TMEM219 antibodies and their therapeutic uses

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to the IGFBP3 receptor, i.e., TMEM219, a method for producing the antibody or an antigen-binding fragment thereof, a pharmaceutical composition containing the antibody, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to the IGFBP3 receptor, i.e., TMEM219, a method for producing the antibody or an antigen-binding fragment thereof, a pharmaceutical composition containing the antibody, and uses thereof. [Background technology]

[0002] IGFBP3 / TMEM219 axis Insulin-like growth factor binding proteins (IGFBPs) are a family of seven binding proteins that regulate the bioavailability of insulin-like growth factors (IGFs). Among them, IGFBP3 is the most abundant, present in almost all tissues, and has a higher affinity for IGFs; in fact, approximately 80–90% of IGFs bind to IGFBP3 in a ternary complex with the acid-labile subunit (ALS) (1).

[0003] In addition to its ability to regulate IGF availability, IGFBP3 has also been shown to have IGF-independent functions (2). Indeed, it can associate with cell surface proteins, cell surface receptors with essential signaling capabilities, intracellular proteins, and nuclear proteins (transcriptional regulators), thereby affecting cell proliferation and directly inducing apoptosis (2). Among death receptors, the single-span membrane protein TMEM219 has been shown to bind highly to IGFBP-3 (3). Binding of IGFBP3 to TMEM219 induces caspase-8-mediated apoptosis in various cells, including cancer cells (i.e., prostate and breast cancer cells) (3), but also in stem cells (i.e., colon stem cells) (4). Blocking or enhancing the IGFBP3 / TMEM219 axis by various strategies has been shown to prevent or increase cell death, respectively. To the best of our knowledge, there are no commercially available monoclonal antibodies against TMEM219 or IGFBP3 that can prevent IGFBP3 / TMEM219 binding and abolish the IGF-I-independent, caspase-8-mediated deleterious effects of IGFBP3 binding to TMEM219 on target tissues / cells.

[0004] IGFBP3 / TMEM219 axis in diabetes Both type 1 (T1D) and type 2 diabetes (T2D) are characterized by β-cell loss, resulting in reduced insulin secretion, dysregulated blood glucose levels, and polysaccharidosis (5, 6). Despite different pathogenic mechanisms, the autoimmune response in T1D and insulin resistance / inflammation in T2D both result in a gradual loss of β-cell mass. In fact, it is becoming clear that the autoimmune activation that occurs does not appear to be sufficient to fully explain β-cell loss in T1D (5). Furthermore, the inability of immunotherapy to treat T1D (7) has highlighted that (i) autoimmunity may not be the only factor involved in T1D pathogenesis and (ii) alternative strategies targeting different disease mechanisms, such as β-cell loss, are needed to establish effective treatments for T1D. The observation that scattered β cells are detected in individuals with long-term T1D (8) supports the idea that either new β cells must arise to maintain β-cell turnover (5, 9) or that destroyed β cells may be "different" or prone to death (10). This may suggest that up- / down-regulated expression of surface β-cell receptors may play an important role in their recognition by the immune system and, more importantly, that other non-immunological determinants may regulate β-cell fate and function. Therefore, preventing non-immunological β-cell destruction in T1D and the progressive loss of β cells in T2D may tip the balance between β-cell development and destruction toward the restoration of an appropriate β-cell population, paving the way for novel therapeutic approaches that can halt or delay the initial stages of disease. The IGFBP3 receptor, TMEM219, is expressed in β-cell lines and human / mouse pancreatic islets, and its ligation has been shown to be toxic to β-cells. Interestingly, it has also been observed that mice transgenic for human IGFBP3 develop polysaccharidosis, exhibit reduced islet mass, and show a reduced response to insulin-glucose stimulation (11), whereas those knocked down for IGFBP3 do not show any alterations in glucose metabolic control (12).

[0005] In humans, Drogan and colleagues recently published that high circulating concentrations of IGFBP3 are associated with the development of T2D. (13) Furthermore, a recent study by the Diabimmune research group demonstrated that IGFBP3 levels correlate with autoantibody positivity and the chance for seroconversion in children at risk for T1D, thus suggesting a role for circulating IGFBP3 in the early development of β-cell autoimmunity. (14)

[0006] The IGFBP3 receptor, TMEM219, has been previously described as a death receptor, the activation of which triggers caspase-8-mediated apoptosis in target cells, resulting in their loss (4).

[0007] IGFBP3 / TMEM219 axis in inflammatory bowel disease Intestinal stem cells (ISCs) reside at the base of the crypts in the small and large intestine and regulate crypt renewal and turnover. In particular, ISCs can differentiate along the crypts to give rise to goblet cells, enterocytes, and enteroendocrine cells (4).

[0008] Inflammatory bowel disease (IBD), encompassing two clinical entities, Crohn's disease (CD) and ulcerative colitis (UC), is an immune-mediated chronic condition affecting approximately 2.5 million people in Europe and 1 million people in the United States (15). Although the pathogenesis of IBD is still under investigation, recent evidence suggests that reduced differentiation of ISCs into Paneth cells in ileal CD and into goblet cells in UC may play an important role in the development of the disease. In particular, both local signaling and inflammatory pathways in the mucosa maintain the number and function of ISCs in response to external stimuli, thereby maintaining intestinal homeostasis (16). Indeed, Yancu et al. recently published results supporting the role of IGFBP-3 in CD. Indeed, they demonstrated that knockout of IGFBP3 has a role in modulating inflammation in a dextran-sodium-sulfate (DSS) colitis mouse model (17).

[0009] We recently found that the insulin-like growth factor binding protein 3 (IGFBP3) receptor, i.e., the TMEM219 receptor, is expressed in ISCs, and that interaction with the circulating hormone IGFBP3 regulates ISC fate and function in models of intestinal damage in diabetes and diabetic enteropathy. (4) Because diabetic enteropathy and IBD share common features, such as alterations in intestinal stem cell (ISC) homeostasis and altered mucosal morphology, these results may add important insights into the still-unknown pathogenesis of IBD and potentially lead to the introduction of novel therapeutic approaches for IBD treatment.

[0010] Currently available therapies for IBD are based on the use of anti-inflammatory drugs and immunotherapeutic strategies, which are complicated by several adverse effects and questionable long-term efficacy. Surgery has also been used successfully in advanced stages of the disease, especially in UC (15). Disease relapse, primarily in CD, is also frequent, thus highlighting the need for different therapeutic approaches. Consequently, the identification of novel therapeutic targets and strategies in the treatment of IBD is of great clinical importance and necessary for the health of the community.

[0011] WO2016193497 and WO2016193496 (incorporated herein by reference in their entireties) describe ecto-TMEM, the extracellular domain of TMEM219, which functions as an effective therapeutic agent. However, receptor constructs are less desirable as therapeutic agents than antibodies. Thus, there remains a need for additional therapeutic agents, such as antibodies or their derivatives, that mimic the effects of ecto-TMEM. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2016193497 [Patent Document 2] WO2016193496 [Patent Document 3] WO2002 / 020565 [Patent Document 4] US20040132028 [Patent Document 5] U.S. Patent Application Publication No. 2003 / 0157108 [Patent Document 6] US2004 / 0093621 [Patent Document 7] WO2003 / 011878 [Patent Document 8] U.S. Patent No. 6,602,684 [Patent Document 9] US2005 / 0123546 [Patent Document 10] WO1997 / 30087 [Patent Document 11] WO1998 / 58964 [Patent Document 12] WO1999 / 22764 [Patent Document 13] U.S. Patent No. 7,521,541 [Patent Document 14] U.S. Patent No. 7,083,784 [Patent Document 15] U.S. Patent No. 8,323,962 [Patent Document 16] U.S. Patent No. 5,215,534 [Patent Document 17] U.S. Patent No. 9,248,242 [Patent Document 18] U.S. Patent No. 9,427,531 [Patent Document 19] U.S. Patent No. 9,566,395 [Non-patent literature]

[0013] [Non-Patent Document 1] Chothia et al. (1992) J. Mol. Biol. 227:799~817 [Non-patent document 2] Tomlinson, (1992) J. Mol. Biol. 227:776~798

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[0014] Disclosed herein are antibodies that specifically bind to human ecto-TMEM (the extracellular domain of TMEM) with high affinity and can reduce or eliminate IGFBP3 binding to its cognate receptor, TMEM219, without activating the TMEM219 pathway upon binding. Such neutralizing antibodies are useful in treating disorders in which IGFBP3 binding to TMEM219 contributes to the pathophysiology of diseases such as diabetic bowel disease, inflammatory bowel disease (IBD), e.g., ulcerative colitis and Crohn's disease, and type 1 or type 2 diabetes. Such neutralizing antibodies provide advantageous therapeutic agents with therapeutic activity similar to that of the receptor-based ligand trap ecto-TMEM219. [Means for solving the problem]

[0015] In a first aspect, there is provided an isolated antibody or antigen-binding fragment thereof that binds to human TEMP219 and inhibits or reduces binding of IGFBP3 to the TMEM219 receptor.

[0016] Preferably, the isolated antibody or antigen-binding fragment thereof inhibits, reduces, or neutralizes activation of the TMEM219 receptor induced by binding of IGFBP3.

[0017] IGFBP3-induced activation of the TMEM219 receptor can be measured by any method known in the art or described below. In particular, IGFBP3-induced activation of the TMEM219 receptor can be measured by measuring increased apoptosis as described therein, or reduced minigut growth, as known in the art and described in several publications (4, 18, 27, 28).

[0018] Preferably, the isolated antibody or antigen-binding fragment thereof does not activate the TMEM219 pathway upon binding to human TMEM219.

[0019] In a preferred embodiment, the isolated antibody or antigen-binding fragment thereof is effective in preserving beta cells and / or preventing pancreatic islet destruction in diabetic patients and / or controlling blood glucose levels in an in vivo model.

[0020] In a preferred embodiment, the isolated antibody or antigen-binding fragment thereof is effective in reducing acute colitis in an in vivo model.

[0021] In a preferred embodiment, the isolated antibody or antigen-binding fragment thereof is effective in reducing DSS-induced increases in DAI scores and histological scores, or reducing acute colitis in an in vivo model.

[0022] The present invention provides Increased mini-gut growth in healthy subjects treated with a-IGFBP3; b-increased mini-gut growth in IBD patients; c - Increased mini-gut growth in healthy subjects treated with diabetic enteropathy serum; Increased expression of EphB2 and / or LGR5 in mini-guts of healthy subjects treated with d-IGFBP3; Decreased caspase 8 expression in mini-intestines of healthy subjects treated with e-IGFBP3; reduced β-cell loss in β-cells treated with f-IGFBP3; Increased insulin expression in β-cells treated with g-IGFBP3; inhibiting or reducing h-DSS-induced intestinal cell apoptosis; restores PCNA expression in i-DSS-treated colon; reduced β-cell apoptosis in j-IGFBP3-treated β-cells; k - Reduction of insulitis score in animal models of diabetes; l -reduced development of diabetes in animal models of diabetes; m-protects beta-cell injury in animal models of diabetes; Prevents beta-cell loss in animal models of n-diabetes Also provided is an isolated antibody or antigen-binding fragment thereof having at least one activity selected from the group consisting of:

[0023] Preferably, the increase in a), b), and c) is at least 20%; the increase in d) and e) is at least 30%, preferably at least 50%; the decrease in f) and the increase in g) is at least 10%, the decrease in k) and l) is at least 50%; preferably, the decrease in l) is at least 70%. Preferably, the decrease in j) is at least 30%.

[0024] The present invention provides a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 8, 10, 56, 59, 62, 65, and 68; ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 11, 57, 60, 63, 66, and 69; and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20, 23, 26, 29, 71, 77, 80, 82, and 85; ii. a CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 18, 21, 24, 27, 30, 72, 78, 83, and 86; and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 19, 22, 25, 28, 31, 73, 74, 75, 76, 79, 81, 84, 87, 166, and 167. The present invention provides an isolated antibody or antigen-binding fragment thereof comprising:

[0025] Preferably, the isolated antibody or antigen-binding fragment thereof comprises: - the Kabat, IMGT, Chothia, AbM or Contact CDRs of SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:17 and SEQ ID NO:18 and SEQ ID NO:19 or TC01 or TC05, or - SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:17 and SEQ ID NO:18 and SEQ ID NO:166 or the Kabat, IMGT, Chothia, AbM or Contact CDRs of TC03, or - SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:17 and SEQ ID NO:18 and SEQ ID NO:167 or the Kabat, IMGT, Chothia, AbM or Contact CDRs of TC04, or SEQ ID NO:68 and SEQ ID NO:69 and SEQ ID NO:70 and SEQ ID NO:85 and SEQ ID NO:86 and SEQ ID NO:87 or Kabat, IMGT, Chothia, AbM, or Contact CDRs of TM1 Includes:

[0026] Preferably, the isolated antibody or antigen-binding fragment thereof comprises: a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); and / or b. A light chain variable domain (VL) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org). Includes:

[0027] Preferably, the isolated antibody or antigen-binding fragment thereof comprises the CDRs shown in Tables 2 to 5 (Tables 1 to 8), Tables 8 to 11 (Tables 11 to 14), and Tables 3.1 to 3.4 (Tables 3 to 6).

[0028] Preferably, the antibody or antigen-binding fragment thereof specifically binds to TMEM219.

