TMEM219 antibodies for medical use

Abstract

The present disclosure relates to antibodies or antigen binding fragments thereof that bind to TMEM219 receptor for use in the treatment and / or prevention of colorectal cancer and / or reduction of risk of developing colorectal cancer. Pharmaceutical compositions comprising said antibody for use in the treatment and / or prevention of colorectal cancer are also herein disclosed.

Description

[0001] TMEM219 ANTIBODIES FOR MEDICAL USE

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to antibodies or antigen binding fragments thereof that bind specifically to the IGFBP3 receptor, namely TMEM219, for use in the treatment of colorectal cancer.

[0004] BACKGROUND ART

[0005] Colorectal cancer (CRC) represents the third most commonly diagnosed type of cancer both in males and females (6% of all cancer subtypes) after lung (11 .4%), breast cancer in women (11 .6%) and prostate cancer in men (7.3%). However, colorectal cancer is the second in terms of mortality (9.4%) after lung (18%), and the incidence is increasing globally despite a substantial geographical variety between different countries (1 ). In fact, colorectal cancer can be considered a marker of a country’s socioeconomical development given that the most implicated risk factors, apart from family history, are linked to lifestyle and unhealthy dietary patterns such as high alcohol, red meat and processed food consumption, cigarette smoking, low physical activity, hyperlipidemia, obesity and diabetes (1 ,2,3).

[0006] Recent studies highlighted how CRC incidence in some countries has decreased from 1980s to 2019 linking it to a change in lifestyle, recommended colonoscopy screening and subsequent early lesion removal (4). On the other hand, even though the trend has improved for adults above 50 years, CRC new diagnoses are increasing for adults aged <50 years (1 ,4) as well as the incidence over the years has been constantly rising in many countries in Eastern Europe, Southeastern and South-Central Asia, and South America (1 ), probably due to a shift towards a western lifestyle (2).

[0007] Chronic inflammation, such as chronic inflammation that occurs in inflammatory bowel disease (IBD), is generally recognised as an important risk factor for the development of colitis-associated colorectal cancer (CAC), a specific type of CRC (5). In fact, data show that IBD patients present both a higher risk of CAC / CRC development (2-fold increase) and an overall worse outcome compared to non-IBD patients (1 .7-fold increase of death cases) (5).

[0008] Over the years, mouse models, such as the azoxymethane / dextran sulfate sodium model (AOM / DSS model), have been considered very informative to investigate the tumorigenesis. In order for tumorigenesis to happen, two events are necessary. The first is a tumor-initiating event that involves mutations or epigenetic alterations and leads to oncogene hyperactivation or inactivation of tumor suppressor genes. This step is usually followed by tumor promotion, in which the cells harbouring those mutations outgrow the healthy ones (6).

[0009] Inflammation can trigger a series of detrimental mechanisms, such as DNA damage initiation and epigenetic silencing of important tumor suppressor genes in intestinal epithelial cells (lECs) (6). Moreover, chronic inflammation increases oxidative stress, leads to exposure to genotoxic compounds released by inflammatory cells, causes the intestinal barrier breakdown which leads to the interaction between lECs and pathogenic microorganisms (6). Finally, chronic inflammation triggers excessive tissue regeneration and dedifferentiation of non-stem cells into stem-like ones as a defence mechanism due to the presence of damaged tissue. This leads to tumor promotion, through the proliferation and clonal expansion of mutated cells (6). In this process, IBD patient colon can develop anatomic abnormalities such as polyps and colon shortening, therefore routine screening through colonoscopy is one of the best ways to prevent and monitor the disease. However, recent studies showed that, in contrast to sporadic CRC, CAC evolves from polymorphous non-adenoma-like dysplastic lesions instead of polypoid adenoma, making the detection through colonoscopy much more difficult (5).

[0010] The most effective treatment is the complete surgical removal of the lesions and local metastases; unfortunately, despite the extensive colonoscopy screening programs, nearly a quarter of CRCs are diagnosed at an advanced stage, requiring adjuvant chemo-, targeted / immune- or radiation therapy (7,8). In the first case, current guidelines include both a single-agent and multiple-agent therapy, the latter recommended for Stage II and III colon cancers7. In fact, fluoropyrimidine (5-Fll) is either employed by itself or in combination with oxaliplatin (OX), irinotecan (IRI), leucovorin (LV) and capecitabine (CAP or XELODA or XEL) (7,8,9). On the other hand, adjuvant radiation therapy only seems to be effective in certain high-risk group of CRC, especially if in combination with surgery and chemotherapy (7). Both chemo- and radiotherapy present important side effects such as systemic toxicity, low tumor-specific selectivity, low success rates and high resistance. On the other hand, novel therapies are being developed to target specific dysregulated pathways or mutated proteins / receptors, helping regulate cell proliferation, differentiation and migration and alter the tumor microenvironment (7). Targeted therapies consist mainly in monoclonal antibodies or small molecules such as bevacizumab or aflibercept (anti-VEGF mAb or small molecule respectively), cetuximab or panitumumab (anti-EGFR, affecting tyrosine kinase signaling at the surface of the cell membrane), ramucirumab (anti-VEGFR2) or BRAF- inhibitors, among others (7,8). Potential challenges regarding the targeted therapies include for example adverse events, cellular bypass mechanisms between pathways leading to acquired resistance and efficacy differences among people (8).

[0011] In addition to CRC treatments, a few therapies are currently under investigation for CAC. The first and the most successful ones are 5-aminosalicylic acid and immunomodulators such as thiopurines, which are usually used for the treatment of mild to moderate IBD and have been associated with a reduction in CAC, albeit with some conflicting results (5). In addition to that, the chemopreventive effect of thalidomide is also under investigation. This is due to its therapeutic effect in the treatment of moderate UC and CD, linked to the suppression of macrophage polarization in the tumor microenvironment, reducing inflammation and promoting mucosal healing (10). There are conflicting results in the use of biological agents commonly used for treatment of IBD such as anti-TNFa, JAK inhibitor, IL-12 / IL-23 antagonist and anti-integrin a4|37, even though in most cases they appear to have no protective effect against CAC (5). Finally, meta-analysis and cohort studies focused on other IBD treatments such as corticosteroids, folic acid, Non-Aspirin Nonsteroidal Anti-inflammatory Drugs (NA- NSAIDs), calcium supplements and statins showed no statistical effect (5,11 ).

[0012] WO2021 094620 discloses antibodies against TMEM219 receptor and their use for the treatment of diabetes and intestinal or bowel disorders. However, as mentioned above, most of known treatments for IBD proved ineffective for the treatment of colorectal cancer or provided conflicting results.

[0013] In view of the above, it is evident that, despite many technical advances in the field, a molecule for use in the treatment of colorectal cancer, in particular of colitis-associated colorectal cancer, is still needed.

[0014] SUMMARY

[0015] It has now been found that an anti-TMEM219 antibody in an AOM / DSS model of colon carcinogenesis significantly reduces tumor density, size and progression following induction of chronic inflammation.

[0016] Accordingly, in a first aspect, it is herein disclosed an antibody that binds to TMEM219 receptor, or an antigen binding fragment thereof, for use in the prevention and / or treatment of colorectal cancer, and / or for use in reducing risk of developing colorectal cancer, such as colitis-associated colorectal cancer (CAC), in patients with inflammatory bowel disease (IBD). In an embodiment, said antibody inhibits or reduces the binding of IGFBP3 to said TMEM219 receptor.

[0017] The isolated antibody or antigen binding fragment thereof inhibits, reduces, or neutralizes the activation of the TMEM219 receptor induced by binding of IGFBP3.

[0018] Activation of the TMEM219 receptor induced by IGFBP3 may be measured by any known method in the art or as described below. In particular, IGFBP3-induced activation of a TMEM219 receptor may be measured by measuring apoptosis increase as described herein or decrease in minigut growth as known in the art and described in several publications (23,24, 25, 26).

[0019] The isolated antibody or antigen binding fragment thereof as herein disclosed does not activate TMEM219 pathway upon binding to human TMEM219.

[0020] In an embodiment, said colorectal cancer is selected from the group consisting of colon cancer, bowel cancer, and rectal cancer. In a preferred embodiment, said cancer is colitis-associated colorectal cancer.

[0021] In an embodiment the antibody as herein disclosed comprises: a. a heavy chain variable domain (VH) comprising: i. a CDR1 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 8, 1 , 10, 56, 59, 62, 65 and 68; ii. a CDR2 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 2, 11 , 57, 60, 63, 66 and 69; and iii. a CDR3 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, 3, 7, 9, 12, 13, 58, 61 , 64, 67 and 70; and / or b. a light chain variable domain (VL) comprising: i. a CDR1 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 17, 14, 20, 23, 26, 29, 71 , 77, 80, 82 and 85; ii. a CDR2 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 18, 15, 21 , 24, 27, 30, 72, 78, 83 and 86; and iii. a CDR3 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 16, 22, 25, 28, 31 , 73, 74, 75, 76, 79, 81 , 84, 87 and 166.

[0022] In a further embodiment, said antibody as herein disclosed comprises (a) a heavy chain variable region (VH) comprising a VHCDR1 , a VHCDR2, a VHCDR3, and (b) a light chain variable region (VL) comprising a VLCDR1 , a VLCDR2, and a VLCDR3, wherein: - VHCDR1 consists of the sequence of SEQ ID NO: 8, VHCDR2 consists of the sequence of SEQ ID NO: 5, VHCDR3 consists of the sequence of SEQ ID NO: 6, VLCDR1 consists of the sequence of SEQ ID NO: 17, VLCDR2 consists of the sequence of SEQ ID NO: 18 and VLCDR3 consists of the sequence of SEQ ID NO: 19 or IMGT, Chothia, AbM, or Contact CDRs of TC01 , TC04, TC05; or wherein:

[0023] - VHCDR1 consists of the sequence of SEQ ID NO: 8, VHCDR2 consists of the sequence of SEQ ID NO: 5, VHCDR3 consists of the sequence of SEQ ID NO:

[0024] 6, VLCDR1 consists of the sequence of SEQ ID NO: 17, VLCDR2 consists of the sequence of SEQ ID NO: 18 and VLCDR3 consists of the sequence of SEQ ID NO: 166 or IMGT, Chothia, AbM, or Contact CDRs of TC03; or wherein:

[0025] - VHCDR1 consists of the sequence of SEQ ID NO: 1 , VHCDR2 consists of the sequence of SEQ ID NO: 2, VHCDR3 consists of the sequence of SEQ ID NO: 3, VLCDR1 consists of the sequence of SEQ ID NO: 14, VLCDR2 consists of the sequence of SEQ ID NO: 15, and VLCDR3 consists of the sequence of SEQ ID NO: 16 or IMGT, Chothia, AbM, or Contact CDRs of TA02; or wherein:

[0026] - VHCDR1 consists of the sequence of SEQ ID NO: 1 , VHCDR2 consists of the sequence of SEQ ID NO: 2, VHCDR3 consists of the sequence of SEQ ID NO:

[0027] 7, VLCDR1 consists of the sequence of SEQ ID NO: 20, VLCDR2 consists of the sequence of SEQ ID NO: 21 , and VLCDR3 consists of the sequence of SEQ ID NO: 22 or IMGT, Chothia, AbM, or Contact CDRs of TC02; or wherein:

[0028] - VHCDR1 consists of the sequence of SEQ ID NO: 8, VHCDR2 consists of the sequence of SEQ ID NO: 5, VHCDR3 consists of the sequence of SEQ ID NO: 9, VLCDR1 consists of the sequence of SEQ ID NO: 23, VLCDR2 consists of the sequence of SEQ ID NO: 24, and VLCDR3 consists of the sequence of SEQ ID NO: 25 or IMGT, Chothia, AbM, or Contact CDRs of TD01 ; or wherein:

[0029] - VHCDR1 consists of the sequence of SEQ ID NO: 10, VHCDR2 consists of the sequence of SEQ ID NO: 11 , VHCDR3 consists of the sequence of SEQ ID NO:

[0030] 12, VLCDR1 consists of the sequence of SEQ ID NO: 26, VLCDR2 consists of the sequence of SEQ ID NO: 27, and VLCDR3 consists of the sequence of SEQ ID NO: 28 or IMGT, Chothia, AbM, or Contact CDRs of TE01 ; or wherein:

