (v/v) chlorhexidine gluconate/70 % (v/v) isopropyl alcohol solution. Under aseptic conditions, a 15-mm midline incision was made immediately caudal to the dorsal hump at the level of the renal angle. Using blunt dissection, a skin flap was raised and the skin retracted laterally in order to expose the implantation site. A superficial stay suture was placed in the dorsal musculature immediately caudal to the lowest rib using a 4/0 braided absorbable suture. After tenting the muscle fibres, an intramuscular pocket was created using a combination of sharp and blunt dissection until it was just large enough to accommodate the BE piece. The BE biopsy coated in matrigel was then placed in the intramuscular (IM) pocket prior to suture closing. One or two separate transplantation sites were used per mouse. The skin was closed using 3/0 braided absorbable suture. Implants were cultured for a period of three months in order to form the in vivo organoid structures. During this period of time, mice received ip treatment three time per week of C4C4 or C8C8 (treatment groups) or saline (control group). Mice were closely monitored for any sign of discomfort and/or stress throughout the experiment.

Harvesting and Processing of the in vivo organoids

Mice were culled using C0 ₂ inhalation after three months. Immediately following culling, mice were shaved and their skin prepared. After identifying the non-absorbable marking suture, the muscle was incised around the site of xenograft leaving a 2mm wide margin. Following this, the muscle was retracted medially and any obvious lump or cyst carefully harvested. Samples were fixed in 10% buffered formalin to be assessed both histologically and immunohistochemically.

Histology and Immunohistochemistry (IHC)

Sections of formalin-fixed paraffin-embedded tissues from all of the original patient-derived biopsies and the in vivo organoids were stained with hematoxylin and eosin (H&E) and Alcian Blue. To confirm that the in vivo structures were of human origin, we performed IHC using an anti-human mitochondria antibody [113-1] (1:1000) (ab92824, Abeam). The in vivo organoid structures were stained for the squamous markers CK5 (1:200) (ab52635; Abeam) and p63 (1:100) (sc-8431; Santa Cruz Biotechnology, Inc.).

Results BE samples harvested endoscopically from the BE segment of 7 different patients were implanted intramuscularly in 21 NSG mice. Samples were implanted either with or without C4C4 (anti-BMP4 llama derived antibody) or C8C8 (anti-BMP2/4 llama derived antibody). Samples were allowed to grow for a period of three month, during which mice received ip injection three times per week of either saline (control) or C4C4 or C8C8 (treatment groups). All xenograft structure and well as the original patient biopsy were subjected to detailed histopathological analysis.

As previously described all samples were successfully engrafted. Barrett's organoids were lined by a functional epithelial layer containing goblet cells (Figure 2F) and recapitulated the crypt and villous regions seen within Barrett's glands. The organoid structures were confirmed to be composed of human tissue, as shown by the anti-human mitochondrial antibody. (Figure 2B, 2E)

The in vivo organoids show the same degree of differentiation (Figure 3A-F) and expression of molecular markers of intestinal differentiation (CK8, CDX2 and villin) as the original biopsy. Following treatment with BMP2/4 inhibitor, C8C8, organoids demonstrate a tendency to form a multi-layered epithelium that expressed the squamous marker p63 and CK5 (Figure 3K-L). The epithelial layer of the treated organoids also lack the presence of goblet cells (Figure 3J). Simultaneously, treatment with C4C4, were only BMP4 is inhibit, the same differentiation doesn't occur and the in vivo organoids resemble the control biopsy and lacking the expression of the squamous markers p63 and CK5. These results demonstrate that organoids are generated with a squamous-like phenotype following treatment with BMP2/4 inhibitors. These pre-clinical results may be translated to the clinical setting in order to prevent the development of esophageal adenocarcinoma.