[0029] Preferably, it is: Increased mini-gut growth in healthy subjects treated with a-IGFBP3; b-increased mini-gut growth in IBD patients; c - Increased mini-gut growth in healthy subjects treated with diabetic enteropathy serum; Increased expression of EphB2 and / or LGR5 in miniguts of healthy subjects treated with d-IGFBP 3; Decreased caspase 8 expression in mini-intestines of healthy subjects treated with e-IGFBP3; reduced β-cell loss in β-cells treated with f-IGFBP3; Increased insulin expression in β-cells treated with g-IGFBP3; inhibiting or reducing h-DSS-induced intestinal cell apoptosis; restores PCNA expression in i-DSS-treated colon; Decreased β-cell apoptosis in β-cells treated with j-IGFBP3 and (iii) at least one activity selected from the group consisting of:

[0030] Preferably, the increase in a), b), and c) is at least 20%; the increase in d) and e) is at least 50%; the decrease in f) and the increase in g) is at least 10%.

[0031] Preferably, the isolated antibody or antigen-binding fragment thereof comprises: a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 1, 8, 10, 56, 59, 62, 65, and 68; ii. a CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 2, 11, 57, 60, 63, 66, and 69; and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 14, 20, 23, 26, 29, 71, 77, 80, 82, and 85; ii. a CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 15, 21, 24, 27, 30, 72, 78, 83, and 86; and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 16, 22, 25, 28, 31, 73, 74, 75, 76, 79, 81, 84, 87, 166, and 167. Includes:

[0032] Preferably, the isolated antibody or antigen-binding fragment thereof comprises: - the Kabat, IMGT, Chothia, AbM, or Contact CDRs of SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:17 and SEQ ID NO:18 and SEQ ID NO:19 or TC01 or TC05, or - SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:17 and SEQ ID NO:18 and SEQ ID NO:166 or the Kabat, IMGT, Chothia, AbM or Contact CDRs of TC03, or - SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:17 and SEQ ID NO:18 and SEQ ID NO:167 or the Kabat, IMGT, Chothia, AbM or Contact CDRs of TC04, or SEQ ID NO:68 and SEQ ID NO:69 and SEQ ID NO:70 and SEQ ID NO:85 and SEQ ID NO:86 and SEQ ID NO:87 or Kabat, IMGT, Chothia, AbM, or Contact CDRs of TM1 Includes:

[0033] Preferably, the isolated antibody or antigen-binding fragment thereof comprises: a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); and / or b. A light chain variable domain (VL) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org); and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of sequences defined using the abysis tool analysis (www.abysis.org). Includes:

[0034] Preferably, the isolated antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable domain sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 32 to SEQ ID NO: 37, or SEQ ID NO: 88 to SEQ ID NO: 95, or SEQ ID NO: 168, SEQ ID NO: 169, and SEQ ID NO: 170; or b. a light chain variable domain sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 43, or SEQ ID NO: 96 to SEQ ID NO: 103, or SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173; c. The light chain variable domain of (a) and the heavy chain variable domain of (b). Includes:

[0035] More 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 an antigen-binding fragment thereof, and preferably, the isolated antibody is TC01, TC05, TC03, TC04, or TM1, or an antigen-binding fragment thereof, as reported in Tables 4 (Table 7), 7 (Table 10), and 10-13 (Tables 13-16) and Tables 3.1-3.4 (Tables 3-6). Preferably, the isolated antibody is TC01.

[0036] More preferably, the isolated antibody is TC01 comprising SEQ ID NO:33 and SEQ ID NO:39, TC03 comprising SEQ ID NO:168 and SEQ ID NO:171, TC04 comprising SEQ ID NO:169 and SEQ ID NO:172, TC05 comprising SEQ ID NO:170 and SEQ ID NO:173, TA02 comprising SEQ ID NO:32 and SEQ ID NO:38, TC01 comprising SEQ ID NO:33 and SEQ ID NO:39, TC02 comprising SEQ ID NO:34 and SEQ ID NO:40, TD01 comprising SEQ ID NO:35 and SEQ ID NO:41, TE01 comprising SEQ ID NO:36 and SEQ ID NO:42, TG02 comprising SEQ ID NO:37 and SEQ ID NO:43, TE02.1 comprising SEQ ID NO:88 and SEQ ID NO:96, TE02.2 comprising SEQ ID NO:89 and SEQ ID NO:97, TE02.3 comprising SEQ ID NO:90 and SEQ ID NO:98, TE03 comprising SEQ ID NO:91 and SEQ ID NO:99, TE04 comprising SEQ ID NO:92 and SEQ ID NO:100, TE07 comprising SEQ ID NO:93 and SEQ ID NO:101, TE10 comprising SEQ ID NO:94 and SEQ ID NO:102, TM1 comprising SEQ ID NO:95 and SEQ ID NO:103.

[0037] Preferably, the isolated antibody or antigen-binding fragment of the present invention is 10 -7 Affinity constant of M or less, preferably 2 × 10 ー8 It binds to human TMEM219 with an affinity constant of less than M.

[0038] The present invention provides (a) specifically binds to an epitope on IGFBP3 that is the same as or similar to the epitope recognized by monoclonal antibodies TC01, TC03, TC04, TC05, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, and TE10, as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); or (b) cross-competes for binding with monoclonal antibodies TC01, TC03, TC04, TC05, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, and TE10, as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); or (c) exhibits the same or similar binding affinity or specificity, or both, as any of TC01, TC03, TC04, TC05, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, and TE10, as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); or (d) has one or more biological properties of an antibody molecule described herein, e.g., an antibody molecule selected from any of TC01, TC03, TC04, TC05, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, and TE10, as defined in Tables 2-19 (Tables 1-22) and Tables 3.1-3.4 (Tables 3-6); or (e) having one or more pharmacokinetic properties of an antibody molecule described herein, e.g., an antibody molecule selected from TC01, TC03, TC04, TC05, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10, as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); Also provided is an isolated antibody or antigen-binding fragment thereof.

[0039] Preferably, the isolated antibody or antigen-binding fragment thereof of the present invention is a human antibody or a humanized antibody.

[0040] More preferably, the isolated antibody or antigen-binding fragment thereof of the present invention is an IgG2 or IgG4 antibody, preferably an IgG2κ antibody, an IgG2λ antibody, an IgG4κ antibody, or an IgG4λ antibody, and preferably, the IgG2 or IgG4 is human IgG2 or human IgG4.

[0041] The present invention provides an isolated polynucleotide comprising at least one sequence encoding an antibody or antigen-binding fragment thereof as defined above, preferably said polynucleotide is a cDNA.

[0042] The present invention provides a vector comprising a polynucleotide as defined above, 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.

[0043] The present invention further provides an isolated cell comprising a polynucleotide as defined above or a vector as defined above, preferably the isolated cell is a hybridoma or a Chinese hamster ovary (CHO) cell or a human embryonic kidney cell (HEK293).

[0044] The present invention further provides an antibody or antigen-binding fragment thereof or an isolated polynucleotide or vector or an isolated cell as defined above for use as a medicament, preferably for use in the treatment of diabetes, intestinal and / or bowel disorders, malabsorption syndrome, cachexia, or diabetic bowel disease, preferably wherein the diabetes is type I or type II diabetes, and preferably wherein the intestinal and / or bowel disorder is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease, or ileus.

[0045] The present invention also provides a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof or the isolated polynucleotide or vector or the isolated cell as defined above and a pharmaceutically acceptable carrier, preferably for use in the treatment of diabetes, intestinal and / or bowel disorders, malabsorption syndromes, cachexia, or diabetic bowel disease, wherein preferably the intestinal and / or bowel disorder is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease, or ileus.

[0046] The present invention provides a method for inhibiting the binding of IGFBP3 to a TMEM219 receptor, comprising the step of contacting TMEM219 with an antibody or composition as defined above.

[0047] The present invention provides a method for treating diabetes, preferably type 1 or type 2 diabetes, intestinal and / or intestinal disorders, malabsorption syndrome, cachexia, or diabetic enteropathy, wherein preferably the intestinal and / or intestinal disorder is inflammatory bowel disease, IBD, celiac disease, Crohn's disease, or ileus, the method comprising the step of administering to a subject in need thereof a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof, or isolated polynucleotide, or vector, or isolated cell defined above, and a pharmaceutically acceptable carrier, or the isolated antibody or antigen-binding fragment thereof, or isolated polynucleotide, or vector, or isolated cell defined above, to a subject in need thereof.

[0048] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof, comprising the steps of obtaining a cell as defined above and producing the antibody or antigen-binding fragment thereof.

[0049] In some embodiments, the combination includes an inhibitor of IGFBP3 (e.g., an anti-TMEM antibody molecule described herein). Accordingly, disclosed herein are compositions and methods for detecting IGFBP3, as well as methods for treating various disorders, including diabetes and intestinal and / or bowel disorders, using anti-TTMEM antibody molecules and combinations thereof.

[0050] Thus, in one aspect, the present invention provides a method for producing a composition comprising: - high affinity, e.g., at least about 4x10 6 M -1 , preferably 10 7 M -1 , typically about 10 8 M -1 , more typically about 10 9 M -1 From 10 10 M -1 binds to TMEM219, e.g., human TMEM219, with an affinity constant of 0.01 or higher; - inhibiting or reducing the binding of IGFBP3 to its receptor TMEM; - specifically binds to an epitope on TMEM219 that is the same as or similar to the epitope recognized by the monoclonal antibodies TC01, TC03, TC04, TC04, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); - cross-compete for binding with the monoclonal antibodies TC01, TC03, TC04, TC04, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); - exhibiting the same or similar binding affinity or specificity, or both, as any of TC01, TC03, TC04, TC04, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); - exhibit the same or similar binding affinity or specificity, or both, as the antibody molecules (e.g., heavy chain variable regions and light chain variable regions) described in Tables 2 to 19 (Tables 1 to 22); - exhibits the same or similar binding affinity or specificity, or both, as an antibody molecule (e.g., heavy chain variable region and light chain variable region) having an amino acid sequence shown in Tables 4, 5, 10, 11, 16, 17 (Tables 7, 8, 13, 14, 19, 20); - exhibit the same or similar binding affinity or specificity, or both, as antibody molecules (e.g., heavy chain variable regions and light chain variable regions) encoded by the nucleotide sequences set forth in Tables 6-7 and 12, 13, 16, and 17 (Tables 9-10 and 15, 16, 19, and 20); - binds to TMEM219 the same epitope or an overlapping epitope as a second antibody molecule (wherein the second antibody molecule is an antibody molecule described herein, for example an antibody molecule selected from TC01, TC03, TC04, TC04, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6)); - have one or more biological properties of an antibody molecule described herein, e.g., an antibody molecule selected from any of TC01, TC03, TC04, TC04, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); - have one or more pharmacokinetic properties of an antibody molecule described herein, e.g., an antibody molecule selected from any of TC01, TC03, TC04, TC04, TA02, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2 to 19 (Tables 1 to 22) and Tables 3.1 to 3.4 (Tables 3 to 6); - inhibit one or more activities of IGFBP3, for example, resulting in one or more of: at least a 20% increase in the development of mini-guts from tissue samples from IBD patients compared to untreated samples; and / or at least a 20% increase in the development of mini-gut growth in the presence of IGFBP3 compared to untreated samples; or at least a 20% increase in the development of mini-gut growth in the presence of diabetic enteropathy serum compared to untreated samples; - induces an increase in EphB2 and LGR5 of at least 50% compared to IGFBP3-treated samples; or a decrease in caspase 8 expression levels of at least 50% compared to IGFBP3-treated samples; or - inhibits one or more activities of IGFBP3, e.g., resulting in one or more of a reduction in beta cell loss or an increase in insulin; the reduction in beta cell loss or the increase in insulin is at least 10% compared to an IGFBP3-treated sample; (xvi) inhibiting, reducing, or neutralizing one or more activities of IGFBP3, resulting in blocking or reducing IGFBP3-induced apoptosis; - Binds to human TMEM219 and cross-reacts with cynomolgus monkey TMEM219 The present invention features antibody molecules (e.g., isolated or recombinant antibody molecules) having one or more of:

[0051] Nucleic acid molecules encoding the antibody molecules, expression vectors, host cells, and methods for making the antibody molecules are also provided. Immunoconjugates, multi- or bispecific antibody molecules, and pharmaceutical compositions comprising the antibody molecules are also provided.

[0052] Without being bound by theory, it is believed that the IGFBP3 / TMEM219 axis is dysfunctional in inflammatory bowel disease (IBD), thus causing ISC loss and altered mucosal barrier function, which further allows for microbial invasion, leading to immune response activation and inflammation, and perpetuating it. It is believed that the use of agents that block IGFBP3-TMEM219 interaction in IBD may protect ISCs, maintain the integrity of the intestinal barrier, and consequently prevent the development of local inflammation.

[0053] Furthermore, activation of TMEM219 signaling increases β-cell apoptosis by upregulating caspase-8 expression and reducing insulin expression. IGFBP3 is elevated in the serum of patients with pre-T1D and pre-T2D, as well as in newly diagnosed and long-standing diabetic patients, and TMEM219 is expressed in β-cells. Expression or overexpression of TMEM219 favors β-cell destruction, affecting β-cell mass, and the resulting glycolysis / inflammation executes processes during the development and progression of diabetes. Altered glycemic control and inflammation in the pre-diabetic state favor increased hepatic production of IGFBP3, which may target TMEM219 expressed in pancreatic β-cells, triggering a loop in which TMEM219 overexpression parallels increased IGFBP3 release. TMEM219 may then trigger β-cell death; consequently, targeting the IGFBP3 / TMEM219 axis may prevent such cell death.