[0031] - VHCDR1 consists of the sequence of SEQ ID NO: 8, VHCDR2 consists of the sequence of SEQ ID NO: 5, VHCDR3 consists of the sequence of SEQ ID NO:

[0032] 13, VLCDR1 consists of the sequence of SEQ ID NO: 29, VLCDR2 consists of the sequence of SEQ ID NO: 30, and VLCDR3 consists of the sequence of SEQ ID NO: 31 or IMGT, Chothia, AbM, or Contact CDRs of TG02; or wherein:

[0033] - VHCDR1 consists of the sequence of SEQ ID NO: 56, VHCDR2 consists of the sequence of SEQ ID NO: 57, VHCDR3 consists of the sequence of SEQ ID NO: 58, VLCDR1 consists of the sequence of SEQ ID NO: 71 , VLCDR2 consists of the sequence of SEQ ID NO: 72, and VLCDR3 consists of the sequence of SEQ ID NO: 73 or IMGT, Chothia, AbM, or Contact CDRs of TE02.1 ; or wherein:

[0034] - VHCDR1 consists of the sequence of SEQ ID NO: 56, VHCDR2 consists of the sequence of SEQ ID NO: 57, VHCDR3 consists of the sequence of SEQ ID NO: 58, VLCDR1 consists of the sequence of SEQ ID NO: 71 , VLCDR2 consists of the sequence of SEQ ID NO: 72, and VLCDR3 consists of the sequence of SEQ ID NO: 74 or IMGT, Chothia, AbM, or Contact CDRs of TE02.2; or wherein:

[0035] - VHCDR1 consists of the sequence of SEQ ID NO: 56, VHCDR2 consists of the sequence of SEQ ID NO: 57, VHCDR3 consists of the sequence of SEQ ID NO: 58, VLCDR1 consists of the sequence of SEQ ID NO: 71 , VLCDR2 consists of the sequence of SEQ ID NO: 72, and VLCDR3 consists of the sequence of SEQ ID NO: 75 or IMGT, Chothia, AbM, or Contact CDRs of TE02.3; or wherein:

[0036] - VHCDR1 consists of the sequence of SEQ ID NO: 59, VHCDR2 consists of the sequence of SEQ ID NO: 60, VHCDR3 consists of the sequence of SEQ ID NO: 61 , VLCDR1 consists of the sequence of SEQ ID NO: 71 , VLCDR2 consists of the sequence of SEQ ID NO: 72, and VLCDR3 consists of the sequence of SEQ ID NO: 76 or IMGT, Chothia, AbM, or Contact CDRs of TE03; or wherein:

[0037] - VHCDR1 consists of the sequence of SEQ ID NO: 56, VHCDR2 consists of the sequence of SEQ ID NO: 57, VHCDR3 consists of the sequence of SEQ ID NO: 58, VLCDR1 consists of the sequence of SEQ ID NO: 77, VLCDR2 consists of the sequence of SEQ ID NO: 78, and VLCDR3 consists of the sequence of SEQ ID NO: 79 or IMGT, Chothia, AbM, or Contact CDRs of TE04; or wherein:

[0038] - VHCDR1 consists of the sequence of SEQ ID NO: 62, VHCDR2 consists of the sequence of SEQ ID NO: 63, VHCDR3 consists of the sequence of SEQ ID NO: 64, VLCDR1 consists of the sequence of SEQ ID NO: 80, VLCDR2 consists of the sequence of SEQ ID NO: 78, and VLCDR3 consists of the sequence of SEQ ID NO: 81 or IMGT, Chothia, AbM, or Contact CDRs of TE07; or wherein:

[0039] - VHCDR1 consists of the sequence of SEQ ID NO: 65, VHCDR2 consists of the sequence of SEQ ID NO: 66, VHCDR3 consists of the sequence of SEQ ID NO: 67, VLCDR1 consists of the sequence of SEQ ID NO: 82, VLCDR2 consists of the sequence of SEQ ID NO: 83, and VLCDR3 consists of the sequence of SEQ ID NO: 84 or IMGT, Chothia, AbM, or Contact CDRs of TE10; or wherein:

[0040] - VHCDR1 consists of the sequence of SEQ ID NO: 68, VHCDR2 consists of the sequence of SEQ ID NO: 69, VHCDR3 consists of the sequence of SEQ ID NO: 70, VLCDR1 consists of the sequence of SEQ ID NO: 85, VLCDR2 consists of the sequence of SEQ ID NO: 86 and VLCDR3 consists of the sequence of SEQ ID NO: 87 or IMGT, Chothia, AbM, or Contact CDRs of TM1 .

[0041] Above disclosed VHCDRs and VLCDRs are defined according to Kabat.

[0042] Preferably the isolated antibody or antigen binding fragment thereof comprises the CDRs as indicated in any of Tables 1 -6.

[0043] In another embodiment, the disclosed antibody comprises: a. a heavy chain variable domain sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 33, SEQ ID NO: 168, SEQ ID NO: 32, SEQ ID NO: 34 to SEQ ID NO: 37 or SEQ ID NO: 88 to SEQ ID NO: 95; or b. a light chain variable domain sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 39, SEQ ID NO: 171 , SEQ ID NO: 172, SEQ ID NO: 38, SEQ ID NO: 40 to SEQ ID NO:43, or SEQ ID NO: 96 to SEQ ID NO: 103; or c. the heavy chain variable domain of (a) and the light chain variable domain of (b).

[0044] Preferably, the disclosed antibody comprises (a) a VH sequence of the amino acid sequence and (b) a VL sequence of the amino acid sequence, wherein:

[0045] - VH consists of the sequence of SEQ ID NO: 33 and VL consists of the sequence of SEQ ID NO: 39; or wherein:

[0046] - VH consists of the sequence of SEQ ID NO: 168 and VL consists of the sequence of SEQ ID NO: 171 ; or wherein:

[0047] - VH consists of the sequence of SEQ ID NO: 168 and VL consists of the sequence of SEQ ID NO: 172; or wherein:

[0048] - VH consists of the sequence of SEQ ID NO: 170 and VL consists of the sequence of SEQ ID NO: 39; or wherein:

[0049] - VH consists of the sequence of SEQ ID NO: 32 and VL consists of the sequence of SEQ ID NO: 38; or wherein: - VH consists of the sequence of SEQ ID NO: 34 and VL consists of the sequence of SEQ ID NO: 40; or wherein:

[0050] - VH consists of the sequence of SEQ ID NO: 35 and VL consists of the sequence of SEQ ID NO: 41 ; or wherein:

[0051] - VH consists of the sequence of SEQ ID NO: 36 and VL consists of the sequence of SEQ ID NO: 42; or wherein:

[0052] - VH consists of the sequence of SEQ ID NO: 37 and VL consists of the sequence of SEQ ID NO: 43; or wherein:

[0053] - VH consists of the sequence of SEQ ID NO: 88 and VL consists of the sequence of SEQ ID NO: 96; or wherein:

[0054] - VH consists of the sequence of SEQ ID NO: 88 and VL consists of the sequence of SEQ ID NO: 97; or wherein:

[0055] - VH consists of the sequence of SEQ ID NO: 88 and VL consists of the sequence of SEQ ID NO: 98; or wherein:

[0056] - VH consists of the sequence of SEQ ID NO: 91 and VL consists of the sequence of SEQ ID NO: 99; or wherein:

[0057] - VH consists of the sequence of SEQ ID NO: 88 and VL consists of the sequence of SEQ ID NO: 100; or wherein:

[0058] - VH consists of the sequence of SEQ ID NO: 93 and VL consists of the sequence of SEQ ID NO: 101 ; or wherein:

[0059] - VH consists of the sequence of SEQ ID NO: 94 and VL consists of the sequence of SEQ ID NO: 102; or wherein:

[0060] - VH consists of the sequence of SEQ ID NO: 95 and VL consists of the sequence of SEQ ID NO: 103. also herein disclosed an antibody wherein:

[0061] - the nucleotide sequence encoding VH comprises SEQ ID NO: 45 and the nucleotide sequence encoding VL comprises SEQ ID NO: 51 ; or wherein:

[0062] - the nucleotide sequence encoding VH comprises SEQ ID NO: 174 and the nucleotide sequence encoding VL comprises SEQ ID NO: 177; or wherein:

[0063] - the nucleotide sequence encoding VH comprises SEQ ID NO: 175 and the nucleotide sequence encoding VL comprises SEQ ID NO: 178 or wherein:

[0064] - the nucleotide sequence encoding VH comprises SEQ ID NO: 176 and the nucleotide sequence encoding VL comprises SEQ ID NO: 179 or wherein: - the nucleotide sequence encoding VH comprises SEQ ID NO: 44 and the nucleotide sequence encoding VL comprises SEQ ID NO: 50; or wherein:

[0065] - the nucleotide sequence encoding VH comprises SEQ ID NO: 46 and the nucleotide sequence encoding VL comprises SEQ ID NO: 52; or wherein:

[0066] - the nucleotide sequence encoding VH comprises SEQ ID NO: 47 and the nucleotide sequence encoding VL comprises SEQ ID NO: 53; or wherein:

[0067] - the nucleotide sequence encoding VH comprises SEQ ID NO: 48 and the nucleotide sequence encoding VL comprises SEQ ID NO: 54; or wherein:

[0068] - the nucleotide sequence encoding VH comprises SEQ ID NO: 49 and the nucleotide sequence encoding VL comprises SEQ ID NO: 55; or wherein:

[0069] - the nucleotide sequence encoding VH comprises SEQ ID NO: 104 and the nucleotide sequence encoding VL comprises SEQ ID NO: 112; or wherein:

[0070] - the nucleotide sequence encoding VH comprises SEQ ID NO: 105 and the nucleotide sequence encoding VL comprises SEQ ID NO: 113; or wherein:

[0071] - the nucleotide sequence encoding VH comprises SEQ ID NO: 106 and the nucleotide sequence encoding VL comprises SEQ ID NO: 114; or wherein:

[0072] - the nucleotide sequence encoding VH comprises SEQ ID NO: 107 and the nucleotide sequence encoding VL comprises SEQ ID NO: 115; or wherein:

[0073] - the nucleotide sequence encoding VH comprises SEQ ID NO: 108 and the nucleotide sequence encoding VL comprises SEQ ID NO: 116; or wherein:

[0074] - the nucleotide sequence encoding VH comprises SEQ ID NO: 109 and the nucleotide sequence encoding VL comprises SEQ ID NO: 117; or wherein:

[0075] - the nucleotide sequence encoding VH comprises SEQ ID NO: 110 and the nucleotide sequence encoding VL comprises SEQ ID NO: 118; or wherein:

[0076] - the nucleotide sequence encoding VH comprises SEQ ID NO: 111 and the nucleotide sequence encoding VL comprises SEQ ID NO: 119.

[0077] Still preferably the isolated antibody is TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 or antigen binding fragment thereof, preferably the isolated antibody is TC01 , TC05, TC03, TC04 or TM1 or antigen binding fragment thereof, as reported in Tables 1-8. Preferably the isolated antibody is TC01 .

[0078] In an embodiment the disclosed antibody is a human or a humanized antibody and / or is an lgG1 or lgG2 or lgG4 antibody, preferably an lgG1 kappa antibody, an lgG1 lambda antibody, an lgG2 kappa antibody, an lgG2 lambda antibody, an lgG4 kappa antibody or an lgG4 lambda antibody.

[0079] In an embodiment said lgG1 antibody comprises at least one amino-acid substitution in the heavy chain constant region (Fc) of the lgG1 antibody, wherein the at least one amino acid substitution is capable of reducing Fc receptor binding; or wherein the at least one amino acid substitution is capable of reducing effector cell function of the antibody; or wherein the at least one amino acid substitution is capable of extending the half-life of the antibody.

[0080] In an embodiment said at least one amino acid substitution capable of extending the half-life of the antibody is selected from the following group: M252Y, S254T or T256E, where the numbering of the residues is according to the Ell numbering system.

