IGFBP3 antibodies and their therapeutic uses

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to IGFBP3. The antibody inhibits or reduces the binding of IGFBP3 to the TMEM219 receptor. The present invention also relates to a method for producing the antibody, a pharmaceutical composition containing the antibody, and its use.

Description

Technical Field

[0001] The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to human IGFBP3, a production method thereof, a pharmaceutical composition containing the antibody, and uses thereof. Background Art

[0002] IGFBP3 / TMEM219 axis

[0003] The insulin-like growth factor binding proteins (IGFBPs) are a family of seven binding proteins that regulate the bioavailability of insulin-like growth factor (IGF). IGFBP3 is the most abundant of these, present in almost all tissues and possessing a high affinity for IGF; in fact, approximately 80-90% of IGFs bind to IGFBP3 in a ternary complex with the acid-labile subunit (ALS) (1).

[0004] In addition to its ability to regulate IGF availability, IGFBP3 has also been shown to have IGF-independent functions (2). Indeed, it is able to associate with cell surface proteins, cell surface receptors with global signal transduction capabilities, intracellular and nuclear proteins (transcription factors), thereby affecting cell growth and directly inducing apoptosis (2). Among the death receptors, TMEM219, a single transmembrane protein, has been shown to bind strongly 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) (3), but also stem cells (i.e., colon stem cells) (4). Blocking or enhancing the IGFBP3 / TMEM219 axis using different strategies has been shown to prevent or increase cell death, respectively. To our knowledge, there are currently no commercially available monoclonal antibodies against TMEM219 or IGFBP3 that can block IGFBP3 / TMEM219 binding at target tissues / cells where IGFBP3 binds to TMEM219, halting IGF-I-independent and caspase-8-mediated deleterious effects.

[0005] IGFBP3 / TMEM219 axis in diabetes

[0006] Both type 1 diabetes (T1D) and type 2 diabetes (T2D) are characterized by β-cell loss, leading to decreased insulin secretion, uncontrolled blood glucose levels, and hyperglycemia (5, 6). Regardless of the different etiological mechanisms, either autoimmune responses in T1D or insulin resistance / inflammation in T2D lead to a progressive reduction in β-cell mass. In fact, it is clear that the autoimmune activation that occurs does not seem to be sufficient to fully explain β-cell loss in T1D (5). Moreover, the failure of immunotherapy to cure T1D (7) highlights that: (i) autoimmunity may not be the only factor in T1D pathology, and (ii) alternative strategies targeting different disease mechanisms (such as β-cell loss) are needed to establish effective treatments for T1D. The detection of scattered β-cells in individuals with long-standing T1D (8) confirms that new β-cells must continue to appear to maintain β-cell turnover (5, 9), or that destroyed β-cells may be "different" and susceptible to death (10). This may suggest that upregulation / downregulation of surface β-cell receptor expression may have a key role in making it visible to the immune system and, more importantly, that other non-immunological determinants may regulate β-cell fate and function. Therefore, preventing non-immune β-cell destruction in TID and progressive β-cell loss in T2D may tilt the balance between β-cell production and destruction toward restoring the appropriate β-cell population, paving the way for new therapeutic approaches capable of aborting or delaying the first stages of the disease.

[0007] Studies have shown that the IGFBP3 receptor TMEM219 is expressed in β-cell lines and human / mouse pancreatic islets, and its ligation is toxic to β-cells. Interestingly, it has also been observed that human IGFBP3 transgenic mice develop hyperglycemia, show reduced islet mass, and exhibit a reduced response to insulin-glucose stimulation (11), while those mice with IGFBP3 knockdown do not show any changes in glucose metabolism control (12).

[0008] In humans, Drogan and colleagues recently published an article suggesting that elevated circulating levels of IGFBP3 are associated with the development of T2D (13). Furthermore, a recent study by the Diabimmune Research Group showed that IGFBP3 levels correlated with autoantibody positivity and the timing of seroconversion in children at risk for T1D, suggesting a role for circulating IGFBP3 in the early development of β-cell autoimmunity (14).

[0009] TMEM219, the IGFBP3 receptor, has been described as a death receptor whose activation triggers caspase 8-mediated apoptosis in target cells, leading to their loss (4).