[0054] The anti-TMEM antibody molecules disclosed herein can be used (alone or in combination with other drugs or therapies) to treat, prevent, and / or diagnose disorders, such as diabetes, and intestinal and / or bowel disorders, malabsorption syndromes, inflammatory bowel disease, cachexia, IBD, celiac disease, diabetic enteropathy, etc. Additionally, disclosed herein are methods and compositions that include combinations of two, three, or more therapeutic agents selected from one, two, or all of the following categories (i)-(iii): (i) drugs that treat diabetes; (ii) anti-inflammatory agents; or (iii) immunotherapeutic agents.

[0055] Additional therapeutic agents include insulin, insulin glargine as detailed in Vandana, 2014 (19, incorporated by reference), biguanides, glucosidase inhibitors, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists as detailed in George et al. 2013 (20, incorporated by reference), drugs used to prevent diabetes, aspirin, anticoagulants and platelet antiaggregants (e.g., enoxaparin, heparin, sulodexide, etc.); cholesterol-lowering drugs (e.g., statins, bile acid sequestrants, ezetimibe, Marsha et al. 2011 (21, incorporated by reference); other antihypertensive agents (e.g., thiazides, ACE inhibitors, beta and alpha blockers, etc.); antiapoptotic agents, anti-inflammatory agents, corticosteroids, and immunosuppressants (22, incorporated by reference), adjuvant therapy in organ transplantation, protective agents in cell therapy approaches, painkillers, antibiotics, probiotics, TNF-α blockers (23, incorporated by reference), SGLT2 inhibitors (e.g., gliflozin derivatives, etc.), integrin inhibitors (24, incorporated by reference), and other agents for treating diabetes.

[0056] Methods for measuring increased mini-gut growth compared to mini-gut growth in the presence of IGFBP3 and / or diabetic enteropathy serum are known in the art and described in several publications (4, 18, 27, 28).

[0057] Methods for measuring increased and / or decreased expression of EphB2, LGR5, or caspase-8 compared to expression in the presence of IGFBP3 are known in the art, and include, for example, quantitative RT-PCR, real-time RT-PCR, microarrays, Northern blotting, RNA-Seq (29, 30), or those described in the Methods section below.

[0058] Methods for measuring reduced beta cell loss compared to beta cell loss in the presence of IGFBP3 are known in the art and include, for example, cell proliferation assays (CFSE staining, Calcein / PI staining, trypan blue exclusion, BrdU staining, MTT), apoptosis assays (TUNEL, caspase activation and detection, Annexin V binding), or those described in the Methods section below.

[0059] Methods for measuring increased insulin levels compared to insulin levels in the presence of IGFBP3 are known in the art and include Western blotting, ELISA, and mass spectrometry (31-33).

[0060] Methods for measuring reduced apoptosis compared to apoptosis in the presence of IGFBP3 are known in the art and include DNA fragmentation, caspase activation assays, mitochondrial membrane permeability, Annexin V binding (34), or those described in the Methods section below.

[0061] In some embodiments, the antibody molecule binds to IGFBP3 with high affinity, e.g., with a KD that is about the same as, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher or lower than, the KD of a murine or chimeric anti-TMEM antibody molecule or a commercial anti-TMEM antibody molecule. In some embodiments, the KD of the murine or chimeric anti-TMEM antibody molecule is less than about 0.4, 0.3, 0.2, 0.1, or 0.05 nM, as measured, for example, by Biacore or KinExA (KindexA) assays. In some embodiments, the KD of the murine or chimeric anti-TMEM219 antibody molecule is less than about 0.2 nM. In other embodiments, the KD of the murine or chimeric anti-IGFBP3 antibody molecule is less than about 10, 5, 3, 2, or 1 nM, e.g., as measured by binding on cells expressing IGFBP3 (e.g., 300.19 cells). In some embodiments, the KD of the murine or chimeric anti-IGFBP3 antibody molecule is less than about 1 nM.

[0062] Methods for measuring binding to TMEM219 are known in the art as protein-protein interaction assays, examples of which include ELISA, co-immunoprecipitation, surface plasmon resonance, FRET-Förster resonance energy transfer (35), or those described in the Methods section below.

[0063] In some embodiments, the expression level of the antibody molecule is higher than the expression level of a murine or chimeric antibody molecule, e.g., a murine anti-TMEM antibody molecule, a commercial anti-TMEM antibody molecule, or a chimeric anti-TMEM antibody molecule, e.g., at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher. In some embodiments, the antibody molecule is expressed in HEK293 cells, CHO cells, or any suitable mammalian cell line known in the art.

[0064] In some embodiments, the anti-TMEM219 antibody molecule reduces one or more TMEM-associated activities with an IC50 (concentration at 50% inhibition) that is about the same as or lower than the IC50 of a murine anti-TMEM antibody molecule, a commercial anti-TMEM antibody molecule, or a chimeric anti-TMEM antibody molecule, e.g., a murine anti-TMEM antibody molecule, a commercial anti-TMEM antibody molecule, or a chimeric anti-TMEM antibody molecule described herein, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower.

[0065] In some embodiments, the anti-TMEM antibody molecules described herein have improved stability, e.g., at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times more stable in vivo or in vitro than a murine, commercial, or chimeric anti-TMEM antibody molecule, such as HPA051870, as defined in the Materials section below.

[0066] In one embodiment, the anti-TMEM antibody molecule is a humanized antibody molecule.

[0067] In another embodiment, the anti-TMEM antibody molecule comprises at least one antigen-binding region, e.g., a variable region or antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10 as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, or 13 (Tables 9-10, 15, or 16); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0068] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, three, or four variable regions from an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, 13 (Tables 9-10, 15, 16); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0069] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10 as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, or 13 (Tables 9-10, 15, or 16); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0070] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14), or encoded by the nucleotide sequence in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0071] In yet another embodiment, the anti-TMEM antibody molecule comprises a heavy chain constant region for IgG4, e.g., human IgG4. In one embodiment, the human IgG4 comprises a substitution at position 228 (e.g., a Ser to Pro substitution). In one embodiment, the human IgG4 comprises a substitution at position 235 (e.g., a Leu to Glu substitution). In one embodiment, the human IgG4 comprises 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 yet another embodiment, the anti-TMEM antibody molecule comprises a heavy chain constant region for IgG1, e.g., human IgG1. In one embodiment, the human IgG1 comprises a substitution at position 297 (e.g., an Asn to Ala substitution). In one embodiment, the human IgG1 comprises a substitution at position 250, a substitution at position 428, or both (e.g., a Thr to Gln substitution at position 250 and / or a Met to Leu substitution at position 428). In one embodiment, the human IgG1 includes a substitution at position 234, a substitution at position 235, or both (eg, a Leu to Ala substitution at position 234 and / or a Leu to Ala substitution at position 235).

[0072] In yet another embodiment, the anti-TMEM antibody molecule comprises a κ light chain constant region, e.g., a human κ light chain constant region. In one embodiment, the light chain constant region comprises an amino acid sequence set forth in Table 8, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical thereto).

[0073] In another embodiment, the anti-TMEM antibody molecule comprises a heavy chain constant region for IgG4, e.g., human IgG4, and a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the human IgG1 or IgG4 comprises substitutions in the variable regions to reduce aggregation, reduce charge heterogeneity, increase affinity, and modulate antigen binding; mutational elimination of instability hotspots in the CDRs of putative N-glycosylation sites in the variable regions, as described in (26) (incorporated by reference).

[0074] In another embodiment, the anti-TMEM antibody molecule comprises a heavy chain variable domain and constant region, a light chain variable domain and constant region, or both, comprising the amino acid sequence of any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10 as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or encoded by the nucleotide sequence in Tables 6-7, 12, or 13 (Tables 9-10, 15, or 16); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences. The anti-TMEM antibody molecule also includes a leader sequence from the heavy chain or light chain, or both, as appropriate.

[0075] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three complementarity determining regions (CDRs) from the heavy chain variable region of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10, as defined in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14), or encoded by the nucleotide sequence in Tables 6, 7, 12, or 13 (Tables 9, 10, 15, or 16); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0076] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs (or collectively, all of the CDRs) from a heavy chain variable region comprising an amino acid sequence set forth in Tables 2-5, 8-11 (Tables 1-8, 11-14) or encoded by a nucleotide sequence set forth in Tables 6-7, 12, 13 (Tables 9-10, 15, 16). In one embodiment, one or more CDRs (or collectively, all of the CDRs) have one, two, three, four, five, six, or more alterations, e.g., amino acid substitutions or deletions, relative to the amino acid sequence set forth in Tables 2-5, 8-11 (Tables 1-8, 11-14) or the amino acid sequence encoded by the nucleotide sequence set forth in Tables 6-7, 12, 13 (Tables 9-10, 15, 16).

[0077] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs from a light chain variable region of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10 as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, or 13 (Tables 9-10, 15, or 16); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0078] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs (or, collectively, all of the CDRs) derived from a light chain variable region comprising an amino acid sequence set forth in Tables 2-5, 8-11 (Tables 1-8, 11-14) or an amino acid sequence encoded by a nucleotide sequence set forth in Tables 6-7, 12, 13 (Tables 9-10, 15, 16). In one embodiment, one or more CDRs (or, collectively, all of the CDRs) have one, two, three, four, five, six, or more alterations, e.g., amino acid substitutions or deletions, relative to the amino acid sequence set forth in Tables 2-5, 8-11 (Tables 1-8, 11-14) or the amino acid sequence encoded by the nucleotide sequence set forth in Tables 6-7, 12, 13 (Tables 9-10, 15, 16). In certain embodiments, the anti-TMEM3 antibody molecule comprises a substitution in a light chain CDR, for example, one or more substitutions in CDR1, CDR2, and / or CDR3 of the light chain.

[0079] In another embodiment, the anti-TMEM antibody molecule comprises at least one, two, three, four, five, or six CDRs (or collectively, all of the CDRs) derived from a heavy chain variable region and a light chain variable region comprising an amino acid sequence set forth in Tables 2-5, 8-11 (Tables 1-8, 11-14) or an amino acid sequence encoded by a nucleotide sequence set forth in Tables 6-7, 12, 13 (Tables 9-10, 15, 16). In one embodiment, one or more CDRs (or collectively, all of the CDRs) have one, two, three, four, five, six, or more alterations, e.g., amino acid substitutions or deletions, relative to the amino acid sequence set forth in Tables 2-5, 8-11 (Tables 1-8, 11-14) or the amino acid sequence encoded by a nucleotide sequence set forth in Tables 6-7, 12, 13 (Tables 9-10, 15, 16).

[0080] In one embodiment, the anti-TMEM antibody molecule comprises all six CDRs from an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10 as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, or 13 (Tables 9-10, 15, or 16), or closely related CDRs, e.g., CDRs that are identical or have at least one amino acid change, but no more than two, no more than three, or no more than four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In one embodiment, the anti-TMEM antibody molecule can comprise any CDR described herein. In certain embodiments, the anti-TMEM antibody molecule comprises a substitution in a light chain CDR, e.g., one or more substitutions in CDR1, CDR2, and / or CDR3 of the light chain. In another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs according to Kabat et al. (e.g., Table 1) from the heavy chain variable region of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, and TE10 as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, and 13 (Tables 9-10, 15, and 16). at least one, two, or three CDRs according to the Kabat definition set out in Tables 2-5 (Tables 1-8); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid change, but no more than two, no more than three, or no more than four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to one, two, or three CDRs set out in Tables 2-5, 8-11 (Tables 1-8, 11-14) according to Kabat et al.

[0081] In another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs according to Kabat et al. from the light chain variable region of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10 as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, 13 (Tables 9-10, 15, 16) (e.g., Tables 2-5, 8-11). at least one, two, or three CDRs according to the Kabat definition set out in Tables 1-8, 11-14); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences, or a sequence having at least one amino acid change, but no more than two, no more than three, or no more than four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), to one, two, or three CDRs according to Kabat et al. as set out in Tables 2-5, 8-11 (Tables 1-8, 11-14).

[0082] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., Tables 1-14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1109, 1110, 1120, 1130, 1140, 1140, 1150, 1160, 1170, 1180, 1190, 120 at least one, two, three, four, five, or six CDRs according to the Kabat definition set out in Tables 2-5, 8-11 (Tables 1-8, 11-14); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid change, but no more than two, no more than three, or no more than four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), to one, two, three, four, five, or six CDRs according to Kabat et al. as set out in Tables 2-5, 8-11 (Tables 1-8, 11-14).

[0083] In yet another embodiment, the anti-TMEM antibody molecule comprises all six CDRs according to Kabat et al. (e.g., Tables 1-14, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-43, 1-44, 1-45, 1-46, 1-47, 1-48, 1-49, 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-9 all six CDRs according to the Kabat definition set forth in Tables 2-5, 8-11 (Tables 1-8, 11-14); or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid change, but no more than two, no more than three, or no more than four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), for all six CDRs according to Kabat et al. set forth in Tables 2-5, 8-11 (Tables 1-8, 11-14). In one embodiment, an anti-TMEM antibody molecule can comprise any CDR described herein.