[0081] In an embodiment the antibody as herein disclosed comprises:

[0082] - a light chain comprising a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 171 , SEQ ID NO: 172, SEQ ID NO: 38, SEQ ID NO: 40 to SEQ ID NO: 43, or SEQ ID NO: 96 to SEQ ID NO: 103; and a light constant domain comprising or consisting of a sequence of SEQ ID NO: 123 and / or

[0083] - a heavy chain comprising a heavy chain variable domain comprising a sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 168, SEQ ID NO: 32, SEQ ID NO: 34 to SEQ ID NO: 37, SEQ ID NO: 88, SEQ ID NO: 91 , or SEQ ID NO: 93 to SEQ ID NO: 95; and a heavy chain constant domain comprising or consisting of a sequence of SEQ ID NO: 120 or of SEQ ID NO: 180.

[0084] Preferably, the antibody as herein disclosed comprises:

[0085] - a light chain comprising a light chain variable domain comprising or consisting of a sequence of SEQ ID NO: 39 and a light chain constant domain comprising or consisting of a sequence of SEQ ID NO: 123 and / or

[0086] - a heavy chain comprising a heavy chain variable domain comprising or consisting of a sequence of SEQ ID NO: 33 and a heavy chain constant domain comprising or consisting of a sequence of SEQ ID NO: 120 or of SEQ ID NO: 180. Even more preferably, the disclosed antibody comprises a light chain comprising or consisting of a sequence of SEQ ID NO: 4 and / or a heavy chain comprising or consisting of a sequence of SEQ ID NO: 125 or of SEQ ID NO: 126.

[0087] It is also herein disclosed an isolated polynucleotide comprising at least one sequence that encodes the antibody or antigen binding fragment thereof as disclosed herein.

[0088] Preferably said polynucleotide is a cDNA, or a vector comprising said polynucleotide. Preferably said vector is selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, an expression vector and a recombinant expression vector, or an isolated cell comprising said polynucleotide or said vector.

[0089] Preferably, said isolated cell being a hybridoma or a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney cell (HEK293), for use in the prevention and / or treatment of colorectal cancer.

[0090] It is also herein disclosed a pharmaceutical composition comprising an antibody that binds to TMEM219 receptor, or an antigen-binding fragment thereof, as herein disclosed and at least one pharmaceutically acceptable carrier for use in the prevention and / or treatment of colorectal cancer and / or for use in reducing risk of developing colorectal cancer, such as colitis-associated colorectal cancer (CAC), in patients with inflammatory bowel disease (IBD). In a preferable embodiment said pharmaceutical composition further comprises a second therapeutic agent.

[0091] The disclosure provides a method of prevention and / or treatment of colorectal cancer, and / or for use in reducing risk of developing colorectal cancer, such as colitis-associated colorectal cancer (CAC), in patients with inflammatory bowel disease (IBD), the method comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount an antibody that binds to TMEM219, for example as defined above, or the isolated polynucleotide or the vector or the isolated cell as defined above, and at least one pharmaceutically acceptable carrier, or administering to a subject in need thereof an antibody that binds to TMEM219 or the isolated polynucleotide or the vector or the isolated cell as defined above.

[0092] In an embodiment, the anti-TMEM219 antibody molecule for use for the prevention and / or treatment of colorectal cancer, and / or for use in reducing risk of developing colorectal cancer, such as colitis-associated colorectal cancer (CAC), in patients with inflammatory bowel disease (IBD), is used in combination with at least one further therapeutic agent. The additional therapeutic agent may be any active ingredient which is known to be effective in the prevention and / or treatment of cancer, for example it can be a chemotherapeutic agent or a radiotherapy agent. In a preferred embodiment, it is an agent commonly used or useful in the prevention and / or treatment of colorectal cancer. For example it can be selected from fluoropyrimidine (5-Fll), oxaliplatin (OX), irinotecan (IRI), leucovorin (LV), capecitabine (CAP or XELODA or XEL), anti-VEGF monoclonal antibodies or anti-VEGF small molecules, such as bevacizumab or aflibercept, cetuximab, panitumumab, nimotuzumab, necitumumab (anti-EGFR, affecting tyrosine kinase signaling at the surface of the cell membrane), ramucirumab (anti-VEGFR2), regorafenib (tyrosine kinase inhibitor targeting VEGFR, platelet-derived growth factor, fibroblast growth factor, and BRAF) or BRAF-inhibitors (encorafenib), 5-aminosalicylic acid, immunomodulators such as thiopurines and immune checkpoint inhibitors (pembrolizumab).

[0093] DETAILED DESCRIPTION

[0094] Figures

[0095] Figure 1. Schematic representation of the AOM / DSS murine model of colitis-associated cancer.

[0096] Figure 2. Schematic representation of the AOM / DSS murine model of colitis-associated cancer, including dosing regimen.

[0097] Figure 3. Schematic representation of proximal and distal colon used in this study.

[0098] Figure 4. Schematic representation of the AOM / DSS murine model of colitis-associated cancer, including the multistep tumor progression, represented on the timeline and based on the GIN-adenoma-carcinoma sequence. GIN: Gastrointestinal intraepithelial neoplasia; LGA: Low-grade adenoma; HGA: High-grade adenoma; K: Adenocarcinoma. Dark grey dotted lines delineate specific tumoral lesions.

[0099] Figure 5. Percentage of body weight loss (A) and Disease Activity Index (DAI) (B) quantified at the indicated time points. Values are mean ± SEM (n=8 per group). The vertical dotted line represents the initiation of Ent001 (alias TC01 ) or anti-TNFalpha administration.

[0100] Figure 6. DAI quantified at sacrifice (Day 56). Values are mean ± SEM (n=8 mice / groups). P:

[0101] **** p<0.0001 vs AOM / DSS + vehicle and

[0102] AAAAp<0.0001 vs AOM / DSS + EntOOl by one-way ANOVA with multiple comparisons. Figure 7. Colon length at sacrifice (Day 56). Values are mean ± SEM (n=8 per group). P:

[0103] **** p<0.0001 vs AOM / DSS+ vehicle;

[0104] AAAp<0.001 vs AOM / DSS + EntOOl by one-way ANOVA with multiple comparisons.

[0105] Figure 8. Graph representing the total histological score of colitis at sacrifice (Day 56) calculated by Rachimilewitz scoring system. Values are mean ± SEM (n=7 per group) vs AOM / DSS+ vehicle.

[0106] Figure 9. Graphs represent histological grading of colitis at sacrifice (Day 56). (A) Total histological score (B) Severity of inflammation score (C) Extent of inflammation score (D) Regeneration score, (E) Crypt damage. Values are mean ± SEM (n=7 per group). P:

[0107] **** p<0.0001 vs AOM / DSS+ vehicle; by one-way ANOVA with multiple comparisons.

[0108] Figure 10. Representative H&E-stained images of distal colons from the indicated groups of mice at the end of the experiment (Day 56). M: Mucosa; SM; Submucosa; Mu: Muscular layer or muscularis propria.

[0109] Figure 11. Total number of tumors per mouse analyzed at sacrifice (Day 56). Values are mean ± SEM (n=7 per group). P:

[0110] ****p<0,0001 vs AOM / DSS + Vehicle; by one-way ANOVA with multiple comparisons.

[0111] Figure 12. Number of tumors per mouse divided by tumor grade at sacrifice (Day 56).

[0112] Values are mean ± SEM (n=7 per group). P:

[0113] ****p<0.0001 and

[0114] ** p<0.01 vs AOM / DSS + Vehicle; by two-way ANOVA with multiple comparisons.

[0115] GIN: glandular intraepithelial neoplasia; LGA: Low-grade adenoma; HGA: High-grade adenoma; K: carcinoma.

[0116] Figure 13. Tumor size at sacrifice (Day 56). Values are mean ± SEM (n=7 per group). P:

[0117] ****p<0.0001 and

[0118] **p<0.01 vs AOM / DSS + Vehicle by two-way ANOVA with multiple comparisons.

[0119] LGA: Low-grade adenoma; HGA: High-grade adenoma; K: carcinoma. Figure 14. Representative H&E images of colons showing different LGA, HGA and K sizes in the indicated groups. Dotted red lines delineate tumors. Scale bar: 100 pm.

[0120] Figure 15. Number of Brdll+ nuclei / mm2in inflammatory areas at sacrifice (Day 56). Values are mean ± SEM (n=4 per group). P:

[0121] ****<0.0001 vs AOM / DSS + Vehicle by one-way ANOVA with multiple comparisons.

[0122] Figure 16. Representative Brdll-stained images of colons showing Brdll-positive epithelial cells in inflammatory areas of AOM / DSS -treated mice in the indicated groups. Scale bar: 100 pm.

[0123] Figure 17. Number of Brdll-positive nuclei / LGA area in AOM / DSS-treated mice at sacrifice (Day 56). Values are mean ± SEM (n=4 per group except for the Ent001 group in which only 3 mice displayed LGA). P:

[0124] **p<0.01 vs AOM / DSS + Vehicle by one-way ANOVA with multiple comparisons.

[0125] Figure 18. Representative Brdll-stained images of colons showing Brdll-positive epithelial cells in LGA tumors, in the indicated groups.

[0126] Figure 19. Ratio of Cleaved Caspase 8 over total Caspase 8 in colon lysate at sacrifice (Day 56). Values in the upper panel were obtained by densitometric analysis using Image J (pool of n=4 colon lysates). The ratio between cleaved and total Caspase 8 has been normalized over actin, as loading control.

[0127] Description

[0128] The inventors found TMEM219 as a therapeutic target for reducing incidence and severity of colorectal cancer consequent to chronic inflammation, in particular colitis- associated colorectal cancer.

[0129] Accordingly, an anti-TMEM219 antibody can be advantageously used for the prevention and / or treatment of colorectal cancer, and / or reducing risk of developing colorectal cancer, such as colitis-associated colorectal cancer (CAC), in patients with inflammatory bowel disease (IBD)These data suggest that treatment of IBD with an anti-TMEM219 antibody offers superior reduction in risk of developing CRC in patients with IBD, and therefore may be preferred as the initial treatment for IBD, such as Crohn’s disease and ulcerative colitis (UC), or may be advantageously deployed as second line (2L) treatment in patients with IBD, either in patients whose response to initial treatment has decreased or in patients whose continue to respond to initial treatment but who are deemed at increased risk of developing CRC. For “colorectal cancer” or “CRC” is intended a cancer which begins in and / or affects at least a portion of the colon and / or of the rectum. The cancer can be at any stage, for example at stage 0, stage I, stage II, stage III or stage IV according to classifications known in the field. Colorectal cancer comprises colon cancer, bowel cancer, and rectal cancer. The cancer can also be a metastasis of another cancer. In an embodiment, the cancer is colitis-associated colorectal cancer. For “colitis-associated colorectal cancer” or “CAC” it is intended a type of colorectal cancer which is preceded by clinically detectable inflammatory bowel disease (IBD), such as Crohn's disease (CD) or Ulcerative colitis.

[0130] “Tumor” and “cancer” are herein used as synonyms.

[0131] For “prevention” is intended that administration of the agent decreases the chance or risk of developing a disease or condition, for example decreases the chance of developing a cancer, in particular a colorectal cancer.

[0132] For “treatment” is intended that administration of the agent improves or cures or reverts a condition or a disease, i.e. it improves or cures or reverts a cancer. “Treatment” includes “reduction in progression” whereby administration of the agent slows down or stops progression of a disease that has already begun. For example, in some embodiments the agent disclosed is administered to a subject who already has cancer and, upon treatment as disclosed herein, the cancer does not develop to a more advanced stage. “Treatment” also encompasses that the disease, such as cancer, is not completely cured but reverts to a less advanced stage.

[0133] Preferably, colorectal cancer is selected from the group consisting of colon cancer, bowel cancer, and rectal cancer.

[0134] Preferably, colorectal cancer is colitis-associated colorectal (CAC) cancer.

[0135] For “antibody that binds to TMEM219” is intended an antibody able to bind and / or block and / or neutralize, at least partially, TMEM219 receptor. Preferably it is an antibody that specifically binds TMEM219.

[0136] TMEM219 is a known receptor of IGFBP3, an insulin-like growth factor binding protein. Anti-TMEM219 antibody or anti-TMEM antibody or antibody against TMEM219 or antibody that binds to TMEM219 are herein used as synonyms.

[0137] By “antibody that specifically binds” TMEM219 is intended that the antibody will not substantially cross react with another, nonhomologous, human polypeptide. By “not substantially cross react” is intended that the antibody or fragment has a binding affinity for a non-homologous protein which is less than 10%, more preferably less than 5%, and even more preferably less than 1 %, of the binding affinity for TMEM219.