References CALPE, S., WAGNER, K., EL KHATTABI, M., RUTTEN, L, ZIMBERLIN, C, DOLK, E., VERRIPS, C. T.,

MEDEMA, J. P., SPITS, H. & KRISHNADATH, K. K. 2015. Effective Inhibition of Bone

Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies. Mol Cancer Ther, 14, 2527-40.

READ, M., LIU, D., DUONG, C. P., CULLINANE, C, MURRAY, W. K., FENNELL, C. M., SHORTT, J.,

WESTERMAN, D., BURTON, P., CLEMONS, N. J. & PHILLIPS, W. A. 2016. Intramuscular Transplantation Improves Engraftment Rates for Esophageal Patient-Derived Tumor

Xenografts. Ann Surg Oncol, 23, 305-11.

Example 2 A: Inhibition of BMPs by Noggin drives the development of squamous epithelium in an ablation model.

In our novel ablation mouse model we also studied if inhibition of the BMP pathway, by using Noggin an inhibitor for BMPs that at least inhibits BMP2 and 4 and could result in squamous re-epithelization instead of columnar epithelium. A total of 44 C57BL/6J mice were used. All mice were given PPI to reduce the acidity in the stomach through damage healing and to mimic the human situation. Mice stomach were ablated just proximal to the squamocolumnar junction (SCJ) and sacrificed and treated for 14 days with Noggin. We observed that after ablation stomachs were properly closed and no leakage of stomach content was observed. Stomachs were completely healed. During the healing process we treated the mice with either saline (control group) or Noggin (treatment group). At day 14 we were able to investigate the re-population of the ablation area. In the control group we observed that, after ablation, the stomach healed normally being able to recognize the columnar part, the SCJ and the neo-squamous cells. Like in the animals treated with BMP2/4, treatment with Noggin resulted in re-epithelization with squamous cells observed at the ablation proximal to the original SCJ, where normally columnar cells reside. The presence of neo-squamous epithelium was validated by immunohistochemistry

(IHC). Stainings were negative for the columnar marker K19 and positive for the squamous markers p63 and K5.

B: Deletion of noggin results in multi-layered glands at the SCJ in mice To study the effects of BMPs in adult mice we developed a conditional noggin knockout mouse.

Hereto, Rosa26-cre mice were crossed with loxp-noggin-loxp mice (fig4A). Upon tamoxifen injection, immunohistochemistry (IHC) for noggin was negative confirming the deletion of noggin.

We found no major differences in morphology and histology of the esophagus, intestine and colon of the conditional noggin knockout mice. However, 8- 12 weeks after deletion of noggin, mice development columnar metaplasia that resembles the human Barrett's esophagus at the SCJ. The metaplastic epithelium seem to arise from multi-lineage glands (MLGs) (Fig4B). As shown before (Mari 2014), the MLGs consist of an outer layer staining positive for squamous markers, including K5, K14 and p63 (Fig4). The inner layer stains positive for columnar markers including K19, K8, PAS and Alcian Blue (Fig4D).

In the Noggin-/- mice, the ki67+ proliferating cells which gave rise to the columnar metaplasia were mainly observed in the columnar layer of the MLGs.

Inhibition of BMP2 and 4 drives the development of squamous epithelium in a conditional Noggin knockout model

To investigate if we could divert the multi layered epithelium in the noggin KO model towards squamous epithelium we treated the Noggin-/- mice with a the anti-bmp2/4 inhibitor that specifically inhibits BMP2 and 4. After 8 weeks of systemic treatment the mice BMP signalling was decreased demonstrated by decreased pSmadl/5/8 staining (Fig4E). Inhibition of BMP2/4 resulted in increased proliferative activity in the squamous layer. After 8 weeks we found expansion of the squamous epithelium with papillae like structures appearing from the multi-lineage glands (Fig4C,D, E). These results indicate that inhibition of BMP2 and 4 is sufficient to increase proliferation and regeneration of squamous epithelium in a model with increased activity of several BMPs due to absence of Noggin.

References

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