[0084] In another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three Chothia or Kabat hypervariable loops (e.g., at least one, two, or three hypervariable loops according to the Chothia or Kabat definitions set out in Tables 2-5, 8-11 (Tables 1-8, 11-14)) from the heavy chain variable region of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, 13 (Tables 9-10, 15, 16); or at least the amino acids from those hypervariable loops that contact TMEM; or Table 2-5, 8-11 (Tables 1-8, 11-14), the hypervariable loops according to Chothia et al., each having at least one amino acid change, but no more than two, no more than three, or no more than four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0085] In another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three Chothia hypervariable loops (e.g., at least one, two, or three hypervariable loops according to the Chothia definitions set out in Tables 2-5, 8-11 (Tables 1-8, 11-14)) of the light chain variable region of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14), or an antibody encoded by the nucleotide sequence in Tables 6-7, 12, 13 (Tables 9-10, 15, 16); or at least the amino acids from those hypervariable loops that contact TMEM; or Table 2-5, 8-11 (Tables 1-8, 11-14), the hypervariable loops according to Chothia et al., each having at least one amino acid change, but no more than two, no more than three, or no more than four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0086] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, three, four, five, or six hypervariable loops from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14); or an antibody encoded by a nucleotide sequence in Tables 6-7, 12, 13 (Tables 9-10, 15, 16) (e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5, 8-11 (Tables 1-8, 11-14)); at least one, two, three, four, five, or six hypervariable loops according to the Chothia definition set out in Tables 2-5, 8-11 (Tables 1-8, 11-14); or at least amino acids from those hypervariable loops that contact TMEM; or amino acids having at least one amino acid change, but no more than two, no more than three, or no more than four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to one, two, three, four, five, or six hypervariable loops according to Chothia et al. shown in Tables 2-5, 8-11 (Tables 1-8, 11-14).

[0087] In one embodiment, the anti-TMEM antibody molecule comprises all six hypervariable loops (e.g., all six hypervariable loops according to the Chothia definition set out in Tables 2-5, 8-11) or closely related hypervariable loops) of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5, 8-11, or encoded by the nucleotide sequences in Tables 6, 7, 12, 13. For example, hypervariable loops that are identical or have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) for all six hypervariable loops according to Chothia et al. shown in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14). In one embodiment, the anti-TMEM antibody molecule can comprise any hypervariable loop described herein.

[0088] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three hypervariable loops having the same canonical structure as the corresponding hypervariable loops in an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14), or encoded by the nucleotide sequence in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16), e.g., the same canonical structure as at least loop 1 and / or loop 2 of the heavy and / or light chain variable domain of an antibody described herein. For descriptions of canonical structures of hypervariable loops, see, e.g., Chothia et al. (1992) J. Mol. Biol. 227:799-817; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798. These structures can be identified by inspection of the tables provided in these references.

[0089] In certain embodiments, the anti-TMEM antibody molecule comprises a combination of CDRs or hypervariable loops defined according to Kabat et al. and Chothia et al.

[0090] In one embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs or hypervariable loops from a heavy chain variable region of an antibody described herein, e.g., an antibody selected from any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14), or encoded by the nucleotide sequence in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16), according to the definition of Kabat and Chothia (e.g., at least one, two, or three CDRs or hypervariable loops set out in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14)); or Table or sequences substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or sequences having at least one amino acid alteration, but no more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to one, two, or three CDRs or hypervariable loops according to Kabat and / or Chothia as shown in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14).

[0091] For example, an anti-TMEM antibody molecule can comprise a VH CDR1 according to Kabat et al., or a VH hypervariable loop 1 according to Chothia et al., or a combination thereof, e.g., as shown in Tables 2-5, 8-11 (Tables 1-8, 11-14). An anti-TMEM antibody molecule can further comprise a VH CDR2-3 according to Kabat et al. and a VL CDR1-3 according to Kabat et al., as shown in Tables 2-5, 8-11 (Tables 1-8, 11-14). Thus, in some embodiments, framework regions are defined based on a combination of CDRs defined according to Kabat et al. and hypervariable loops defined according to Chothia et al. For example, an anti-TMEM antibody molecule can comprise a VH FR1 defined based on VH hypervariable loop 1 according to Chothia et al., and a VH FR2 defined based on VH CDR1-2 according to Kabat et al., e.g., as shown in Tables 2-5, 8-11, 6, 7, 12, and 13 (Tables 1-8, 11-14, 9, 10, 15, and 16). An anti-TMEM antibody molecule can further comprise VH FR3-4 defined based on VH CDR2-3 according to Kabat et al., and VL FR1-4 defined based on VL CDR1-3 according to Kabat et al.

[0092] An anti-TMEM antibody molecule can contain any combination of CDRs or hypervariable loops according to the Kabat and Chothia definition. In one embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs according to the Kabat and Chothia definition (e.g., at least one, two, or three CDRs according to the Kabat and Chothia definition set out in Table 3.3 or 3.4 (Table 5 or 6)) from the light chain variable region of an antibody described herein, e.g., an antibody selected from TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10, as defined in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14), or encoded by the nucleotide sequence in Tables 6, 7, 12, or 13 (Tables 9, 10, 15, or 16). Preferred anti-TMEM antibodies are TC01 and TM1 as defined in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14), or encoded by the nucleotide sequences in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16). In certain embodiments, e.g., embodiments comprising a variable region, CDR (e.g., a Chothia CDR or Kabat CDR), or other sequence referred to herein, e.g., in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14), the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, or an antibody molecule comprising an antigen-binding fragment of an antibody, e.g., a half antibody or an antigen-binding fragment of a half antibody. In one embodiment, the antibody molecule is a bispecific antibody molecule having a first binding specificity for IGFBP3 and a second binding specificity for TNF-α, an integrin, IL1, IL12 and IL23, CD3, CD20, CD80, or CD86.

[0093] In one embodiment, the anti-TMEM antibody molecule is (i) a heavy chain variable region (VH) comprising: a VHCDR1 amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 8, 10, 56, 59, 62, 65, and 68; a VHCDR2 amino acid sequence selected from any one of SEQ ID NOs: 2, 5, 11, 57, 60, 63, 66, and 69; and a VHCDR3 amino acid sequence selected from any one of SEQ ID NOs: 3, 6, 7, 9, 12, 13, 58, 61, 64, 67, and 70; and (ii) a light chain variable region (VL) comprising a VLCDR1 amino acid sequence selected from any one of SEQ ID NOs: 14, 17, 20, 23, 26, 29, 71, 77, 80, 82, and 85; a VLCDR2 amino acid sequence selected from any one of SEQ ID NOs: 15, 18, 21, 24, 27, 30, 72, 78, 83, and 86; and a VLCDR3 amino acid sequence selected from any one of SEQ ID NOs: 16, 19, 22, 25, 28, 31, 73, 74, 75, 76, 79, 81, 84, and 87. Includes:

[0094] In one embodiment, the light or heavy chain variable framework of the anti-TMEM antibody molecule (e.g., a region comprising at least FR1, FR2, FR3, and optionally FR4) comprises: (a) a light or heavy chain variable framework comprising at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, and preferably 100% of human light or heavy chain variable framework amino acid residues, e.g., light or heavy chain variable framework residues derived from a human mature antibody, a human germline sequence, or a human consensus sequence; (b) a human light or heavy chain variable framework comprising at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, and preferably 100% of human light or heavy chain variable framework residues derived from a human mature antibody, a human germline sequence, or a human consensus sequence; The light or heavy chain variable framework may be selected from (a) a light or heavy chain variable framework that comprises 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of the amino acid residues of a human framework derived from, for example, a human mature antibody, a human germline sequence, or a human consensus sequence; (b) a non-human framework (e.g., a rodent framework); or (c) 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) comprises a light or heavy chain variable framework sequence that is at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical or identical to the framework of the VL or VH segment of a human germline gene.

[0095] In certain embodiments, the anti-TMEM antibody molecule comprises a heavy chain variable domain with at least one, two, three, four, five, six, seven, ten, fifteen, twenty, or more alterations, e.g., amino acid substitutions or deletions.

[0096] In one embodiment, the heavy or light chain variable region, or both, of the anti-TMEM antibody molecule comprises an amino acid sequence encoded by a nucleic acid sequence described herein, or a nucleic acid or complement thereof that hybridizes to a nucleic acid sequence described herein (e.g., a nucleic acid sequence shown in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16) under, e.g., low stringency, medium stringency, or high stringency conditions, or other hybridization conditions described herein.

[0097] In another embodiment, the anti-TMEM antibody molecule comprises at least one, two, three, or four antigen-binding regions, e.g., variable regions, having an amino acid sequence set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or a sequence that differs from a sequence set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14) in no more than 1, no more than 2, no more than 5, no more than 10, or no more than 15 amino acid residues). In another embodiment, the anti-TMEM antibody molecule comprises a VH and / or VL domain encoded by a nucleic acid having a nucleotide sequence set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or a sequence that differs in no more than 3, no more than 6, no more than 15, no more than 30, or no more than 45 nucleotides from a sequence set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14)).

[0098] In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs derived from a heavy chain variable region having an amino acid sequence set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three, or more substitutions, insertions, or deletions, e.g., conservative substitutions). In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, or three CDRs derived from a light chain variable region having an amino acid sequence set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three, or more substitutions, insertions, or deletions, e.g., conservative substitutions). In yet another embodiment, the anti-TMEM antibody molecule comprises at least one, two, three, four, five, or six CDRs from heavy and light chain variable regions having an amino acid sequence set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three, or more substitutions, insertions, or deletions, e.g., conservative substitutions).

[0099] In yet other embodiments, the anti-TMEM antibody molecule has a heavy chain constant region (Fc) selected from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly selected from, e.g., IgG1, IgG2, IgG3, and IgG4, and more particularly, an IgG1 or IgG4 (e.g., human IgG1, IgG2, or IgG4) heavy chain constant region. In one embodiment, the heavy chain constant region is human IgG1. In another embodiment, the anti-TMEM antibody molecule has a light chain constant region selected from, e.g., a kappa or lambda light chain constant region. In one embodiment, the constant region is altered, e.g., mutated, to alter the properties of the anti-TMEM antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, complement function, half-life, aggregation, and stability). In certain embodiments, the anti-TMEM antibody molecule comprises a mutated human IgG4.

[0100] In one embodiment, the anti-TMEM antibody molecule is isolated or recombinant.

[0101] In one embodiment, the anti-TMEM antibody molecule is a humanized or human antibody molecule.

[0102] The invention also features nucleic acid molecules comprising one or both nucleotide sequences encoding the heavy and light chain variable regions, CDRs, hypervariable loops, and framework regions of an anti-TMEM antibody molecule, as described herein. In certain embodiments, the nucleotide sequences encoding the anti-TMEM antibody molecule are codon-optimized. For example, the invention features first and second nucleic acids encoding the heavy and light chain variable regions, respectively, of an anti-TMEM antibody molecule selected from one or more of any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, and TE10, e.g., as defined in Tables 2-5 (Tables 1-8), 8-11 (Tables 11-14), or encoded by a nucleotide sequence in Tables 6, 7, 12, and 13 (Tables 9, 10, 15, and 16), or a sequence substantially identical thereto. For example, the nucleic acid can include a nucleotide sequence set forth in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16), or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or a sequence that differs in no more than 3, no more than 6, no more than 15, no more than 30, or no more than 45 nucleotides from a sequence set forth in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16)).

[0103] In other embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable domain and / or a heavy chain constant region comprising the amino acid sequence of any of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, TE10, as defined in Tables 2-5, 8-11 (Tables 3-8, 11-14), or encoded by the nucleotide sequence in Tables 6-7, 12, 13 (Tables 9-10, 15, 16); or a sequence substantially identical (e.g., at least about 85%, 90%, 95%, 99% or more identical) to any of the foregoing sequences.

[0104] In other embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain variable domain and / or a light chain constant region comprising any of the amino acid sequences of TA02, TC01, TC02, TD01, TE01, TG02, TM1, TE02.1, TE02.2, TE02.3, TE03, TE04, TE07, or TE10 as defined in Tables 2-5, 8-11 (Tables 3-8, 11-14); or a nucleotide sequence in Tables 6-7, 12, or 13 (Tables 9-10, 15, or 16); or a sequence substantially identical (e.g., at least about 85%, 90%, 95%, 99% or more identical) to any of the foregoing sequences.

[0105] The aforementioned nucleotide sequences encoding the anti-TMEM heavy and light chain variable domains and constant regions can be present on separate nucleic acid molecules or on the same nucleic acid molecule, which in certain embodiments includes a nucleotide sequence encoding a leader sequence.

[0106] 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 set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three, or more substitutions, insertions, or deletions, e.g., conservative substitutions).

[0107] 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 set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three, or more substitutions, insertions, or deletions, e.g., conservative substitutions).

[0108] 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 set forth in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three, or more substitutions, insertions, or deletions, e.g., conservative substitutions).

[0109] In another embodiment, the nucleic acid molecule comprises one or more heavy chain framework regions (e.g., VHFW1(type a), VHFW1(type b), VHFW1(type c), VHFW1(type d), VHFW1(type e), VHFW1(type f), VHFW1(type g), VHFW1(type i ... VHFW3(Type a), VHFW3(Type b), VHFW3(Type c), VHFW3(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 combination of frameworks described herein. For example, the nucleic acid molecule can include a nucleotide sequence set forth in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16), or a sequence substantially identical thereto (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto, or a sequence that differs in no more than 3, no more than 6, no more than 15, no more than 30, or no more than 45 nucleotides from a sequence set forth in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16)).

[0110] In another embodiment, the nucleic acid molecule comprises one or more light chain framework regions of any of E01, E02, E08, E14, E19, E20, E23, E24, or M1 as defined in Tables 2-5, 8-11 (Tables 3-8, 11-14), or a sequence substantially identical thereto (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 described herein). For example, the nucleic acid molecule can include a nucleotide sequence set forth in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16), or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or a sequence that differs in no more than 3, no more than 6, no more than 15, no more than 30, or no more than 45 nucleotides from a sequence set forth in Tables 6, 7, 12, 13 (Tables 9, 10, 15, 16)).

[0111] In another embodiment, the nucleic acid molecule comprises one or more heavy chain framework regions and one or more light chain framework regions described herein. The heavy and light chain framework regions can be present on the same vector or on separate vectors.