[0138] In various embodiments, an antibody that "specifically binds" TMEM219, as used herein, includes antibodies that bind TMEM219 or the extracellular portion thereof, such as ecto- TMEM219, with a KD of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or about 0.5 nM, as measured with an Octet biolayer interferometry device or in a surface plasmon resonance assay, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ) or kinetic exclusion assays or any known method in the art.

[0139] The term "antibody" herein is used in the broadest sense understood in the art, including all polypeptides described as antibodies.

[0140] For example, the term “antibody”, as used herein, encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as the fragment exhibits the desired antigen-binding activity (antigen-binding fragments). The term has its broadest art- recognized meaning and includes all known formats, including, without limitation: bivalent monospecific monoclonal antibodies, bivalent bispecific antibodies, trivalent trispecific antibodies, F(ab) fragments, F(ab)’2 fragments, scFv fragments, diabodies, single domain antibodies, including camelid VHH single domain antibodies, tandAbs, and flexibodies.

[0141] The terms "antigen-binding fragment" of an antibody or equivalently "antigen-binding portion" of an antibody and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that comprises a portion of an antibody and that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0142] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.

[0143] In particular embodiments, an antigen-binding fragment of an antibody comprises at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody include: (i) VH- CH1 ; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1 -CH2-CH3; (vi) VH- CH2-CH3; (vii) VH-CL; (viii) VL-CH1 ; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1 -CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may in various embodiments consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody may in various embodiments comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0144] The term “antigen-binding fragment” of an antibody further includes single domain antibodies.

[0145] A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. In some embodiments, the single-domain antibody is derived from the variable domain of the antibody heavy chain from camelids (also termed nanobodies, or VHH fragments). In some embodiments, the single-domain antibody is an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, and bivalent nanobodies), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0146] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0147] The antibody or binding molecule as herein disclosed can further be linked to an active substance, preferably a nanoparticle or a radionucleotide.

[0148] As used herein, the term "antigen binding molecule" refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are antibodies, including antigen-binding antibody fragments, and scaffold antigen binding proteins.

[0149] The term "antigen binding moiety" refers to the portion of an antigen binding molecule that specifically binds to an antigenic determinant. Antigen binding moieties include antibodies and antigen-binding fragments thereof, such as scFv, that are capable of specific binding to an antigen on a target cell. In a particular aspect, the antigen binding moiety is able to direct the entity to which it is attached, such as a cell, to a target site. Anti-TMEM219 antibodies are known in the field. For example, anti-TMEM219 antibodies are described in WO2021094620.

[0150] Preferred anti-TMEM219 antibodies for the use as herein disclosed are TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as disclosed in WO2021094620 and herein in Tables 1 -8, or encoded by the nucleotide sequences in Tables 9-10. A preferred anti-TMEM219 antibody is TC01 , also herein named as EntOOI .

[0151] In a preferred embodiment, said anti-TMEM219 antibody for the use as herein disclosed is TC01 and it comprises a light chain comprising a light variable region comprising or consisting of a sequence of SEQ ID NO. 39 and a light constant region comprising or consisting of a sequence of SEQ ID NO. 123 and / or a heavy chain comprising a heavy variable region comprising or consisting of a sequence of SEQ ID NO. 33 and a heavy constant region comprising or consisting of a sequence of SEQ ID NO. 120 or of SEQ ID NO. 180. The terms “region” and “domain” are herein used as synonyms.

[0152] In a particular embodiment, said anti-TMEM219 antibody is TC01 , and it comprises at least: a light chain comprising or consisting of the following sequence:

[0153] QAVLTQPPSVSVSPGQTASITCSGDKLGNKNAYWYQQKPGQSPVLVMYQSTRRPS GIPERFSASNSGNTATLTISGTQAMDEADYYCQAWDSSSGWEVFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO. 4) and / or a heavy chain comprising or consisting of the following sequence:

[0154] QIQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNG NTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWGRWLAHDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO. 125) or of the following sequence:

[0155] QIQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNG NTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWGRWLAHDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO. 126)

[0156] In an embodiment, the anti-TMEM219 antibody molecule comprises at least one antigen-binding region, e.g., a variable region or an antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody chosen from any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1-8 or encoded by the nucleotide sequences in Tables 9-10; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0157] In yet another embodiment, the anti-TMEM219 antibody molecule comprises at least one, two, three or four variable regions from an antibody described herein, e.g., an antibody chosen from any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1- 8 or encoded by the nucleotide sequence in Tables 9-10; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0158] In yet another embodiment, the anti-TMEM219 antibody molecule comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody chosen from any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1- 8 or encoded by the nucleotide sequence in Tables 9-10; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0159] In yet another embodiment, the anti-TMEM219 antibody molecule comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody chosen from any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1- 8 or encoded by the nucleotide sequence in Tables 9-10; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

[0160] In yet another embodiment, the anti-TMEM219 antibody molecule includes a heavy chain constant region for an lgG4, e.g., a human lgG4. In one embodiment, the human lgG4 includes a substitution at position 228 (e.g., a Ser to Pro substitution). In one embodiment, the human lgG4 includes a substitution at position 235 (e.g., a Leu to Glu substitution). In one embodiment, the human lgG4 includes a substitution at position 228 (e.g., a Ser to Pro substitution) and a substitution at position 235 (e.g., a Leu to Glu substitution). In still another embodiment, the anti-TMEM219 antibody molecule includes a heavy chain constant region for an IgG 1 , e.g., a human IgG 1 . In one embodiment, the human lgG1 includes a substitution at position 297 (e.g., an Asn to Ala substitution). In one embodiment the human lgG1 includes a substitution at position 250, a substitution at position 428, or both (e.g., a Thr to Gin substitution at position 250 and / or a Met to Leu substitution at position 428). In one embodiment, the human lgG1 includes a substitution at position 234, a substitution at position 235, or both (e.g., a Leu to Ala substitution at position 234 and / or a Leu to Ala substitution at position 235).

[0161] In yet another embodiment, the anti-TMEM219 antibody molecule includes a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the light chain constant region comprises an amino sequence set forth in Table 11 , or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

[0162] In another embodiment, the anti-TMEM219 antibody molecule includes a heavy chain constant region for an lgG4, e.g., a human lgG4, and a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the human lgG1 or lgG4 includes a substitution at the variable region to decrease aggregation, reduce charge heterogeneity, increase affinity and modulate antigen binding; removal by mutation of instability hotspot in the CDR, putative N-glycosylation sites in the variable region as described in (27), incorporated by reference. In one embodiment, the heavy chain constant region comprises an amino sequence set forth in Table 11 , or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

[0163] In another embodiment, the anti-TMEM219 antibody molecule includes a heavy chain variable domain and a constant region, a light chain variable domain and a constant region, or both, comprising the amino acid sequence of any of TC01 , TC03, TC04, TC05, TA02, TC01 , TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1-8, 11 or encoded by the nucleotide sequences in Tables 9- 10; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences. The anti-TMEM219 antibody molecule, optionally, comprises a leader sequence from a heavy chain, a light chain, or both.

[0164] In yet another embodiment, the anti-TMEM219 antibody molecule includes at least one, two, or three complementarity determining regions (CDRs) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1 -8 or encoded by the nucleotide sequence in Tables 9-10; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences. In yet another embodiment, the anti-TMEM219 antibody molecule includes at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising an amino acid sequence shown in Tables 1-8 or encoded by a nucleotide sequence shown in Tables 9-10. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Tables 1 -8 or encoded by a nucleotide sequence shown in Tables 9-10.

[0165] In yet another embodiment, the anti-TMEM219 antibody molecule includes at least one, two, or three CDRs from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any of any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1-8 or encoded by the nucleotide sequence in Tables 9-10; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequence.

[0166] In yet another embodiment, the anti-TMEM219 antibody molecule includes at least one, two, or three CDRs (or collectively all the CDRs) from a light chain variable region comprising an amino acid sequence shown in Tables 1-8 or encoded by a nucleotide sequence shown in Tables 9-10. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Tables 1-8, or encoded by a nucleotide sequence shown in Tables 9-10. In certain embodiments, the anti-TMEM219 antibody molecule includes a substitution in a light chain CDR, e.g., one or more substitutions in a CDR1 , CDR2 and / or CDR3 of the light chain. In another embodiment, the anti-TMEM219 antibody molecule includes at least one, two, three, four, five or six CDRs (or collectively all the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Tables 1 -8 or encoded by a nucleotide sequence shown in Tables 9-10. In one embodiment, one or more of the CDRs (or collectively all the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Tables 1 -8, or encoded by a nucleotide sequence shown in Tables 9-10.

[0167] In one embodiment, the anti-TMEM219 antibody molecule includes all six CDRs from an antibody described herein, e.g., an antibody chosen from any of any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1 -8, or encoded by the nucleotide sequences in Tables 9-10, or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0168] In one embodiment, the anti-TMEM219 antibody molecule may include any CDR described herein. In certain embodiments, the anti-TMEM219 antibody molecule includes a substitution in a light chain CDR, e.g., one or more substitutions in a CDR1 , CDR2 and / or CDR3 of the light chain. In another embodiment, the anti-TMEM219 antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in Tables 3, 5, 6) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1 -8 or encoded by the nucleotide sequence in Tables 9-10; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Tables 3, 5, 6.

[0169] The assignment of amino acids to each VL and VH domain is in accordance with any conventional definition of CDRs. The CDRs of the present disclosure have been annotated with abYsis annotation tool from http: / / www.abysis.org / , using the Kabat, Chothia, AbM (Martin), Contact and IMGT numbering schemes. Those skilled in the art of antibodies will appreciate that these conventions consist of nonsequential numbering in specific regions of an immunoglobulin sequence, enabling a normalized reference to conserved positions in immunoglobulin families. Accordingly, the positions of any given immunoglobulin as defined by Kabat or Chothia or AbM (Martin) or Contact or IMGT numbering will not necessarily correspond to its sequential sequence.

[0170] Conventional definitions include, the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991 ), the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196: 901 -917, 1987; Chothia et al., Nature 342: 878-883, 1989); the AbM definition used by Oxford Molecular’s antibody modelling software; and, the Contact definition of Martin et al. (bioinfo.org. uk / abs); the IMGT definition (Lefranc, M.-P. and Lefranc, G. The Immunoglobulin FactsBook Academic Press, London, UK (2001 ); Lefranc, M.-P. and Lefranc, G. The T cell receptor FactsBook Academic Press, London, UK (2001 )); the Eu numbering system, also referred herein, relates to the sequence numbering used in the description of the human IgGI myeloma protein called Eu (Edelman GM et al., Proc Natl Acad Sci USA 63(1 ):78- 85, 1969). Thus, in any immunoglobulin molecule such as an antibody or fragment thereof or an antibody-like molecule, then amino acid in a given position number relates to, or corresponds to, the corresponding amino acid residue position number in the Eu sequence. Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. The present disclosure can use CDRs defined according to any of these numbering systems, although preferred embodiments use Kabat defined CDRs.

[0171] In one embodiment, the anti-TMEM219 antibody molecule comprises:

[0172] (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence chosen from any one of SEQ ID NO: 1 , 8, 10, 56, 59, 62, 65 and 68; a VHCDR2 amino acid sequence chosen from any one of SEQ ID NO: 2, 5, 11 , 57, 60, 63, 66 and 69; and a VHCDR3 amino acid sequence chosen from any one of SEQ ID NO: 3, 6, 7, 9, 12, 13, 58, 61 , 64, 67 and 70; and

[0173] (ii) a light chain variable region (VL) comprising a VLCDR1 amino acid sequence chosen from any one of SEQ ID NO: 14, 17, 20, 23, 26, 29, 71 , 77, 80, 82 and 85, a VLCDR2 amino acid sequence chosen from any one of SEQ ID NO: 15, 18, 21 , 24, 27, 30, 72, 78, 83 and 86, and a VLCDR3 amino acid sequence chosen from SEQ ID NO: 16, 19, 22, 25, 28, 31 , 73, 74, 75, 76, 79, 81 , 84 and 87.