[0112] In another aspect, this application features host cells and vectors containing nucleic acids described herein or nucleic acids modified for codon optimization according to known methods. The nucleic acids can be present in a single vector or in separate vectors present in the same host cell or in separate host cells. The host cell can be a eukaryotic cell, such as a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, such as E. coli. For example, the mammalian cell can be a cultured cell or cell line. Exemplary mammalian cells include lymphoid cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocytes, and cells derived from transgenic animals, such as mammary epithelial cells.

[0113] In one aspect, the invention features a method of providing an antibody molecule described herein. The method includes providing a TMEM antigen (e.g., an antigen comprising at least a portion of a TMEM epitope); obtaining an antibody molecule that specifically binds to the TMEM polypeptide; and assessing whether the antibody molecule specifically binds to the TMEM polypeptide or the effectiveness of the antibody molecule in modulating, e.g., inhibiting, the activity of a TMEM. The method can further include administering the antibody molecule to a subject, e.g., a human or non-human animal.

[0114] In another aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising a pharmaceutically acceptable carrier, excipient, or stabilizer and at least one of the anti-TMEM antibody molecules described herein. In one embodiment, the composition, e.g., pharmaceutical composition, comprises a combination of the antibody molecule and one or more agents, e.g., a therapeutic agent described herein or another antibody molecule. In one embodiment, the antibody molecule is conjugated to a label or a therapeutic agent.

[0115] The anti-TMEM antibody molecules disclosed herein can inhibit, reduce, or neutralize one or more of the activities of IGFBP3 listed above, and thus can be used to treat or prevent disorders in which inhibition, reduction, or neutralization of IGFBP3-induced activity in a subject is desired.

[0116] Use of anti-TMEM antibody molecules The antibodies of the invention find use in methods of treating a variety of disorders or conditions, such as diabetes, as well as enteropathy, malabsorption syndromes, inflammatory bowel disease, cachexia, Crohn's disease, ulcerative colitis, celiac disease, diabetic enteropathy, and the like.

[0117] Thus, in another aspect, a method of modulating the IGFBP3 / TMEM219 axis in a subject is provided. The method comprises administering to the subject an anti-TMEM antibody molecule disclosed herein (e.g., a therapeutically effective amount of the anti-TMEM antibody molecule), alone or in combination with one or more agents or procedures, such that the IGFBP3 / TMEM219 axis is modulated in the subject. In one embodiment, the antibody molecule inhibits, reduces, neutralizes, or blocks IGFBP3 / TMEM219 axis activity in the subject. The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient with or at risk of having a disorder described herein). In one embodiment, the subject is in need of inhibiting, reducing, neutralizing, or blocking the IGFBP3 / TMEM219 axis. In one embodiment, the subject has or is at risk of having a disorder described herein, e.g., diabetes, or inflammatory bowel disease (IBD), malabsorption syndrome, irritable bowel disease, cachexia, celiac disease, diabetic enteropathy described herein. [Brief explanation of the drawings]

[0118] [Figure 1]Effect of newly generated anti-TMEM mAbs on DSS-induced colitis in mice. Experimental timeline for mouse studies of DSS-induced colitis: C57BL / 6 mice received 2.5% DSS in drinking water for 5 days and daily intraperitoneal administration of 0.5 mg / mouse of anti-TMEMm Abs starting 3 days before colitis induction and continuing until euthanasia 7 days after the last DSS administration. [Figure 2] (A) Disease Activity Index (DAI), a scoring system used to determine the severity of colitis in mice, and (B) Histological score, a diagnostic system used to evaluate the severity and extent of inflammation, including the intensity of cellular infiltrates in the mucosa, their extension in the submucosa, the presence of epithelial lesions, and mucosal regeneration, were assessed 7 days after the last administration of DSS during wound healing / beginning of the chronic phase. Values are mean ± SEM. * denotes p ≤ 0.05, and ** denotes p ≤ 0.01 relative to the DSS group. [Figure 3] Paraffin sections of the colon obtained at euthanasia, i.e., 7 days after the last DSS administration, were stained with May-Grunwald-Giemsa for histological examination (A). Apoptotic cells were detected by TUNEL assay (B) and counterstained with DAPI. Cell proliferation was detected by PCNA assay (C). [Figure 4] Effect of newly generated anti-TMEM219 mAbs on diabetes development in a T1D mouse model. Experimental timeline of the NOD mouse study. [Figure 5](A) Effect of anti-TMEM219 mAb on preventing the onset of diabetes in 22-week-old NOD mice, and (B) effect of anti-IGFBP3 mAbs on maintaining blood glucose levels. Diabetes prevention achieved by the use of anti-TMEM219 mAb was observed in 100% of mice. * indicates p<0.05 by Mantel-Cox analysis vs. untreated. Diabetes-free mice were defined as mice with normoglycemia. Diabetes was defined as a blood glucose level >250 mg / dl on three consecutive measurements. Diabetes-free mice did not have a blood glucose level >250 mg / dl on three consecutive measurements. [Figure 6] Serial paraffin sections of pancreatic tissue obtained at euthanasia were prepared and stained with H&E. The area and morphology of islets were analyzed by light microscopy. (A) Representative images are shown; original magnification: 20x. (B) Insulitis scores are shown. In (B), the degree of cellular infiltration was scored from 0 to 4. Insulitis was scored by examining a minimum of 30 islets per animal. * indicates p < 0.05 by Mann-Whitney test. [Figure 7] Serial paraffin sections of pancreatic tissue obtained at euthanasia were prepared and show immunohistochemical staining for insulin (brown). Representative images are shown; original magnification: 20x. [Figure 8] Effect of anti-TMEM219 mAb on downregulating CASP8 expression in β-cell lines exposed to IGFBP3. ** denotes p<0.01 vs. IGFBP3, *** denotes p<0.001 vs. IGFBP3 (n=3 experiments). [Figure 9] Effect of anti-TMEM219 mAb on downregulating CASP8 expression in β-cell lines exposed to IGFBP3-enriched T1D serum. * indicates p<0.05 vs. T1D, ** indicates p<0.01 vs. T1D (n=3 experiments). [Figure 10]Effect of anti-TMEM219 mAb on downregulating CASP8 expression in human islets exposed to IGFBP3. N=3 experiments. *** indicates P<0.001 vs. IGFBP3. [Figure 11] Effect of anti-TMEM219 mAb on reducing apoptosis in human islets exposed to IGFBP3. N=3 experiments. *** indicates p<0.001 vs. IGFBP3, * indicates p<0.05. [Figure 12] Effect of anti-TMEM219 mAb in reducing apoptosis in human islets exposed to T1D serum. N=3 experiments. ** indicates P<0.01 vs. T1D serum, * indicates p<0.05 vs. T1D serum. [Figure 13] Effect of anti-TMEM219 mAb on downregulating CASP8 expression in beta cell lines exposed to T1D serum. N=3 experiments. ** indicates P<0.001 vs. T1D serum. [Figure 14] Effect of anti-TMEM219 mAb on apoptosis of β-cell lines. ** indicates p<0.01 for IGFBP3 vs. untreated. Experiments were performed in triplicate on β-lox5 cells cultured for 72 hours. [Figure 15] Effect of anti-TMEM219 mAb on CASP8 mRNA expression in β-cell lines. **** indicates p<0.0001 for IGFBP3 vs. untreated TM1, TC01; ** indicates p<0.01 for IGFBP3 vs. commercial anti-TMEM219. Experiments were performed in triplicate in β-lox5 cells cultured for 72 hours. [Figure 16] Effect of newly generated anti-TMEM219 mAb on diabetes development in a diabetic mouse model. Experimental timeline for the low-dose streptozotocin model of diabetes. [Figure 17] Line graph showing blood glucose levels measured in B6 mice injected with multiple low doses of streptozotocin (ldSTZ, 50 mg / Kg), treated with anti-TMEM219 mAb, or left untreated (n=5). [Figure 18]Blood glucose measured at 60 min during IPGTT (1 g / Kg) in B6 mice injected with ld-STZ with or without anti-TMEM219 mAb on day 10 (n=5). * indicates p<0.05, ** indicates p<0.01. [Figure 19] Representative H&E staining of contiguous islet tissue sections (n=3) obtained from B6 mice injected with ldSTZ and treated with or without anti-TMEM219 mAb. Original magnification 20x, scale bar 100 μm. DETAILED DESCRIPTION OF THE INVENTION

[0119] The antibodies of the present invention specifically bind to human TMEM219. As described herein, the antibodies of the present invention are collectively referred to as "anti-TMEM or anti-TMEM219 antibodies." All such antibodies are encompassed by the description herein. Each antibody can be used alone or in combination in the methods of the present invention.

[0120] By "an antibody that specifically binds to" TMEM219 is intended that the antibody does not substantially cross-react with another non-homologous human peptide. By "does not substantially cross-react," it is intended that the antibody or fragment has binding affinity for a non-homologous protein that is less than 10%, more preferably less than 5%, and even more preferably less than 1% of the binding affinity for TMEM219.

[0121] In various embodiments, as used herein, an antibody that "specifically binds to" TMEM219 is an antibody that specifically binds to TMEM219, as determined by an Octet Biolayer Interferometry device or, for example, by a BIAcore™ system (Biacore Life Sciences division of GE Healthcare). or less than about 0.5 nM, as measured in a surface plasmon resonance assay using a ELISA kit (Diagnostics Laboratory, Piscataway, NJ) or in an equilibrium binding exclusion assay, or by any method known in the art.

[0122] The term "antibody" is used herein in the broadest sense understood in the art to include all polypeptides described as antibodies in (25), which is incorporated herein by reference.

[0123] For example, the term "antibody" as used herein includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (antigen-binding fragments), so long as the fragment exhibits the desired antigen-binding activity. The term has its broadest meaning recognized in the art and encompasses all known formats, including, but not limited to, bivalent monospecific monoclonal antibodies, bivalent bispecific antibodies, trivalent trispecific antibodies, F(ab) fragments, F(ab)' fragments, scFv fragments, diabodies, single domain antibodies such as camelized VHH signal domain antibodies, TandAbs, and flexibodies.

[0124] As used herein, terms such as "antigen-binding fragment" of an antibody or equivalently "antigen-binding portion" of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that comprises a portion of an antibody and specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from intact antibody molecules using, for example, any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding antibody variable and, where appropriate, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, by using chemical or molecular biology techniques to align one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, or modify, add, or delete amino acids.

[0125] When using intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, where each variable domain can specifically bind to a separate antigen or to a different epitope on the same antigen.

[0126] In certain 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 in 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 directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may, in various embodiments, consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies may, in various embodiments, comprise homodimers or heterodimers (or other multimers) of any of the configurations of variable and constant domains listed above in non-covalent association with each other (e.g., via disulfide bonds) and / or with one or more monomeric VH or VL domains.

[0127] The term "antigen-binding fragment" of an antibody further includes single domain antibodies.

[0128] Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. In some embodiments, single domain antibodies are derived from the variable domain of an antibody heavy chain from a camelid (also referred to as a nanobody or VHH fragment). In some embodiments, single domain antibodies are spontaneous human heavy chain variable domains (aVH) or VNAR fragments derived from sharks.

[0129] 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 amino acid residues that mimic a 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 by the term "antigen-binding fragment" as used herein.

[0130] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally will contain at least one CDR 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 positioned in any suitable orientation relative to each other. 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.

[0131] The antibodies or binding molecules of the present invention can further be linked to an active agent, preferably a nanoparticle or a radionucleotide.

[0132] As used herein, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, including antigen-binding antibody fragments, and scaffold antigen-binding proteins.

[0133] The term "antigen-binding moiety" refers to a 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 scFvs, that can specifically bind to an antigen on a target cell. In certain embodiments, an antigen-binding moiety can direct an entity to which it is attached, such as a cell, to a target site. In addition, antigen-binding moieties that can specifically bind to a target cell antigen include binding domains based on scaffold antigen-binding proteins, as defined herein below, such as designed repeat proteins or designed repeat domains, such as designed ankyrin repeat proteins (DARPins) (see, e.g., WO2002 / 020565) or lipocalins (anticalins).

[0134] Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. They can be engineered to bind to different target antigens by randomizing residues in the first α-helix and β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003), and J. Mol. Biol. 369, 1015-1028 (2007), as well as US20040132028.

[0135] In certain embodiments, the antibodies and antigen-binding molecules provided herein are modified to increase or decrease the degree of glycosylation of the antigen-binding portion. Glycosylation variants of the molecule can be conveniently obtained by altering the amino acid sequence to create or remove one or more glycosylation sites. When the antigen-binding molecule contains an Fc region, the carbohydrate attached thereto can be altered. In one aspect, variants of antigen-binding molecules are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. Such fucosylation variants can have improved ADCC function; see, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) or US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Additional variants of the antigen-binding molecules of the present invention include those with bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, e.g., WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved ADCC function, as described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0136] In certain embodiments, it may be desirable to create cysteine-engineered variants of the antibodies or antigen-binding molecules of the present invention, e.g., "thioMAbs," in which one or more residues of the molecule are substituted with cysteine residues. In certain embodiments, the substituted residues occur at accessible sites in the molecule. By replacing these residues with cysteine, reactive thiol groups are consequently positioned at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antigen-binding molecules can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0137] In certain embodiments, the antibodies or antigen-binding molecules provided herein can be further modified to contain additional nonproteinaceous moieties known and readily available in the art. Suitable moieties for derivatization of the antibodies or antigen-binding molecules include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when two or more polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used in therapy under defined conditions.