[0174] In one embodiment, the light or the heavy chain variable framework (e.g., the region encompassing at least FR1 , FR2, FR3, and optionally FR4) of the anti-TMEM219 antibody molecule can be chosen from: (a) a light or heavy chain variable framework including at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% of the amino acid residues from a human light or heavy chain variable framework, e.g., a light or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (b) a light or heavy chain variable framework including from 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of the amino acid residues from a human light or heavy chain variable framework, e.g., a light or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (c) a non-human framework (e.g., a rodent framework); or (d) a non-human framework that has been modified, e.g., to remove antigenic or cytotoxic determinants, e.g., deimmunized, or partially humanized. In one embodiment, the light or heavy chain variable framework region (particularly FR1 , FR2 and / or FR3) includes a light or heavy chain variable framework sequence at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical or identical to the frameworks of a VL or VH segment of a human germ line gene.

[0175] In certain embodiments, the anti-TMEM219 antibody molecule comprises a heavy chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more changes, e.g., amino acid substitutions or deletions.

[0176] In one embodiment, the heavy or light chain variable region, or both, of the anti- TMEM219 antibody molecule includes an amino acid sequence encoded by a nucleic acid sequence described herein or a nucleic acid that hybridizes to a nucleic acid sequence described herein (e.g., a nucleic acid sequence as shown in Tables 9-10) or its complement, e.g., under low stringency, medium stringency, or high stringency, or other hybridization conditions described herein.

[0177] In another embodiment, the anti-TMEM219 antibody molecule comprises at least one, two, three, or four antigen-binding regions, e.g., variable regions, having an amino acid sequence as set forth in Tables 7-8 or a sequence substantially identical thereto, e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 1 , 2, 5, 10, or 15 amino acid residues from the sequences shown in Tables 7-8. In another embodiment, the anti-TMEM219 antibody molecule includes a VH and / or a VL domain encoded by a nucleic acid having a nucleotide sequence as set forth in Tables 9-10 or a sequence substantially identical thereto, e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in Tables 9-10.

[0178] In yet other embodiments, the anti-TMEM219 antibody molecule has a heavy chain constant region (Fc) chosen from, e.g., the heavy chain constant regions of IgG 1 , lgG2, lgG3, lgG-4, IgM, lgA1 , lgA2, IgD, and IgE; particularly, chosen from, e.g., the heavy chain constant regions of IgG 1 , lgG2, lgG3, and lgG4, more particularly, the heavy chain constant region of lgG1 or lgG4 (e.g., human lgG1 , lgG2 or lgG4). In one embodiment, the heavy chain constant region is human lgG1. In another embodiment, the anti- TMEM219 antibody molecule has a light chain constant region chosen from, e.g., the light chain constant regions of kappa or lambda. In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the anti-TMEM219 antibody molecule (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, complement function, half-life, aggregation and stability). In certain embodiments, the anti-TMEM antibody molecules comprise a human lgG4 mutated. The constant region of an antibody is important in the ability of an antibody to fix complement and mediate celldependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity. In certain embodiments, the constant region is an lgG1 , lgG2, lgG3, lgG4 constant region.

[0179] In one embodiment, the anti-TMEM219 antibody molecule is isolated or recombinant.

[0180] In one embodiment, the anti-TMEM219 antibody molecule is a humanized or human antibody molecule.

[0181] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies may in various embodiments nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in some embodiments, CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences are derived from the germline of another mammalian species, such as a mouse, which have been grafted onto human framework sequences. The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295, incorporated herein by reference in its entirety,) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0182] An "isolated antibody," as used herein, means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody." In various embodiments, the isolated antibody also includes an antibody in situ within a recombinant cell. In other embodiments, isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In various embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0183] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0184] The disclosure also features a nucleic acid molecule that comprises one or both nucleotide sequences that encode heavy and light chain variable regions, CDRs, hypervariable loops, framework regions of the anti-TMEM219 antibody molecules, as described herein, for use for the prevention and / or treatment of colorectal cancer. In certain embodiments, the nucleotide sequence that encodes the anti-TMEM219 antibody molecule is codon optimized. For example, a first and second nucleic acids encoding heavy and light chain variable regions, respectively, of an anti-TMEM219 antibody molecule chosen from one or more of, e.g., any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1 -8 or encoded by the nucleotide sequence in Tables 9-10, or a sequence substantially identical thereto can be used. For example, the nucleic acid can comprise a nucleotide sequence as set forth in Tables 9-10, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in Tables 9-10).

[0185] In other embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain variable domain and / or a heavy chain constant region comprising the amino acid sequence of any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1-8 or encoded by the nucleotide sequence in Tables 9-10; or a sequence substantially identical (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical) to any of the aforesaid sequences.

[0186] In other embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a light chain variable domain and / or a light chain constant region comprising the amino acid sequence of any of TC01 , TC03, TC04, TC05, TA02, TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1 - 8 or the nucleotide sequence in Tables 9-10, or a sequence substantially identical (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical) to any of the aforesaid sequences.

[0187] The aforesaid nucleotide sequences encoding the anti-TMEM219 heavy and light chain variable domain and constant regions can be present in a separate nucleic acid molecule, or in the same nucleic acid molecule. In certain embodiments, the nucleic acid molecules comprise a nucleotide sequence encoding a leader sequence.

[0188] In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding at least one, two, or three CDRs, or hypervariable loops, from a heavy chain variable region having an amino acid sequence as set forth in Tables 1-8, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three or more substitutions, insertions or deletions, e.g., conserved substitutions).

[0189] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding at least one, two, or three CDRs, or hypervariable loops, from a light chain variable region having an amino acid sequence as set forth in Tables 1 -8 or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three or more substitutions, insertions or deletions, e.g., conserved substitutions).

[0190] In yet another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding at least one, two, three, four, five, or six CDRs, or hypervariable loops, from heavy and light chain variable regions having an amino acid sequence as set forth in Tables 1 -8 or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three or more substitutions, insertions or deletions, e.g., conserved substitutions).

[0191] In another embodiment, the nucleic acid molecule includes one or more heavy chain framework region (e.g., any of VHFW1 (type a), VHFW1 (type b), VHFW1 (type c), VHFW1 (type d), VHFW2 (type a), VHFW2 (type a'), VHFW2 (type b), VHFW2 (type c), VHFW2 (type d), VHFW2 (type e), VHFW3 (type a), VHFW3 (type b), VHFW3 (type c), VHFW3 (type d), VHFW3 (type e), or VHFW4, or any combination thereof, e.g., a framework combination as described herein) for any of TA02, TC01 , TC02, TD01 , TE01 , TG02, TM1 , TE02.1 , TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 1 - 8, or a sequence substantially identical thereto. For example, the nucleic acid molecule can comprise a nucleotide sequence as set forth in Tables 9-10, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in Tables 9-10).

[0192] In another embodiment, the nucleic acid molecule includes one or more light chain framework region (e.g., any of VLFW1 (type a), VLFW1 (type b), VLFW1 (type c), VLFW1 (type d), VLFW1 (type e), VLFW1 (type f), VLFW2 (type a), VLFW2 (type c), VLFW3 (type a), VLFW3 (type b), VLFW3 (type c), VLFW3 (type d), VLFW3 (type e), VLFW3 (type f), VLFW3 (type g), or VLFW4, or any combination thereof, e.g., a framework combination as described herein) for of any of E01 , E02, E08, E 14, E 19, E20, E23, E24 or M1 as defined in Tables 1 -8, or a sequence substantially identical thereto. For example, the nucleic acid molecule can comprise a nucleotide sequence as set forth in Tables 9-10, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in Tables 9-10).

[0193] In another embodiment, the nucleic acid molecule includes one or more heavy chain framework region and one or more light chain framework region as described herein. The heavy and light chain framework regions may be present in the same vector or separate vectors.

[0194] Host cells and vectors containing the nucleic acids described herein or modified for codon optimization according to known methods can also be used. The nucleic acids may be present in a single vector or separate vectors present in the same host cell or separate host cell. The host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli. For example, the mammalian cell can be a cultured cell or a cell line. Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell.

[0195] In another aspect, the disclosure provides, compositions, e.g., pharmaceutical compositions, for use for the prevention and / or treatment of colorectal cancer which include a pharmaceutically acceptable carrier, excipient or stabilizer, and at least one anti-TMEM219 antibody, such as the ones described herein. Preferably said pharmaceutical composition includes anti-TMEM219 antibody TC01 , as described herein. In one embodiment, the composition, e.g., the pharmaceutical composition, includes a combination of the antibody molecule and one or more agents, e.g., an anticancer therapeutic agent or other anti-TMEM219 antibody molecule, as described herein. In one embodiment, the antibody molecule is conjugated to a label or a therapeutic agent.

[0196] The present disclosure also includes anti-TMEM219 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. The antibody can be administered to the subject in various embodiments in a formulation comprising suitable carriers, excipients, and other agents to provide improved transfer, delivery, tolerance, and the like, and suitable for an intravenous or subcutaneous injection.

[0197] The injectable preparations may be prepared by methods publicly known. For example, injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 20 or 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injectable preparation thus prepared can be filled in an appropriate ampoule.

[0198] The antibody for the use as herein disclosed can be administered to the subject using any acceptable device or mechanism. For example, the administration can be accomplished using a syringe and needle or with a reusable pen and / or autoinjector delivery device. The methods of the present disclosure include the use of numerous reusable pen and / or autoinjector delivery devices to administer an antibody (or pharmaceutical formulation comprising the antibody). Examples of such devices include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen and / or autoinjector delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present disclosure include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), the HUMIRA™ Pen (Abbott Labs, Abbott Park, IL), the DAI® Auto Injector (SHL Group) and any auto-injector featuring the PUSHCLICK™ technology (SHL Group), to name only a few.

[0199] In one embodiment, the antibody is administered with a prefilled syringe. In another embodiment, the antibody is administered with a prefilled syringe containing a safety system. For example, the safety system prevents an accidental needlestick injury. In various embodiments, the antibody is administered with a prefilled syringe containing an ERIS™ safety system (West Pharmaceutical Services Inc.). See also U.S. patent numbers 5,215,534 and 9,248,242, incorporated herein by reference in their entireties. In another embodiment, the antibody is administered with an auto-injector. In various embodiments, the antibody is administered with an auto-injector featuring the PUSHCLICK™ technology (SHL Group). In various embodiments, the auto-injector is a device comprising a syringe that allows for administration of a dose of the composition and / or antibody to a subject. See also U.S. patent numbers 9,427,531 and 9,566,395 3.

[0200] According to the disclosure, “subject” means a human subject or human patient. In one embodiment, the subject is in need of inhibiting, reducing, neutralizing or blocking colorectal cancer. In one embodiment, the subject has, or is at risk of having, colorectal cancer.

[0201] The content of the antibody or antigen binding fragment thereof in the pharmaceutical composition for the use as disclosed is not limited as far as it is useful for treatment or prevention of colorectal cancer but preferably contains 0.0000001 -10% by weight per total composition. The choice of the carrier may depend upon the route of administration and concentration of the active agent(s) and the carrier may be in the form of a lyophilised composition or an aqueous solution. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to render the composition isotonic. Examples of the carrier include but are not limited to saline, Ringer's solution and dextrose solution. Preferably, acceptable excipients, carriers, or stabilizers are non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt-forming counter- ions, e.g. sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g. serum albumin, or gelatine; hydrophilic polymers, e.g. polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrins; monosaccharides; disaccharides; other sugars, e.g. sucrose, mannitol, trehalose or sorbitol; chelating agents, e.g. EDTA; non-ionic surfactants, e.g. Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and / or preservatives, e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g. methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Suitable carriers and their formulations are described in greater detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co. The composition may also contain at least one further active compound, such as a chemotherapeutic agent. Preferably, the antibody or antigen binding fragment thereof is included in a pharmaceutical composition for the use as herein disclosed in an effective amount. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is to be administered. SEQUENCES

[0202]

[0203] CDR definition is also provided using annotation tool from http: / / www.abysis.org / based on full VH and VL amino acid sequences as defined in Tables 7 and 8.

[0204] Below is shown the example in reference to SEQ ID No. 33 (VH of TC01 ).

[0205] Table 3: Kabat defined CDR sequences

[0206] The VH amino acid sequence of any antibody may also be plugged into the annotation tool and IMGT defined CDR sequences are provided. Below is shown the example in reference to SEQ ID No. 33 (VH of TC01 ).