[0138] In another aspect, a conjugate of an antibody and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, including, but not limited to, a wavelength that does not harm normal cells but heats the nonproteinaceous moiety to a temperature that kills cells in close proximity to the antibody-nonproteinaceous moiety. In another aspect, an immunoconjugate of an antigen-binding molecule provided herein can be obtained. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, such as, but not limited to, a cytotoxic drug.

[0139] The constant region of an antibody is important for the antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Therefore, the antibody isotype can be selected based on whether it is desirable for the antibody to mediate cytotoxicity. In certain embodiments, the constant region is an IgG1, IgG2, IgG3, or IgG4 constant region.

[0140] In various embodiments, the present invention encompasses antibodies with one or more mutations in the hinge region, CH2 region, or CH3 region that may be desirable in production, for example, to improve the yield of the desired antibody form. In some embodiments, for example, the antibodies described herein comprise a human IgG4 constant region. In certain embodiments, the IgG4 constant region has a single amino acid substitution in the hinge region of a human IgG4 hinge that reduced Fab arm exchange (Angal et al. (1993) Molecular Immunology 30:105) to the level typically observed using a human IgG1 hinge.

[0141] In certain embodiments, the antibody comprises one or more mutations in the constant region that increase serum half-life, such as those described in U.S. Pat. Nos. 7,083,784, 8,323,962, and Dall'Aqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Hinton et al., J. Immunology 176:346-356 (2006); Yeung et al., J. Immunology 182:7663-7671 (2009); and Petkova et al., Intn'l Immunology, 18: 1759-1769 (2006), which are incorporated herein by reference in their entireties.

[0142] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, human antibodies featured in the present invention may, in various embodiments, 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), e.g., in the CDRs, and in some embodiments, in CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies derived from the germline of another mammalian species, such as a mouse, in which CDR sequences have been grafted onto human framework sequences.

[0143] The term "recombinant human antibody," as used herein, is intended to include all human antibodies 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 involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, where animals transgenic for human Ig sequences are used, in vivo somatic mutation) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0144] "Isolated antibody," as used herein, refers to 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 the tissue or cell in which it naturally occurs or is produced, is an "isolated antibody." In various embodiments, an isolated antibody also includes an antibody in situ within a recombinant cell. In other embodiments, an isolated antibody is an antibody that has 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.

[0145] 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 the paratope. A single antigen can have two or more epitopes. Thus, different antibodies may bind to different areas on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are created by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are created by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes can include carbohydrate, phosphoryl, or sulfonyl moieties on the antigen.

[0146] The anti-TMEM219 antibodies described herein and useful for the methods featured herein may, in various embodiments, contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains when compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences, for example, from public antibody sequence databases.

[0147] The present invention, in various embodiments, includes antibodies and methods involving the use of antibodies, and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations").

[0148] Numerous antibodies and antigen-binding fragments can be constructed that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues in the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a different germline sequence from the germline sequence from which the antibody was originally derived). Moreover, the antibody can have any combination of two or more germline mutations in the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments having one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Use of antibodies and antigen-binding fragments obtained in this general manner is encompassed within the present invention.

[0149] The present invention also includes methods involving the use of anti-TMEM219 antibodies and anti-TMEM219 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes the use of anti-IL-6R antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0150] The term "bioequivalent" as used herein refers to a molecule that has similar bioavailability (rate and extent of availability) after administration of the same molar dose under similar conditions (e.g., the same route of administration), thereby predicting that the effects, both in terms of efficacy and safety, are substantially the same as those of the compared molecule. Two pharmaceutical compositions containing an anti-IGFBP3 antibody are bioequivalent if they are pharmaceutically equivalent, meaning that they contain the same amount of active ingredient (e.g., an IGFBP3 antibody) in the same dosage form via the same route of administration and meet the same or comparable standards. Bioequivalence can be determined, for example, by in vivo studies comparing the pharmacokinetic parameters of the two compositions. Parameters commonly used in bioequivalence testing include peak plasma concentration (Cmax) and area under the plasma drug concentration-time curve (AUC).

[0151] In certain embodiments, the present invention relates to antibodies and methods comprising administering to a subject an antibody comprising a heavy chain variable region comprising a sequence selected from the group of SEQ ID NO: 32 to SEQ ID NO: 37 or SEQ ID NO: 88 to SEQ ID NO: 95 and a light chain variable region comprising a sequence selected from the group of SEQ ID NO: 38 to SEQ ID NO: 43 or SEQ ID NO: 96 to SEQ ID NO: 103. The disclosure provides pharmaceutical compositions comprising such antibodies and methods of using these compositions.

[0152] In various embodiments, the antibody is administered to a subject in a formulation suitable for intravenous or subcutaneous injection, including suitable carriers, excipients, and other agents to provide improved mobility, delivery, tolerability, etc.

[0153] Injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium customarily used for injections. Aqueous media for injections include, for example, physiological saline, isotonic solutions containing glucose and other auxiliary agents, and the like, which can be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 20 or 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), and the like. Oily media include, for example, sesame oil, soybean oil, and the like, which can be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, and the like. The injectable preparations prepared in this manner can be filled into appropriate ampoules.

[0154] The antibodies according to the invention can be administered to a subject using any acceptable device or mechanism. For example, administration can be achieved using a syringe and needle, or by a reusable pen and / or autoinjector delivery device. The methods of the invention include the use of multiple reusable pens and / or autoinjector delivery devices to administer the antibodies (or pharmaceutical formulations comprising the antibodies). Examples of such devices include, but are not limited to, the AUTOPEN™ (Owen Mumford, Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX75 / 25™ pen, the HUMALOG™ pen, the HUMALIN70 / 30™ pen (Eli Lilly and Company, Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN IV ... STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany).Examples of disposable pens and / or autoinjector delivery devices that have use in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly & Co.), the SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), the HUMIRA™ pen (Abbott Labs, Abbott Park, IL), the DAI® autoinjector (SHL Group), and any autoinjector featuring PUSHCLICK™ technology (SHL Group).

[0155] In one embodiment, the antibody is administered by a pre-filled syringe. In another embodiment, the antibody is administered by a pre-filled syringe that includes a safety system. For example, the safety system prevents accidental needlestick injuries. In various embodiments, the antibody is administered by a pre-filled syringe that includes the ERIS™ safety system (West Pharmaceutical Services). See also U.S. Patent Nos. 5,215,534 and 9,248,242, which are incorporated herein by reference in their entirety.

[0156] In another embodiment, the antibody is administered by an autoinjector. In various embodiments, the antibody is administered by an autoinjector featuring PUSHCLICK™ technology (SHL Group, Inc.). In various embodiments, the autoinjector is a device that includes a syringe that allows for the administration of a dose of the composition and / or antibody to a subject. See also U.S. Pat. Nos. 9,427,531 and 9,566,395, which are incorporated by reference in their entireties.

[0157] According to the present invention, "subject" means a human subject or a human patient. [Example]

[0158] method Recombinant proteins Recombinant human IGFBP3 was obtained from Life Technologies (IGFBP3, Life Technologies, 10430H07H5). The extracellular domain of the TMEM219 receptor, Ecto-TMEM219, was obtained through Genescript's customized protein service. The protein produced in E. coli has the following amino acid sequence:

[0159] Human Ecto-TMEM amino acid sequence THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSR (SEQ ID NO: 125)

[0160] Mouse Ecto-TMEM amino acid sequence THTTGLRSPDIPQDWVSFLRSFGQLSLCPMNETVTGTWQGPHVVGLLTTLNFGDGPDRNKTQTFQAKIHGSQIGLTGSSAGESVLVTARVASGRTPGTCLYFSGVPKVLPSSQPPISCSEEGVGNATLSPVMGEECVRVWSHERLVLTELLTSEELALCGS (SEQ ID NO: 126)

[0161] Development of monoclonal antibodies from naive human phage display libraries Monoclonal anti-TMEM antibodies were selected from a naive human phage display library using human EctoTMEM219 (obtained from Genescript's customized protein service) as the antigen for screening. The EctoTMEM antigen was immobilized on a 96-well ELISA plate by either direct adsorption or capture with an anti-ectoTMEM polyclonal antibody. After washing and blocking the wells with BSA, the antibody-phage library was added. The library was previously cleared of sticky or cross-reactive antibody-phage.

[0162] Phages that displayed antigen-specific antibodies were captured on the plate surface. After washing with PBS-T to remove unbound / weakly binding phages, antigen-specific phages were eluted and amplified. This amplified library subset was reselected for target binding under more stringent conditions, i.e., the number of washing steps was increased to eliminate unbound or weakly binding phages. A total of three selection rounds were performed to enrich antigen-specific antibody-phages.

[0163] At the end of the selection process, the selection output was screened for antigen-specific antibodies by ELISA. For this purpose, monoclonal scFv antibodies were produced from clones of the selection output. These were then tested for specific antigen binding by ELISA. Fifteen target-specific hits were identified. Eleven of them contained unique CDR sequences. These were cloned into mammalian scFv-Fc expression vectors, resulting in genetic fusions of scFv with human IgG4 Fc.

[0164] Six of these antibodies could be produced in scFv-Fc format by transient transfection of HEK293 cells. The antibodies were purified by affinity chromatography (Protein A) and rebuffering in PBS. Protein concentration was determined by UV / VIS spectroscopy, and purity was confirmed by Coomassie staining.

[0165] Hybridoma-based monoclonal antibody development Monoclonal anti-TMEM antibodies were identified using the Trianni Mouse™ (Trianni, Inc.), a transgenic mouse in which relevant human immunoglobulin sequences have been genetically engineered into the animal's genome. Use of such technology has produced chimeric monoclonal antibodies containing a full repertoire of human heavy and light chain variable domains and retaining mouse constant domains.

[0166] Essentially, two groups of Trianni Mice™ (Group 1: ALD / MDP adjuvant and Group 2: SAS / Ribi adjuvant) were immunized with human EctoTMEM219 (Genescript's customized protein service) twice weekly for four weeks, followed by a two-week extension with weekly injections. Lymphocytes (e.g., B cells) were then collected from the mice expressing antibodies, and these cells were fused with myeloid-type cell lines to prepare immortal hybridoma cell lines. These hybridoma cell lines were screened and selected by ELISA to identify hybridoma cell lines producing antibodies specific to human Ecto-TMEM219. Hybridoma cell lines reactive to the antigen of interest were expanded. Sequencing was performed by RNA isolation, followed by cDNA sequencing of the human VH and human VK using Sanger sequencing.

[0167] Antibodies can be expressed in cell lines other than hybridoma cell lines. The antibody coding sequence can be used for transformation of a suitable mammalian host cell.

[0168] Methods for expressing recombinant proteins in CHO cells To produce fully human IgG4 mAbs, the corresponding TCO1 and TM1 cDNAs were cloned into the vitoria vector system using conventional (non-PCR-based) cloning techniques. The vitoria vector plasmids were synthetic genes. Plasmid DNA was prepared under low-endotoxin conditions using anion-exchange chromatography. Sequence accuracy was verified by Sanger sequencing (up to two sequencing reactions per plasmid depending on the size of the cDNA).

[0169] Suspension-adapted CHO K1 cells (evitria) were used for production. Seeds were grown in eviGrow medium, a chemically defined, animal-component-free, serum-free medium. Cells were transfected using eviFect, a custom-made, proprietary transfection reagent from evitria. After transfection, cells were grown in eviMake, an animal-component-free, serum-free medium, at 37°C and 5% CO for 7 days. The supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter).

[0170] The antibody was purified using MabSelect™ SuRe™ with Dulbecco's PBS (Lonza BE17-512Q) as the wash buffer and 0.1 M glycine, pH 3.5 as the elution buffer, followed by size exclusion chromatography on a HiLoad Superdex 200 pg column with the final buffer as the running buffer.

[0171] Monomeric nature was determined by analytical size exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7 μm 7.8×300 mm) with DPBS as the running buffer at 0.8 ml / min.

[0172] The sequences of six novel anti-TMEM antibodies from the human phage display library are reported in Tables 2-7 (Tables 1-11) below.

[0173] [Table 1]

[0174] [Table 2]

[0175] CDR definitions are also provided using the annotation tool at http: / / www.abysis.org / based on the complete VH and VL amino acid sequences defined in Tables 4, 5, 10, and 11 (Tables 7, 8, 13, 14). For example, the VH amino acid sequence of any antibody disclosed herein can be plugged into the annotation tool to provide the Kabat-defined CDR sequences.

[0176] An example is shown below with reference to SEQ ID NO: 33 (VH of TC01).

[0177] [Table 3]

[0178] The VH amino acid sequence of any antibody disclosed herein can also be plugged into the annotation tool to provide the IMGT-defined CDR sequences.

[0179] An example is shown below with reference to SEQ ID NO: 33 (VH of TC01).

[0180] [Table 4]

[0181] Additionally, the VH amino acid sequence of any antibody disclosed herein can also be plugged into the annotation tool to provide "All, side by side" defined CDR sequences.

[0182] An example is shown below with reference to SEQ ID NO: 33 (VH of TC01).

[0183] [Table 5A]

[0184] [Table 5B]

[0185] The CDR definitions are also reported, based on the complete VL amino acid sequence of TC01 (SEQ ID NO: 39), and provided using the annotation tool from http: / / www.abysis.org / .

[0186] [Table 6A]

[0187] [Table 6B]

[0188] [Table 7]

[0189] [Table 8]

[0190] [Table 9A]

[0191] [Table 9B]

[0192] [Table 10]

[0193] The sequences of eight novel hybridoma-based anti-TMEM antibodies are reported in Tables 8-13 (Tables 11-16) below.