[0207] Table 4: IMGT defined CDR sequences In addition, the VH amino acid sequence of any antibody disclosed herein may also be plugged into the annotation tool and the “All, side by side” defined CDR sequences are provided.

[0208] Below is shown the example in reference to SEQ ID No. 33 (VH of TC01 ). Table 5: All, side by side defined CDR sequences

[0209] CDR definition provided using annotation tool from http: / / www.abysis.org / based on full VL amino acid sequence of TC01 (SEQ ID No. 39) is also reported.

[0210] Table 6: All, side by side defined CDR sequences

[0211]

[0212]

[0213]

[0214] EXAMPLES

[0215] Material and methods

[0216] In vivo experimental design

[0217] As shown in Figure 1, after a single intraperitoneal injection of AOM (10 mg / kg body weight in physiological solution), mice were treated with repeated oral cycles of the inflammatory agent, 2% (weight / volume) DSS (molecular mass 40 kDa) (MP Biomedicals), each characterized by 7 days of DSS exposure in drinking water, followed by 7 days of regular drinking water, according to the scheme of Figure 1 (12). The 40 kDa DSS-induced Inflammation model, as the one used in this study, affords a high degree of uniformity and reproducibility of most lesions in the distal colon, where an enormous number of microorganisms live, whereas less inflammation is observed in the proximal site and almost none in the caecum. On the contrary, mice treated with 5 kDa DSS develop relatively patchy lesions mainly in the cecum and upper colon (18-20).

[0218] The design of the study is summarized in Figure 2. EntOOl is the antibody described herein and in WO2021094620 as TC01 . The study included a set of control groups, as outlined below, aimed at testing: 1 ) the effect of intraperitoneal administrations of vehicle (i.e. PBS) in the absence of any other agent (Group 1 ); 2) the effect of intraperitoneal administrations of EntOOl alone in the absence of any other agent (Group 2); 3) the effect of intraperitoneal administrations of EntOOl in combination with the carcinogenic agent AOM without DSS (Group 3); 4) the effect of intraperitoneal administrations of EntOOl in combination with the pro-inflammatory agent DSS without AOM (Group 4) and the effect of intraperitoneal administrations of anti-murine TNFa antibody, used as positive control (Group 6). The other experimental groups in the study aimed at testing the effect of intraperitoneal administrations of EntOOl alone (Group 5) or in combination with the anti-murine TNFa antibody (Group 7).

[0219] 1 ) Vehicle (N=8 mice)

[0220] 2) EntOOl alone (no AOM / no DSS) (N=8 mice)

[0221] 3) AOM + EntOOl (N=8 mice)

[0222] 4) DSS + EntOOl + Vehicle (N=8 mice) 5) AOM / DSS + EntOOl (N=8 mice)

[0223] 6) AOM / DSS + anti-murine TNFalpha antibody (N=8 mice)

[0224] 7) AOM / DSS + anti-murine TNFalpha antibody + EntOOl (N=8 mice)

[0225] Of note, the commercial anti-murine TNFalpha antibody clone MP6-XT22 (eBioscience, San Diego, CA) was used, since it displayed beneficial effects, at the indicated dose, in the DSS-induced model of colitis, as documented by the literature (13).

[0226] Clinical and Physical Examination: Disease Activity Index & Body Weight Loss

[0227] Colitis seventy was monitored using a disease activity index (DAI) score based on daily evaluation of body weight, stool consistency, and presence of blood in the stools; grading of intestinal inflammation was determined according to the criteria proposed by Cooper et al. (14, 15); the DAI was determined by scoring changes in: weight loss (0 = none, 1 = 1 %— 5%, 2 = 5%— 10%, 3 = 10%— 20%, 4 = >20%); stool consistency (0 = normal, 2 = loose, 4 = diarrhoea); and rectal bleeding (0 = normal, 2 = occult bleeding, 4 = gross bleeding). A 5-point (0-4) DAI was thus obtained as the mean of the three parameter scores.

[0228] Tissue sampling

[0229] At Day 56, mice were euthanized by CO2, colons excised (without caecum), and collected to perform the following analyses:

[0230] - Measurement of colon length, as an additional parameter of intestinal inflammation.

[0231] - Histological analysis: whole colons were fixed in formalin and embedded in paraffin, in order to perform histologic inflammatory scores and evaluation of tumor density, grade and size.

[0232] - Quantification of intestinal epithelial cell proliferation: mice (n=4 per group) were administered with 5-Bromo-2'-deoxyuridine (Brdll, from Merck Life Science S.r.l.) by intraperitoneal injection, 1 hour before sacrifice. Formalin-fixed and paraffin-embedded sections were stained using an anti-Brdll antibody and Brdll- positive nuclei were quantified using an imaging system.

[0233] - Quantification of cleaved Caspase 8: as an additional readout to confirm the efficient inhibition of IGFBP3, we quantified IGFBP3-dependent apoptotic molecule cleaved Caspase 8 in the intestinal mucosa of n=4 animals per group, by western blot.

[0234] Assessment of histological colonic lesions

[0235] Grading of intestinal inflammation was confirmed histologically. At sacrifice, colons were fixed in 10% formalin for 24-48 hour at room temperature. Tissues were then dehydrated through 70%, 80%, and 95% alcohol, 45 min each, followed by 3 changes of 100% alcohol, 1 hour each. Colons were then cleared through 2 changes of xylene, 1 hour each, and then immersed in 3 changes of paraffin, 1 hour each. 2-pM paraffin-embedded sections were obtained using a microtome and transferred onto glass slides suitable for immunohistochemistry (Superfrost Plus). Upon staining with hematoxylin (Dako) and eosin (Diapath), a blinded pathologist evaluated the degree of inflammatory cell infiltration and mucosal damage on colon sections, using 2 different protocols:

[0236] 1 ) Rachimilewitz score (16). This scoring system takes into account the ulceration, extent of ulceration, flogosis, extent of flogosis and fibrosis in the whole colon section, without distinguishing into proximal and distal colon. Minimal score was 0, and maximal score was 20, as a sum of all single scores (Total histologic score).

[0237] 2) Histologic score, as described by Dieleman et al. (17). The sections were graded in the proximal and distal colons (Figure 3) by a blinded pathologist with a range from 0 to 3 as to amount of inflammation, depth of inflammation and with a range from 0 to 4 as to the amount of crypt damage or regeneration as indicated in Table 12. These changes were also quantified as to the percentage involvement by the disease process: (1 ) 1-25%; (2) 26-50%; (3) 51-75%; (4) 76-100%. Each section was then scored for each feature separately by establishing the product of the grade for that feature and the percentage involvement (in a range from 0 to 12 for inflammation and for extent, and in a range from 0 to 16 for regeneration and for crypt damage). Total histological score was calculated as the sum of the score products (in a range from 0 to 56). Table 12. Histologic score by Dieleman et al.

[0238] Analysis and grading of tumor lesions

[0239] Typically, colons of animals that have been treated with AOM / DSS are found to have occasional, abnormally large, darkly staining, and slightly raised ‘aberrant crypts’, called glandular intraepithelial neoplasia (GIN), after 3-4 weeks from the AOM injection (around Day 14 to Day 21 of the present study). With longer time intervals between carcinogen administration and inflammation, these aberrant crypts are frequently found as foci, with two to many hundreds of aberrant crypts appearing together in a cluster, after 5-7 weeks (around Day 28 to Day 42 of the present study) from AOM injection. Typically, many aberrant crypts on histological examination show a low and a high level of dysplasia and are then correctly called adenoma, which may evolve to non-metastatic colon carcinomas at 7-8 weeks (around Day 42 to Day 56 of the present study) (Figure 4). Thus, in the current experiment, four grades of mucosal lesions were identified and quantified by a blinded pathologist: glandular intraepithelial neoplasia (GIN), indicating small clusters of dysplastic colonic crypts on the mucosal surface, low grade (LG) and high-grade (HG) adenomas, and in situ carcinoma (K) (12, 21 ). Mice were sacrificed at Day 56, seven days after the fourth DSS cycle, and during the recovery phase in water. This was done because around this time point not only are there higher chances to find different grades of tumoral lesions, including in situ carcinoma, but also because the reduced inflammation during recovery allows a better visualization and quantification of neoplastic lesions.

[0240] BrdU quantification on colon sections

[0241] To achieve labelling of epithelial cells undergoing DNA synthesis, mice (n=4 per group) received an i.p. injection of 100 mg / kg Brdll 1 hour prior to sacrifice. Formalin-fixed, paraffin-embedded colon sections (2-pm) were stained for Brdll after antigen retrieval for 30 minutes at 98°C in citric buffer (10 mM, pH 6.0). Following endogenous peroxidase blocking with peroxidase 1 solution for 20 minutes at RT, tissues were washed in TBS. Upon incubation with the rat anti-BrdU Ab (1 :600; AbD Serotec) for 40 minutes at 4°C, a rat HRP polymer kit (Biocare Medical) was used to detect BrdU using DAB (Biocare Medical) as chromogen. Tissue sections were then counterstained with CAT Hematoxylin (Biocare Medical), dried overnight in a 37°C oven, and mounted in Eukitt Quick Hardening Mounting Media. Images were acquired with a VS120 Dot Slide System (Olympus), and BrdU+ cells were counted in consecutive and randomly selected 10x fields (n = 4 fields of 1 mm2 / section) using the IHC Tool Box of Imaged 1.80p (see examples below). We distinguished inflammatory from tumor areas.

[0242] Non-inflammatory (control groups) and inflammatory areas (colitic and tumor-bearing mice): upon definition of the region of interest (ROI) (1 mm2), containing all mucosa layers (whenever possible), BrdU-positive nuclei with different sizes were selected using the ellipse tool, through the TRAINING-NUCLEI function. Image J automatically processes the image and calculates the number of nuclei within the selected region.

[0243] Tumor areas: in this case, nuclei segmentation and quantification was performed in selected tumor areas calculated in pixels on a 20x magnification, and results are reported as number of BrdU-positive nuclei per tumor area. Quantification of cleaved Caspase 8 by western blot

[0244] Colonic samples (1 entire colon from 1 mouse per group + 1 small specimen from the proximal colon of 3 mice per group) were mechanically homogenized in lysis buffer (Tris 50 mM, 2% SDS, 1 mM Na2VO2) containing proteinase inhibitors (Complete Mini, Roche Diagnostics GmbH, Penzberg, Germany), for protein extraction. Insoluble material was removed by centrifugation for 30 min at 14,000 rpm at 4°C. The concentration of proteins in each lysate was measured using the Bio-Rad protein assay (Bio-Rad Laboratories).

[0245] Next, proteins from each sample were separated on a 10% Tris-glycine polyacrylamide gel at 160 V for 80 minutes and blotted onto PVDF-membranes (Millipore, Schwalbach, Germany) at 80 mA for 90 min. Membrane was cut according to protein weight and nonspecific binding was blocked with Tris-buffered saline (TBS) containing 5% non-fat dried milk and 0.1 % Tween 20, followed by overnight incubation at 4°C with the rabbit anti-mouse Caspase 8 antibody which recognizes the active / cleaved form of Caspase 8 antibody (NB100-56116 from Novus Biological); 1 :2000), and with a rabbit anti Caspase- 8 antibody (Cell Signaling Technology, #4927; 1 :500). Membranes were washed for 1 h with TBS containing 0.1 % Tween 20 and then incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (1 :3000; GE Healthcare). The membranes were then incubated with Immobilon Western Chemilum (Millipore) for 1 -5 minutes, after which bands were detected by Chemidoc (BioRad Laboratories), using Quantity One software. The filter was then stripped with buffer Restore (Pierce) and reprobed with a mouse anti-actin antibody (clone C11 ; 1 :1000; Santa Cruz Biotechnology) for control protein loading. Protein bands were quantified using the densitometry program Image J.

[0246] Statistical analysis

[0247] Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software). Data are presented as mean ± Standard Error of Mean (SEM) and differences were considered statistically significant when P < 0.05. For experiments including more groups a one-way or two-way ANOVA multivariate analysis has been performed accompanied by a post-hoc modification test. RESULTS

[0248] Example 1

[0249] Clinical and Physical Examinations: Disease Activity Index & Body Weight Loss

[0250] After 2 weeks of treatments (Day 18-33, treatment every day), results show that EntOOl significantly inhibits experimental colitis in comparison with control group (AOM / DSS + vehicle), in terms of percentage of body weight loss (Figure 5A) and DAI (Figure 5B). Statistical analyses and p values of specific groups at each time point can be found in following Tables 13 and 14.

[0251] Table 13

[0252] Table 14

[0253] Of note, EntOOl was significantly more effective than anti-murine TNFalpha in reducing body weight loss and DAI, at all-time points between Day 18 and Day 33. Combination of anti-murine TNFalpha + EntOOl was no more effective than EntOOl alone.

[0254] In the following weeks (treatment 2 times per week), results confirmed the statistically significant inhibitory effect of Ent001 on experimental colitis also from Day 33 to Day 56, in comparison with control group (AOM / DSS + vehicle), in terms of body weight loss (Figure 5A) and DAI (Figure 5B). Again, EntOOl was significantly more effective than anti-murine TNFalpha in reducing both these clinical parameters of inflammation, whereas combination of anti-murine TNFalpha + Ent001 was no more effective than EntOOl alone.

[0255] As shown in Figure 6, at sacrifice (Day 56), all treatment groups showed a significantly lower DAI than AOM / DSS + vehicle group. Of note, treatment with EntOOl resulted in a much better control on DAI compared to anti-murine TNFalpha (anti-TNF).

[0256] Example 2

[0257] Post-mortem analysis of colon length

[0258] Colon shortening is a parameter of intestinal inflammation (20). At Day 56 mice were sacrificed, and colon lengths were measured.

[0259] Results confirmed the statistically significant inhibitory effect of EntOOl on the severity of colitis in comparison with control group (AOM / DSS + vehicle) (Figure 7). EntOOl , when administered from Day 18 to Day 55, was significantly more effective than antimurine TNFalpha in reducing colon shortening, and combination of anti-murine TNFalpha + EntOOl was as effective as EntOOl alone.

[0260] Example 3

[0261] Evaluation of colonic inflammation at the histological level

[0262] The protective effects of EntOOl in DSS-induced chronic colitis were confirmed by a blinded pathologist using the histologic score by Rachmilewitz et al (16) and Dieleman et al (17).

[0263] According to the Rachmilewitz score, performed on the whole colon, results show that EntOOl significantly improved colitis (Figure 8) compared to control group (AOM / DSS + vehicle) in terms of total histological score.

[0264] According to the Dieleman score, performed separately on the proximal and distal colon, results show that EntOOl significantly inhibited experimental colitis in comparison with control group (AOM / DSS + vehicle), in terms of total histologic score (Figure 9A), severity of inflammation (Figure 9B), extent of inflammation (Figure 9C), regeneration (Figure 9D) and crypt damage (Figure 9E). In both proximal and distal colons there were no statistically significant differences between groups treated with EntOOl and antimurine TNFalpha. Representative images of inflammatory conditions per group are shown in Figure 10. In healthy mice (e.g. Vehicle), the tissue layers (mucosa, muscularis mucosae, submucosa and muscularis propria) are well structured. Administrations of AOM + EntOOl , or EntOOl alone do not affect the layer composition and there is no inflammatory infiltrate. In DSS- induced colitic mice (e.g. AOM / DSS + vehicle), layer stratification is not maintained anymore, a massive inflammatory infiltrate completely occupies both mucosa and submucosa, and crypts are severely damaged in most part of the colon. The epithelium is eroded indicating the presence of ulcerations.

[0265] Treatment with EntOOl significantly ameliorated experimental colitis: tissue layers are maintained, there is a slight inflammation with inflammatory cells infiltrating both mucosa and submucosa, but both epithelium and crypts are well structured. At the histological level we observed no major differences between groups treated with EntOOl or antimurine TNFalpha or the combination of EntOOl and anti-murine TNFalpha.

[0266] Example 4

[0267] Analysis & grading of tumor lesions

[0268] The objective of this study was to identify a potential effect of modulation, in terms of tumor initiation and promotion, tumor growth, or tumor protection in the AOM / DSS mouse model of colitis-associated cancer.

[0269] When we looked at the total tumor density, i.e. total number of tumors / mouse, we found no tumors in the vehicle (as expected), EntOOl , AOM + EntOOl and DSS + EntOOl groups, indicating that EntOOl antibody does neither induces nor accelerates tumor initiation in the colon (Figure 11). In AOM / DSS treated mice, we observed a certain number of tumors per mouse, that were significantly reduced by treatment with EntOOl and / or anti-murine TNFalpha. In terms of tumor grade, we observed no significative differences in GIN density among the various groups (Figure 12). This is not surprising, since GIN are neoplastic lesions that develop around 3-4 weeks after AOM injection, when we just started to treat mice with EntOOl and / or anti-murine TNFalpha. On the contrary we observed a significant reduction in the density of LGA and HGA in tumorbearing mice treated with EntOOl and / or anti-murine TNFalpha in comparison with AOM / DSS + vehicle control group (Figure 12). Of note, progression from adenoma (LGA / HGA) to carcinoma (K) was observed only for the AOM / DSS + vehicle , with a significantly higher number in the control group (Figure 12), suggesting that EntOOl blocks AOM / DSS-induced tumor progression. When we looked at the tumor size, we found significant reduction in the AOM / DSS + EntOOl group when compared to AOM / DSS + vehicle, both for LGA and HGA (Figure 13), and a trend for the other groups. . These results could be due to a direct effect of EntOOl antibody on intestinal epithelial biologic functions (e.g. proliferation), whereas anti-murine TNFalpha acts only indirectly on tumor growth by modulating inflammation.

[0270] Representative images of tumor size variation in LGA, HGA and K tumors are shown in Figure 14.

[0271] Example 5

[0272] BrdU quantification on colon sections

[0273] In order to evaluate the impact of EntOOl on intestinal epithelial cell proliferation, mice (n=4 per group) received an i.p. injection of 100 mg / kg Brdll 1 hour prior to sacrifice, time that allows incorporation of Brdll solely by proliferating intestinal epithelium and not by other cell types with slower cell cycle (e.g. leukocytes). Formalin-fixed, paraffin- embedded, colon sections were stained using an anti-Brdll antibody. Brdll-positive nuclei were quantified in non-inflammatory (control groups), inflammatory (colitic and tumor-bearing mice) and tumor areas, as described above. In inflammatory areas of AOM / DSS mice, the number of Brdll-positive nuclei was significantly increased compared to Vehicle group, due to accelerated epithelial cell turnover caused by chronic inflammation in combination with DNA damage caused by AOM (22).

[0274] In both non-inflammatory and inflammatory regions, results showed that treatment with EntOOl alone in the presence or absence of AOM, or in DSS-induced colitic mice, did not affect epithelial cell proliferation (Figure 15 and 16). In AOM / DSS mice, treatment with EntOOl and anti-murine TNFalpha significantly reduced the number of Brdll- positive nuclei / mm2when compared with AOM / DSS + vehicle.

[0275] Within tumors (i.e. LGA), treatment with EntOOl significantly reduced the number of Brdll-positive nuclei per tumor area, while AOM / DSS + anti-murine TNFalpha did not (Figure 17 and 18). This data indicates that EntOOl might impact intestinal epithelial cell proliferation.

[0276] Example 6

[0277] Quantification of cleaved Caspase 8

[0278] In order to confirm the efficient inhibition of IGFBP3-dependent Caspase 8 activation by EntOOl , we quantified cleaved Caspase 8 and total Caspase 8 protein in the colon lysates from n=4 animals per group (1 whole colon + 3 small proximal pieces from 3 mice), by western blot. Lysates from the same group were pooled to increase the total amount of proteins. Upon normalization with the loading control (actin), results showed that the ratio of Cleaved Caspase 8 / total Caspase 8 is reduced in mice treated with Ent001 (Figure 19), confirming efficacy for EntOOI .

[0279] Conclusions

[0280] This study showed that in the AOM / DSS model of colon carcinogenesis, EntOOI not only does not induce or accelerate tumor initiation in the colon, but also significantly reduces tumor density, size and progression.

[0281] A potential synergist or antagonistic effect of EntOOI with anti-murine TNFalpha on tumor modulation was also evaluated and results indicated that the combination of anti-murine TNFalpha + EntOOI is as effective as EntOOI alone, and therefore no more effective than treatment with EntOOI alone.

[0282] These data support TMEM219 as a potential therapeutic target for the treatment of colon cancer, in particular of colitis-associated cancer, and for reducing incidence of new colon cancer in IBD patients.

[0283] REFERENCES

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Claims

CLAIMS1. An antibody that binds to TMEM219 receptor or an antigen binding fragment thereof for use in the prevention and / or treatment of colorectal cancer, and / or reducing risk of developing colorectal cancer, such as colitis-associated colorectal cancer (CAC), in patients with inflammatory bowel disease (IBD), wherein said antibody inhibits or reduces the binding of IGFBP3 to said TMEM219 receptor and / or inhibits, reduces, or neutralizes the activation of the TMEM219 receptor induced by binding of IGFBP3 and / or does not activate TMEM219 pathway upon binding to TMEM219 receptor.

2. The antibody for the use according to claim 1 wherein said colorectal cancer is selected from the group consisting of colon cancer, bowel cancer, and rectal cancer.

3. The antibody for the use according to any one of claims 1 -2 wherein said cancer is colitis-associated colorectal cancer.

4. The antibody for the use according to any one of claims 1 -3 wherein said antibody comprises: a. a heavy chain variable domain (VH) comprising: i. a CDR1 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 8, 1 , 10, 56, 59, 62, 65 and 68; ii. a CDR2 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 2, 11 , 57, 60, 63, 66 and 69; and iii. a CDR3 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, 3, 7, 9, 12, 13, 58, 61 , 64, 67 and 70; and / or b. a light chain variable domain (VL) comprising: i. a CDR1 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 17, 14, 20, 23, 26, 29, 71 , 77, 80, 82 and 85; ii. a CDR2 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 18, 15, 21 , 24, 27, 30, 72, 78, 83 and 86; and iii. a CDR3 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 16, 22, 25, 28, 31 , 73, 74, 75, 76, 79, 81 , 84, 87 and 166.