[0194] [Table 11]

[0195] [Table 12]

[0196] [Table 13]

[0197] [Table 14]

[0198] [Table 15A]

[0199] [Table 15B]

[0200] [Table 16A]

[0201] [Table 16B]

[0202] [Table 16C]

[0203] Further antibodies IgG production The amino acid sequence was reverse-translated into DNA and codon-optimized for HEK expression. The optimized DNA sequence was chemically synthesized and cloned into a human IgG4 (S228P L235E mutant) expression vector. Transfection-grade DNA was prepared and used for transient transfection of HEK cells. The produced antibody was purified from HEK culture supernatant by affinity chromatography (Protein A). Protein concentration was determined by UV / VIS spectroscopy, and purity was confirmed by reducing SDS-PAGE analysis.

[0204] [Table 17]

[0205] [Table 18]

[0206] [Table 19]

[0207] [Table 20]

[0208] [Table 21A]

[0209] [Table 21B]

[0210] [Table 21C]

[0211] [Table 22A]

[0212] [Table 22B]

[0213] [Table 23]

[0214] All studies were performed using fully human IgG4 antibodies.

[0215] Example 1 Affinity measurement Octet BLI-based analysis The antibodies have high affinity for the target TMEM. Binding affinity measurements were performed using an Octet instrument (Octet BMIA), a biolayer interferometry (BLI) platform based on biomolecular interaction analysis. To establish the assay, target monoclonal antibodies (30 μg / ml in PBS) were immobilized via Fc on an anti-mouse IgG Fc capture (AMC) biosensor or an anti-human IgG Fc capture (AMC) biosensor, and their interactions with the antigens human and mouse Ecto-TMEM219 (Genescript's customized protein service) were measured at 150 nM. The affinity measurements of anti-TMEM mAbs for the target human and mouse Ecto-TMEM219 are reported in Table 21.

[0216] [Table 24]

[0217] The newly generated anti-TMEM mAb was 2 × 10 -8 The antibody exhibits good human antigen-binding affinity with a KD of less than M. The antibody also exhibits mouse cross-reactivity. This data confirms that mice can be considered a relevant animal species for testing monoclonal antibodies during preclinical development.

[0218] Measurement of antibody binding activity by ELISA The binding activity of purified IgG4 (TC03, TC04, TC05) to recombinant ectoTMEM protein was measured by ELISA. Briefly, ectoTMEM was diluted to 5 μg / ml in PBS and coated onto a 96-well ELISA plate (100 μl / well) for 1 hour at room temperature. After blocking and washing the plate, serial antibody dilutions were added to the plate and incubated for 1 hour at room temperature. After washing the plate, bound antibody was detected with a secondary anti-human-Fc HRP-labeled antibody. After another washing step, a TMB reaction was performed, the reaction was stopped with sulfuric acid, and the absorbance was measured. The EC50 of the saturating antibody was calculated based on the absorbance reading. The EC50 of anti-TMEM mAbs against the target human Ecto-TMEM219 is reported in Table 22.

[0219] [Table 25]

[0220] Example 2 Efficacy of anti-TMEM mAb in an IBD mouse model after intraperitoneal (IP) administration The dextran sulfate sodium (DSS)-induced colitis model in C57BL / 6J mice is a valid animal model for evaluating and confirming the anti-inflammatory and wound-healing properties of drugs in IBD. DSS (administered orally in drinking water) induces severe diarrhea and subsequent inflammation. This model is well-characterized, reliable, reproducible, and approved by regulatory agencies [see, e.g., Eichele and Kharbanda, "Dextran sodium sulfate colitis murine model: an indispensable tool for advancing our understanding of inflammatory bowel disease pathogenesis," World J. Gastroenterol. 23(33):6016-6028 (2017)]. This study was conducted in C57BL / 6J mice. In this particular genetic background, mice develop acute colitis when analyzed 3 days after the last DSS administration or chronic-like inflammation when analyzed 7 days after the last DSS administration.

[0221] Anti-inflammatory and wound healing effects were assessed at the clinical and histological levels using a validated clinical score, the Disease Activity Index (DAI) (Table 23), and a validated score for histological analysis (Table 24).

[0222] animal Male C57BL / 6J mice were obtained from Charles River Laboratories, L'Arbresel, France. The mice were housed at 20±5°C and provided with water and food ad libitum. All experimental protocols were carried out in facilities accredited by the Institut Pasteur in Lille in accordance with government guidelines.

[0223] Establishment and treatment of a DSS-induced murine colitis model Acute colitis was induced in mice by feeding them 2.5% (w / v) DSS (45 kDa; TDB Consultancy AB, Uppsala, Sweden, batch number DB001-41) dissolved in drinking water for 5 days. The mice were randomly divided into five groups: control; DSS + vehicle; DSS + Humira (adalimumab) 0.3 mg / mouse (Abbvie, 1108722), DSS + TM1 0.5 mg / mouse, and DSS + TC01 0.5 mg / mouse. To evaluate the effect of anti-TMEM mAb on DSS-induced acute colitis in C57BL / 6J mice, the mice were treated with the indicated doses of anti-TMEM mAb by daily intraperitoneal administration starting 3 days before colitis induction and continued until euthanasia 7 days after the last DSS administration. The experimental timeline for the animal model is shown in Figure 1.

[0224] The therapeutic properties of TMEM mAb were compared with those of Humira (adalimumab), a positive control approved for the treatment of both Crohn's disease and ulcerative colitis (Taghipour N et al. Gastroenterol Hepatol Bed Bench 2016;9(1):45-52).

[0225] The DSS mouse model cannot gauge relative efficacy in humans because it requires that each antibody—TMEM mAb and the positive control Humira—cross-react with the mouse orthologue of its cognate human antigen (TMEM219 and TNFα, respectively).

[0226] Clinical Scoring In all groups, mouse weight, stool consistency, and bloody stool were recorded daily. The Disease Activity Index (DAI) score was based on weight change, stool consistency, and Hemoccult bleeding according to a standard scoring system. These parameters were assessed on the scale described in Table 23. DAI data are presented as the mean score of these parameters obtained each day. Animals were sacrificed by cervical dislocation under anesthesia. Upon euthanasia, the colons were carefully dissected and their weight and size were measured. The presence of occult blood (OB) was recorded using the Hemoccult method.

[0227] [Table 26]

[0228] As shown in Figure 2A, 7 days after the last DSS administration, the DAI score significantly increased in the group of DSS mice receiving vehicle compared to the healthy control group (group receiving vehicle only) (p-value = 0.0012), indicating the severity of colitis. Compared to DSS mice receiving vehicle only, a significant decrease in DAI score was recorded in colitic mice receiving TCO1 (p-value = 0.002), TM1 (p-value = 0.05), and Humira (p-value = 0.02). This result indicates the strong anti-inflammatory effect of the newly generated anti-TMEM mAb.

[0229] Histological evaluation of colonic lesions To assess the level of inflammation and tissue regeneration, colonic samples were embedded in paraffin and analyzed. For histological evaluation, colonic tissue sections (4 μm) were stained with May-Grunwald-Giemsa and evaluated. Multiparameter scoring (0 to 18) as described by Dieleman et al. (1998) (Table 24) was performed blindly by two investigators. Histological examination scored the severity and extent of inflammation, the intensity of cellular infiltrate in the mucosa, its extension in the submucosa, the presence of epithelial lesions, and tissue regeneration. To detect apoptosis, paraffin-embedded colonic samples were further examined by immunofluorescence staining for deoxynucleotidyl transferase-mediated deoxyuridine triphosphatase (TUNEL) using the TUNEL Assay Kit (Sigma, ref. 11684795910) according to the manufacturer's protocol. The TUNEL assay is an effective method for measuring DNA fragments resulting from apoptotic activation of intracellular endonucleases. Meanwhile, to identify the level of cell proliferation, immunofluorescence staining for proliferating cell nuclear antigen (PCNA) was performed using a PCNA assay kit (Novus, NB600-1331) according to the manufacturer's protocol. PCNA is a cell cycle-related protein that is maximally expressed in the late G1 and S phases of proliferating cells. The sections were counterstained with a nuclear stain using DAPI. Localized fluorescence was detected by fluorescence microscopy.

[0230] [Table 27]

[0231] As shown in Figure 2B, 7 days after the last DSS administration, the histological scores in each group were quantified from May-Grunwald-Giemsa-stained sections (Figure 3A). Compared to the healthy control group without colitis, persistent and significant colonic inflammation was still recorded at the histological level in the group of DSS mice receiving vehicle (p-value = 0.002). After IP administration of 0.5 mg / mouse of TCO1, a significant improvement in the level of inflammation (p-value = 0.01) and mucosal regeneration at the histological level were observed compared to the colitis mice receiving vehicle. Furthermore, IP administration of 0.5 mg / mouse of TM1 showed an improvement in the level of inflammation and mucosal regeneration at the histological level compared to the colitis mice receiving vehicle.

[0232] TCO1 and TM1 inhibit DSS-induced intestinal cell apoptosis in mice, as shown in sections from colon tissue stained with a TUNEL staining kit, a method for detecting DNA fragmentation generated during apoptosis (Figure 3B). The sections were counterstained with DAPI to provide nuclear staining. TUNEL-positive cells were lower in the TCO1 and TM1 groups compared with the DSS group.

[0233] In the group of mice treated with DSS, PCNA expression was inhibited, indicating the severity of colitis, while in the TCO1 and TM1-treated groups, PCNA expression in the colon was maintained and was comparable to that in the control group that did not receive DSS, as shown in sections from colon tissue stained with a PCNA staining kit (Figure 3C).

[0234] The sections were counterstained with DAPI to provide nuclear staining.

[0235] statistical analysis All comparisons were analyzed using a permutation test for two independent samples. Statistics were calculated using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA). Differences were considered statistically significant when p-value was ≤ 0.05.

[0236] Example 3 Efficacy of anti-TMEM mAb in T1D mouse models after intraperitoneal (IP) administration animal Female non-obese diabetic (NOD) mice (10 weeks old) were obtained from Charles River Laboratories (Calco, Varese, Italy) (stock #613). All mice were housed and used in accordance with Italian Law No. 116 / 1992 on animal care and European Community Council Directive EEC / 609 / 86.

[0237] Diabetes monitoring and treatment Overt diabetes (the most advanced stage, characterized by high fasting blood glucose concentrations and classic symptoms) was defined as a blood glucose level above 250 mg / dL for three consecutive measurements. Glycemia was monitored twice a week. The inventors established the following treatment groups: 1) Untreated 2) Ecto-TMEM219 0.1 mg / day (ip) for 10 days 3) Anti-TMEM219 TM1 0.5 mg / day (ip) for 10 days 4) Anti-TMEM219 TC01 0.5 mg / day (ip) for 10 days Ecto-TMEM and antibodies were dissolved in PBS.

[0238] N=10 mice were included in each treatment group. Treatment began on day 1 when the mice were 10 weeks old. Mice were followed up until 22 weeks of age. Mice were assessed for diabetes or harvested at 22 weeks of age. Plasma samples and pancreases were collected for ex vivo analysis. The experimental timeline is shown in Figure 1.

[0239] Insulitis scoring and islet histopathology As previously described, insulitis scoring was performed on 5-µm-thick formalin-fixed, paraffin-embedded, hematoxylin and eosin (H&E)- and insulin-stained pancreatic sections (Vergani A et al. Diabetes 2010; Ben Nasr M et al. Sci Transl Med 2017). Insulitis scoring was performed on hematoxylin and eosin (H&E)-stained pancreatic sections. A score of 0 to 4 was assigned by an experienced pathologist based on islet infiltration. The insulitis score was graded as follows: Grade 0, normal islets; Grade 1, mild mononuclear infiltration (25%) at the periphery; Grade 2, 25–50% infiltration of islets; Grade 3, (50% infiltration of islets); and Grade 4, complete infiltration of islets with no residual parenchyma. At least 30 islets per group were analyzed and pooled from sections obtained from different mice.

[0240] statistical analysis Data are presented as the mean and standard error (SEM) unless otherwise reported. The statistical significance of differences was tested using a two-tailed t-test (Mann-Whitney test). Incidence of diabetes among different groups was analyzed by the log-rank (Mantel-Cox) test. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software, La Jolla, CA). All statistical tests were performed at the 5% significance level.

[0241] As shown in Figures 4 and 5, we evaluated whether 10-day administration of newly generated anti-TMEM219 mAbs prevents the onset of clinical diabetes in NOD mice, a mouse model selected to study autoimmune type 1 diabetes (T1D). Surprisingly, anti-TMEM219 mAbs are effective in keeping blood glucose levels under control over time and preventing the onset of diabetes in the T1D NOD mouse model. Interestingly, 100% of mice treated with antibodies TM1 and TC01 were diabetes-free at 22 weeks, compared with 50% of untreated controls (p<0.05, all vs. untreated).

[0242] Next, NOD mouse pancreatic tissue sections obtained from untreated, TM1-, TC01-, and Ecto-TMEM219-treated mice were analyzed for islet infiltration (insulitis), islet area and morphology (Figure 6A), and insulin staining (Figure 7). Both TM1- and TC01-treated groups showed favorable islet morphology. In addition, area appeared slightly increased in TM1-treated mice, suggesting that treatment with anti-TMEM mAb prevented islet destruction, thereby allowing normal β-cell function. Furthermore, compared with untreated controls, TM1- and TC01-treated mice showed well-preserved insulin positivity, further supporting the preservation of islet function. Indeed, islet infiltration was significantly reduced in treated mice compared with untreated mice, thus supporting the protective role of the antibody (Figure 7).