5. The antibody for the use according to any one of claims 1 -4 wherein said antibody comprises (a) a heavy chain variable region (VH) comprising a VHCDR1 , a VHCDR2, a VHCDR3, and (b) a light chain variable region (VL) comprising a VLCDR1 , a VLCDR2, and a VLCDR3, wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 8, VHCDR2 consists of the sequence of SEQ ID NO: 5, VHCDR3 consists of the sequence of SEQ ID NO: 6, VLCDR1 consists of the sequence of SEQ ID NO: 17, VLCDR2 consists of the sequence of SEQ ID NO: 18 and VLCDR3 consists of the sequence of SEQ ID NO: 19; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 8, VHCDR2 consists of the sequence of SEQ ID NO: 5, VHCDR3 consists of the sequence of SEQ ID NO:6, VLCDR1 consists of the sequence of SEQ ID NO: 17, VLCDR2 consists of the sequence of SEQ ID NO: 18 and VLCDR3 consists of the sequence of SEQ ID NO: 166; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 1 , VHCDR2 consists of the sequence of SEQ ID NO: 2, VHCDR3 consists of the sequence of SEQ ID NO: 3, VLCDR1 consists of the sequence of SEQ ID NO: 14, VLCDR2 consists of the sequence of SEQ ID NO: 15, and VLCDR3 consists of the sequence of SEQ ID NO: 16; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 1 , VHCDR2 consists of the sequence of SEQ ID NO: 2, VHCDR3 consists of the sequence of SEQ ID NO:7, VLCDR1 consists of the sequence of SEQ ID NO: 20, VLCDR2 consists of the sequence of SEQ ID NO: 21 , and VLCDR3 consists of the sequence of SEQ ID NO: 22; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 8, VHCDR2 consists of the sequence of SEQ ID NO: 5, VHCDR3 consists of the sequence of SEQ ID NO: 9, VLCDR1 consists of the sequence of SEQ ID NO: 23, VLCDR2 consists of the sequence of SEQ ID NO: 24, and VLCDR3 consists of the sequence of SEQ ID NO: 25; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 10, VHCDR2 consists of the sequence of SEQ ID NO: 11 , VHCDR3 consists of the sequence of SEQ ID NO: 12, VLCDR1 consists of the sequence of SEQ ID NO: 26, VLCDR2 consists ofthe sequence of SEQ ID NO: 27, and VLCDR3 consists of the sequence of SEQ ID NO: 28; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 8, VHCDR2 consists of the sequence of SEQ ID NO: 5, VHCDR3 consists of the sequence of SEQ ID NO: 13, VLCDR1 consists of the sequence of SEQ ID NO: 29, VLCDR2 consists of the sequence of SEQ ID NO: 30, and VLCDR3 consists of the sequence of SEQ ID NO: 31 ; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 56, VHCDR2 consists of the sequence of SEQ ID NO: 57, VHCDR3 consists of the sequence of SEQ ID NO: 58, VLCDR1 consists of the sequence of SEQ ID NO: 71 , VLCDR2 consists of the sequence of SEQ ID NO: 72, and VLCDR3 consists of the sequence of SEQ ID NO: 73; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 56, VHCDR2 consists of the sequence of SEQ ID NO: 57, VHCDR3 consists of the sequence of SEQ ID NO: 58, VLCDR1 consists of the sequence of SEQ ID NO: 71 , VLCDR2 consists of the sequence of SEQ ID NO: 72, and VLCDR3 consists of the sequence of SEQ ID NO: 74; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 56, VHCDR2 consists of the sequence of SEQ ID NO: 57, VHCDR3 consists of the sequence of SEQ ID NO: 58, VLCDR1 consists of the sequence of SEQ ID NO: 71 , VLCDR2 consists of the sequence of SEQ ID NO: 72, and VLCDR3 consists of the sequence of SEQ ID NO: 75; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 59, VHCDR2 consists of the sequence of SEQ ID NO: 60, VHCDR3 consists of the sequence of SEQ ID NO: 61 , VLCDR1 consists of the sequence of SEQ ID NO: 71 , VLCDR2 consists of the sequence of SEQ ID NO: 72, and VLCDR3 consists of the sequence of SEQ ID NO: 76; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 56, VHCDR2 consists of the sequence of SEQ ID NO: 57, VHCDR3 consists of the sequence of SEQ ID NO: 58, VLCDR1 consists of the sequence of SEQ ID NO: 77, VLCDR2 consists of the sequence of SEQ ID NO: 78, and VLCDR3 consists of the sequence of SEQ ID NO: 79; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 62, VHCDR2 consists of the sequence of SEQ ID NO: 63, VHCDR3 consists of the sequence of SEQ ID NO: 64, VLCDR1 consists of the sequence of SEQ ID NO: 80, VLCDR2 consists of the sequence of SEQ ID NO: 78, and VLCDR3 consists of the sequence of SEQ ID NO: 81 ; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 65, VHCDR2 consists of the sequence of SEQ ID NO: 66, VHCDR3 consists of the sequence of SEQ ID NO: 67, VLCDR1 consists of the sequence of SEQ ID NO: 82, VLCDR2 consists of the sequence of SEQ ID NO: 83, and VLCDR3 consists of the sequence of SEQ ID NO: 84; or wherein:- VHCDR1 consists of the sequence of SEQ ID NO: 68, VHCDR2 consists of the sequence of SEQ ID NO: 69, VHCDR3 consists of the sequence of SEQ ID NO: 70, VLCDR1 consists of the sequence of SEQ ID NO: 85, VLCDR2 consists of the sequence of SEQ ID NO: 86 and VLCDR3 consists of the sequence of SEQ ID NO: 87; wherein said VHCDRs and VLCDRs are defined according to Kabat.

6. The antibody for the use according to any one of claims 1 -5 wherein said antibody comprises: a. a heavy chain variable domain sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 33, SEQ ID NO: 168, SEQ ID NO: 32, SEQ ID NO: 34 to SEQ ID NO: 37 or SEQ ID NO: 88 to SEQ ID NO: 95; or b. a light chain variable domain sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 39, SEQ ID NO: 171 , SEQ ID NO: 172, SEQ ID NO: 38, SEQ ID NO: 40 to SEQ ID NO:43, or SEQ ID NO: 96 to SEQ ID NO: 103 ; or c. the heavy chain variable domain of (a) and the light chain variable domain of (b).

7. The antibody for the use according to any one of claims 1 -6 wherein said antibody comprises (a) a VH sequence of the amino acid sequence and (b) a VL sequence of the amino acid sequence, wherein:- VH consists of the sequence of SEQ ID NO: 33 and VL consists of the sequence of SEQ ID NO: 39; or wherein:- VH consists of the sequence of SEQ ID NO: 168 and VL consists of the sequence of SEQ ID NO: 171 ; or wherein:- VH consists of the sequence of SEQ ID NO: 1689 and VL consists of the sequence of SEQ ID NO: 172; or wherein:- VH consists of the sequence of SEQ ID NO: 170 and VL consists of the sequence of SEQ ID NO: 39; or wherein:- VH consists of the sequence of SEQ ID NO: 32 and VL consists of the sequence of SEQ ID NO: 38; or wherein:- VH consists of the sequence of SEQ ID NO: 34 and VL consists of the sequence of SEQ ID NO: 40; or wherein:- VH consists of the sequence of SEQ ID NO: 35 and VL consists of the sequence of SEQ ID NO: 41 ; or wherein:- VH consists of the sequence of SEQ ID NO: 36 and VL consists of the sequence of SEQ ID NO: 42; or wherein:- VH consists of the sequence of SEQ ID NO: 37 and VL consists of the sequence of SEQ ID NO: 43; or wherein:- VH consists of the sequence of SEQ ID NO: 88 and VL consists of the sequence of SEQ ID NO: 96; or wherein:- VH consists of the sequence of SEQ ID NO: 88 and VL consists of the sequence of SEQ ID NO: 97; or wherein:- VH consists of the sequence of SEQ ID NO: 88 and VL consists of the sequence of SEQ ID NO: 98; or wherein:- VH consists of the sequence of SEQ ID NO: 91 and VL consists of the sequence of SEQ ID NO: 99; or wherein:- VH consists of the sequence of SEQ ID NO: 88 and VL consists of the sequence of SEQ ID NO: 100; or wherein:- VH consists of the sequence of SEQ ID NO: 93 and VL consists of the sequence of SEQ ID NO: 101 ; or wherein:- VH consists of the sequence of SEQ ID NO: 94 and VL consists of the sequence of SEQ ID NO: 102; or wherein:- VH consists of the sequence of SEQ ID NO: 95 and VL consists of the sequence of SEQ ID NO: 103.

8. The antibody for the use according to any one of claims 1 -7 wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 45 and the nucleotide sequence encoding VL comprises SEQ ID NO: 51 ; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 174 and the nucleotide sequence encoding VL comprises SEQ ID NO: 177; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 175 and the nucleotide sequence encoding VL comprises SEQ ID NO: 178 or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 176 and the nucleotide sequence encoding VL comprises SEQ ID NO: 179 or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 44 and the nucleotide sequence encoding VL comprises SEQ ID NO: 50; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 46 and the nucleotide sequence encoding VL comprises SEQ ID NO: 52; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 47 and the nucleotide sequence encoding VL comprises SEQ ID NO: 53; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 48 and the nucleotide sequence encoding VL comprises SEQ ID NO: 54; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 49 and the nucleotide sequence encoding VL comprises SEQ ID NO: 55; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 104 and the nucleotide sequence encoding VL comprises SEQ ID NO: 112; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 105 and the nucleotide sequence encoding VL comprises SEQ ID NO: 113; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 106 and the nucleotide sequence encoding VL comprises SEQ ID NO: 114; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 107 and the nucleotide sequence encoding VL comprises SEQ ID NO: 115; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 108 and the nucleotide sequence encoding VL comprises SEQ ID NO: 116; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 109 and the nucleotide sequence encoding VL comprises SEQ ID NO: 117; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 110 and the nucleotide sequence encoding VL comprises SEQ ID NO: 118; or wherein:- the nucleotide sequence encoding VH comprises SEQ ID NO: 111 and the nucleotide sequence encoding VL comprises SEQ ID NO: 119.

9. The antibody for the use according to any one of claims 1 -8 wherein said antibody is a human or a humanized antibody and / or is an IgG 1 or lgG2 or lgG4 antibody, preferably an lgG1 kappa antibody, an lgG1 lambda antibody, an lgG2 kappa antibody, an lgG2 lambda antibody, an lgG4 kappa antibody or an lgG4 lambda antibody.

10. The antibody for the use according to claim 9 wherein said lgG1 antibody comprises at least one amino-acid substitution in the heavy chain constant region (Fc) of the lgG1 antibody, wherein:- the at least one amino acid substitution is capable of reducing Fc receptor binding; or wherein- the at least one amino acid substitution is capable of reducing effector cell function of the antibody; or wherein- the at least one amino acid substitution is capable of extending the halflife of the antibody.

11. The antibody for the use according to claim 10 wherein the at least one amino acid substitution capable of extending the half-life of the antibody is selected from the following group: M252Y, S254T or T256E, where the numbering of the residues is according to the EU numbering system.

12. The antibody for the use according to any one of claims 1 -11 , wherein said antibody comprises:- a light chain comprising a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 171 , SEQ ID NO: 172, SEQ ID NO: 38, SEQ ID NO: 40 to SEQ ID NO: 43, or SEQ ID NO: 96to SEQ ID NO: 103; and a light constant domain comprising or consisting of a sequence of SEQ ID NO: 123 and / or- a heavy chain comprising a heavy chain variable domain comprising a sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 168, SEQ ID NO: 32, SEQ ID NO: 34 to SEQ ID NO: 37, SEQ ID NO: 88, SEQ ID NO: 91 , or SEQ ID NO: 93 to SEQ ID NO: 95; and a heavy chain constant domain comprising or consisting of a sequence of SEQ ID NO: 120 or of SEQ ID NO: 180.

13. The antibody for the use according to any one of claims 1 -12, wherein said antibody comprises:- a light chain comprising a light chain variable domain comprising or consisting of a sequence of SEQ ID NO: 39 and a light chain constant domain comprising or consisting of a sequence of SEQ ID NO: 123 and / or- a heavy chain comprising a heavy chain variable domain comprising or consisting of a sequence of SEQ ID NO: 33 and a heavy chain constant domain comprising or consisting of a sequence of SEQ ID NO: 120 or of SEQ ID NO: 180; preferably wherein said antibody comprises a light chain comprising or consisting of a sequence of SEQ ID NO: 4 and / or a heavy chain comprising or consisting of a sequence of SEQ ID NO: 125 or of SEQ ID NO: 126.

14. An isolated polynucleotide comprising at least one sequence that encodes the antibody or antigen binding fragment thereof as disclosed in any one of claims 1 -11 , preferably said polynucleotide being a cDNA, or a vector comprising said polynucleotide, preferably said vector being selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, an expression vector and a recombinant expression vector, or an isolated cell comprising said polynucleotide or said vector, preferably said isolated cell being a hybridoma or a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney cell (HEK293), for use in the prevention and / or treatment of colorectal cancer.

15. A pharmaceutical composition comprising an antibody that binds to TMEM219 receptor or an antigen-binding fragment thereof as disclosed in any one of claims 1 -13 and at least one pharmaceutically acceptable carrier for use in theprevention and / or treatment of colorectal cancer, and / or reduction of risk of developing colorectal cancer in patients with inflammatory bowel disease (IBD).

16. The pharmaceutical composition according to claim 15 further comprising a second therapeutic agent.

17. The antibody for use according to any one of claims 1-13 wherein said antibody is used in combination with at least one further therapeutic agent.

18. The antibody for use according to claim 11 or the pharmaceutical composition for use according to claim 16 wherein said therapeutic agent is an active ingredient which is known to be effective in the prevention and / or treatment of cancer, preferably of colorectal cancer, such as a chemotherapeutic agent or a radiotherapy agent, preferably said therapeutic agent is selected from fluoropyrimidine, oxaliplatin, irinotecan, leucovorin, capecitabine, anti-VEGF monoclonal antibodies or anti-VEGF small molecules, such as bevacizumab or aflibercept, cetuximab or panitumumab, ramucirumab, BRAF-inhibitors, such as encorafenib, 5-aminosalicylic acid, immunomodulators, such as thiopurines, and immune checkpoint inhibitors, such as pembrolizumab.

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