[0243] Thus, the present data demonstrate the efficacy of anti-TMEM monoclonal antibodies for the prevention and / or treatment of diabetes.

[0244] Example 4 To confirm that the newly generated monoclonal anti-TMEM219 antibodies could prevent the pro-apoptotic effects of IGFBP3 on TMEM219-expressing cells in the pancreas, we further tested them in vitro in the β cell line Betalox-5. The upregulation of CASP8 induced by IGFBP3 exposure was prevented by the newly generated anti-TMEM219 mAb, resulting in a nearly 30% reduction in CASP8. Furthermore, exposure of β cells to IGFBP3-enriched pooled T1D serum increased CASP8 expression, and the anti-TMEM219 mAb was able to counteract this effect by reducing CASP8 by at least 30%, thereby supporting the beneficial effect of these newly generated monoclonal anti-TMEM219 antibodies in preventing splenic β cell apoptosis (Figures 8 and 9).

[0245] Example 5 A newly generated monoclonal anti-TMEM219 antibody inhibits apoptosis in human pancreatic islets To confirm that the newly generated monoclonal anti-TMEM219 antibodies can prevent the pro-apoptotic effects of IGFBP3 on TMEM219-expressing cells in the pancreas, we further tested them in vitro in human pancreatic islets (Celprogen). The upregulation of CASP8 and apoptosis induced by IGFBP3 exposure were prevented by the newly generated anti-TMEM219 mAb, reducing CASP8 expression by 40% and apoptosis by 30% (Figures 10 and 11). Interestingly, exposure of human islets to pooled T1D serum naturally enriched for IGFBP3 increased CASP8 expression and apoptosis, and the anti-TMEM219 mAb was able to counteract this effect by reducing both CASP8 and apoptosis by approximately 50%, thereby supporting the beneficial effect of the newly generated monoclonal anti-TMEM219 antibodies in preventing islet apoptosis (Figures 12 and 13).

[0246] Example 6 A newly generated monoclonal anti-TMEM219 antibody is not toxic to .BETA.-cells To demonstrate that the newly generated monoclonal anti-TMEM219 antibody does not activate TMEM219 downstream signaling and therefore induces cell apoptosis in the absence of IGFBP3 ligation, we performed two main assays. First, we demonstrated that anti-TMEM219-treated β cells did not undergo apoptosis compared to those challenged with IGFBP3, which induced an approximately 30% increase in cell death (Figure 14). Next, we evaluated CASP8 mRNA expression and demonstrated its expected upregulation (approximately 70%) in β cells cultured with IGFBP3, while CASP8 remained unchanged in anti-TMEM219-cultured β cells (Figure 15). Overall, these data support the absence of any toxic / apoptotic effects of the anti-TMEM219 mAb.

[0247] Example 7 Effect of anti-TMEM219 mAb on streptozotocin-induced β-cell death in a diabetic model We further tested the effects of IGFBP3 / TMEM219 pharmacological blockade with anti-TMEM219 mAb in a second model of beta cell destruction by multiple low doses of streptozotocin (1DSTZ, 50 mg / kg for 5 days) and diabetes.

[0248] Chemically induced diabetes by streptozotocin injection was used to evaluate the effects of strategies targeting primarily the beta cell mass. It is associated with the development of low-grade inflammation but does not result in an autoimmune response, such as that observed in NOD mice. Therefore, the success of a compound in maintaining blood glucose levels depends primarily on protecting the beta cell mass from damage and maintaining insulin secretion.

[0249] Streptozotocin induces β-cell death, as observed in diabetes, when administered using a low-dose regimen of, for example, 50 mg / kg for five consecutive days, and diabetes / hyperglycemia usually develops within the first two weeks of administration ( FIG. 16 ). Therefore, we administered low-dose STZ to B6 mice for five days and simultaneously administered anti-TMEM219 mAb at a dose of 0.5 mg / day for 10 days. Anti-TMEM219 mAb treatment successfully maintained blood glucose levels in treated mice compared with untreated animals ( FIG. 17 ), and also improved blood glucose levels detected at 60 minutes during an intraperitoneal glucose tolerance test (IPGTT) ( FIG. 18 ). Morphological analysis also revealed that the number and extent of pancreatic islets were well preserved in animals treated with anti-TMEM219 mAb compared with untreated mice ( FIG. 19 ). Overall, this confirms that anti-TMEM219 can preserve beta cell mass and maintain blood glucose levels even in models of beta cell destruction, thereby highlighting the benefit of this strategy for protecting pancreatic islets from diabetes-induced beta cell loss and dysfunction.

[0250] Anti-TMEM219 antibodies such as TC01 are effective in protecting beta cells from injury and preventing their loss, as well as preserving their ability to respond to glucose stimulation. Furthermore, inflammation was not detectable in the islets of mice treated with anti-TMEM219 TC01, thus supporting a protective effect on islet morphology.

[0251] Methods of Examples 4 to 7 Recombinant Protein and Intervention Studies Recombinant human IGFBP3 (Life Technologies, 10430H07H5), 50 ng / ml IGFBP3, and 130 ng / ml ecto-TMEM219 were added to the cultures on day +1 of minigut culture. A newly generated anti-TMEM219 monoclonal antibody was added at a 1:1 molecular ratio compared to IGFBP3 at a concentration of 10 μg / ml.

[0252] β cell line (β-lox5) Cells were cultured with DMEM, 10% FBS, 0.02% BSA, 15 mM HEPES, 1x NEA, 1 g / L glucose, and PEN / STREP. Cells are typically seeded in 35 mm wells at a density of 10,000 cells / well. Cells are passaged at 80% confluency. Cells were cultured for 3 days with or without recombinant proteins / antibodies as described in the recombinant protein and intervention studies section.

[0253] human pancreatic islets Human islets of Langerhans (#35002-04) isolated from healthy subjects were also purchased from a commercial source (Celprogen, Torrance, CA) and cultured in standard medium and 10% FBS according to the manufacturer's instructions. To mimic the diabetic condition, human diabetic serum (n=5 / group) obtained from established T1D patients was added to the human islet / β-cell line at a concentration of 10% instead of regular FBS.

[0254] Pancreatic islet / beta cell death To assess apoptosis / cell death in purified human islets and β-cell lines, we used a photometric enzyme immunoassay (Roche Diagnostics GmbH, 11544675001, Mannheim, Germany) that quantifies histone-associated DNA fragments in vitro after inducing cellular stress in cytoplasmic lysates and cell supernatants.

[0255] STZ-induced diabetes research Diabetes was chemically induced by injecting low-dose streptozotocin (50 mg / kg, intraperitoneally; Sigma-Aldrich S0130) for 5 consecutive days, and glycemia was monitored for the following 15 days. A control group consisting of wild-type B6 mice was also injected with low-dose streptozotocin and monitored accordingly. Anti-TMEM219 mAb and ecto-TMEM219 were also administered intraperitoneally at doses of 0.5 mg / day and 0.1 mg / day, respectively, from day 0 to day 10, and glycemia was monitored for the following 15 days. At termination, an intraperitoneal glucose tolerance test (IPGTT) was performed by injecting mice with 1 kg / g of glucose after overnight starvation, and glycemia was monitored at 0, 30, 60, and 120 minutes.

[0256] statistical analysis Data were presented as the mean and standard error of the mean (SEM). Statistical significance of differences was tested using a two-tailed t-test. Significance between two groups was determined by a two-tailed unpaired Student's t-test. Analysis of variance with Bonferroni correction was used for multiple comparisons. Graphs were generated using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA). All statistical tests were performed at the 5% significance level.

[0257] (References) TIFF0007719061000037.tif230169TIFF0007719061000038.tif42168

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that binds to a human TMEM219 receptor and inhibits or reduces binding of insulin-like growth factor 3 (IGFBP3) to the TMEM219 receptor, a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of the amino acid sequence of SEQ ID NO: 4; ii. a CDR2 sequence of the amino acid sequence of SEQ ID NO: 5; and iii. A CDR3 sequence of the amino acid sequence of SEQ ID NO: 6; and b. A light chain variable domain (VL) comprising: i. a CDR1 sequence of the amino acid sequence of SEQ ID NO: 17; ii. a CDR2 sequence of the amino acid sequence of SEQ ID NO: 18; and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 166 An isolated antibody or antigen-binding fragment thereof comprising:

2. 1. An isolated antibody or antigen-binding fragment thereof that binds to a human TMEM219 receptor and inhibits or reduces IGFBP3 binding to the TMEM219 receptor, a. a heavy chain variable domain sequence of the amino acid sequence of SEQ ID NO: 33, 168, 169, or 170; and b. A light chain variable domain sequence of the amino acid sequence of SEQ ID NO: 39, 171, 172, or 173 An isolated antibody or antigen-binding fragment thereof comprising:

3. An isolated antibody or its antigen-binding fragment described in claim 1 or 2, which inhibits, reduces, or neutralizes activation of the TMEM219 receptor induced by IGFBP3 binding and / or does not activate the TMEM219 pathway when binding to the human TMEM219 receptor.

4. are effective in preserving beta cells and / or preventing islet destruction in diabetic patients and / or controlling blood glucose levels in in vivo models; and / or 4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, which is effective in reducing acute colitis in an in vivo model.

5. Increased mini-gut growth in healthy subjects treated with a-IGFBP3; b-increased mini-gut growth in IBD patients; c - Increased mini-gut growth in healthy subjects treated with diabetic enteropathy serum; Increased expression of EphB2 and / or LGR5 in mini-guts of healthy subjects treated with d-IGFBP3; Decreased caspase 8 expression in mini-intestines of healthy subjects treated with e-IGFBP3; 5. The isolated antibody or antigen-binding fragment thereof according to claim 1, having at least one activity selected from the group consisting of:

6. reduced β-cell loss in β-cells treated with f-IGFBP3; Increased insulin expression in β-cells treated with g-IGFBP3; reduced β-cell apoptosis in j-IGFBP3-treated β-cells; 5. The isolated antibody or antigen-binding fragment thereof according to claim 1, having at least one activity selected from the group consisting of:

7. inhibiting or reducing h-DSS-induced intestinal cell apoptosis; restores PCNA expression in i-DSS-treated colon; 5. The isolated antibody or antigen-binding fragment thereof according to claim 1, having at least one activity selected from the group consisting of:

8. k-Decreased insulitis scores in animal models of diabetes; l -reduced development of diabetes in animal models of diabetes; m-protects beta-cell injury in animal models of diabetes; Prevents beta-cell loss in animal models of n-diabetes 5. The isolated antibody or antigen-binding fragment thereof according to claim 1, having at least one activity selected from the group consisting of:

9. 7. An isolated antibody or its antigen-binding fragment according to claim 5 or 6, wherein the increase in a), b), and c) is at least 20%; the increase in d) and the decrease in e) is at least 30%; and the decrease in f) and the increase in g) is at least 10%.

10. - the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, or - the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 166 10. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 9, comprising:

11. 1. An isolated antibody or antigen-binding fragment thereof that binds to a human TMEM219 receptor and inhibits or reduces IGFBP3 binding to the TMEM219 receptor, a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of the following sequences: 【Table 1】 ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of the following sequences: 【Table 2】 iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of the following sequences: 【Table 3】 b. A light chain variable domain (VL) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of the following sequences: 【Table 4】 ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of the following sequences: 【Table 5】 iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of the following sequences: 【Table 6】 An isolated antibody or antigen-binding fragment thereof comprising:

12. a. a heavy chain variable domain sequence of the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 168, SEQ ID NO: 169, or SEQ ID NO: 170; and b. A light chain variable domain sequence of the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173 12. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 11, comprising:

13. The table below 【Table 7】 13. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, comprising the VH and VL regions of antibody TC01, TC03, TC04, or TC05, as defined above.

14. 10- for human TMEM -7 14. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 13, having an affinity constant of M or less.

15. 15. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 14, which is a human antibody or a humanized antibody.

16. 16. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 15, which is an IgG2 or IgG4 antibody.

17. 17. The isolated antibody or antigen-binding fragment thereof of claim 16, wherein the IgG2 or IgG4 antibody is an IgG2κ antibody, an IgG2λ antibody, an IgG4κ antibody, or an IgG4λ antibody.

18. 18. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, comprising the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO:

39.

19. a light chain constant region of the amino acid sequence of SEQ ID NO: 122, SEQ ID NO: 123, or SEQ ID NO: 124; and comprising a heavy chain constant region of the amino acid sequence of SEQ ID NO: 120 or SEQ ID NO: 121; 19. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 18, wherein the heavy chain constant region comprises a heavy chain constant region for human IgG4 comprising a Ser to Pro substitution at position 228 and a Leu to Glu substitution at position 235.

20. 20. An isolated polynucleotide comprising at least one sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 19.

21. 21. The polynucleotide of claim 20, which is a cDNA.

22. 22. A vector comprising the polynucleotide of claim 20 or 21.

23. 23. The vector of claim 22, wherein the 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.

24. 24. An isolated cell comprising a polynucleotide according to claim 20 or 21 or a vector according to claim 22 or 23.

25. 25. The isolated cell of claim 24, which is a hybridoma or a Chinese hamster ovary (CHO) cell or a human embryonic kidney cell (HEK293).

26. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 19, or a polynucleotide described in claim 20 or 21, or a vector described in claim 22 or 23, and a pharmaceutically acceptable carrier.

27. 27. The pharmaceutical composition of claim 26 for use in the treatment of diabetes, intestinal and / or bowel disorders, malabsorption syndromes, cachexia, or diabetic enteropathy.

28. 28. The pharmaceutical composition of claim 27, for the treatment of type I or type II diabetes, inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease, or intestinal obstruction.

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