[0010] IGFBP3 / TMEM219 axis in inflammatory bowel disease

[0011] Intestinal stem cells (ISCs) reside at the base of the crypts of the small and large intestines and control crypt regeneration and turnover. Specifically, ISCs can differentiate along the crypts to generate goblet cells, enterocytes, and enteroendocrine cells (4).

[0012] Inflammatory bowel disease (IBD) is a chronic immune-mediated disease that includes the clinical entities of Crohn's disease (CD) and ulcerative colitis (UC), affecting nearly 2.5 million people in Europe and nearly 1 million in the United States (15). The pathogenesis of IBD is still under investigation, but recent evidence suggests that impaired differentiation of ISCs into Paneth cells in ileal CD and into goblet cells in UC may play a key role in the pathogenesis of IBD. In particular, local signaling and inflammatory pathways in the mucosa are responsive to external stimuli and maintain the number and function of ISCs, thereby maintaining intestinal homeostasis (16). In fact, Yancu et al. recently published results supporting a role for IGFBP-3 in CD. In fact, they demonstrated that knockout of IGFBP3 modulated inflammation in a dextran-sodium-sulfate (DSS) colitis mouse model (17).

[0013] The inventors recently discovered that the insulin-like growth factor binding protein 3 (IGFBP3) receptor, also known as the TMEM219 receptor, is expressed on ISCs and that its interaction with the circulating hormone IGFBP3 controls the fate and function of ISCs in models of diabetic bowel disease and diabetic enteropathy (4). Because diabetic enteropathy and IBD share common features such as altered intestinal stem cell (ISC) homeostasis and mucosal morphology, these results may provide important insights into currently unknown mechanisms of IBD pathogenesis and may lead to the introduction of new therapeutic approaches for the treatment of IBD.

[0014] Currently, effective treatments for IBD are based on the use of anti-inflammatory and immunotherapy strategies, which are exacerbated by adverse reactions and whose long-term efficacy remains questionable. Surgery has also been successfully used in advanced stages of the disease, particularly in UC (15). Disease relapse is also common in CD, highlighting the need for different treatment approaches. Therefore, the identification of new therapeutic targets and strategies for the treatment of IBD is of high clinical relevance and need for public health.

[0015] WO2016193497 and WO2016193496 (incorporated herein by reference in their entirety) describe the TMEM219 extracellular domain, ecto-TMEM, as an effective therapeutic agent. However, receptor constructs are not as ideal as antibodies as therapeutic agents. Therefore, there remains a need for other therapeutic agents, antibodies or derivatives thereof that mimic the effects of ecto-TMEM. SUMMARY OF THE INVENTION

[0017] Disclosed herein are antibodies that bind to human IGF binding protein 3 (IGFBP3) with high affinity and specificity and are capable of reducing or eliminating the binding of IGFBP3 to its cognate receptor, TMEM219. These neutralizing antibodies can be used to treat diseases in which IGFBP3 binding to TMEM219 leads to disease pathology, including 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 similar therapeutic activity to receptor-based ligand traps (exo-TMEM219).

[0018] In a first aspect, an isolated antibody or antigen-binding fragment thereof is provided that binds to human IGFBP3 with an affinity constant less than or equal to 1.1 x 10 -9 M, which inhibits or reduces the binding of IGFBP3 to the TMEM219 receptor. Preferably, the isolated antibody or antigen-binding fragment thereof inhibits, reduces or neutralizes the activation of the TMEM219 receptor induced by the binding of IGFBP3.

[0019] IGFBP3-induced activation of the TMEM219 receptor can be measured by any method known in the art or as described below. In particular, IGFBP3-induced activation of the TMEM219 receptor can be measured by measuring an increase in apoptosis as described herein or a decrease in minigut growth as known in the art and as described herein and in several publications (4, 18, 27, 28).

[0020] In a preferred embodiment, the isolated antibody or antigen-binding fragment thereof effectively controls blood glucose levels in an in vivo model.

[0021] The present invention also provides an isolated antibody or antigen-binding fragment thereof having at least one activity selected from the following:

[0022] Increased mini-intestinal growth in healthy subjects treated with α-IGFBP3

[0023] b-IBD patients have increased mini-intestinal growth;

[0024] c- Increased mini-intestinal growth in healthy subjects treated with diabetic enteropathy serum;

[0025] The expression of EphB2 and / or LGR5 was increased in the mini intestines of healthy subjects treated with d-IGFBP3;

[0026] e-Reduction of caspase-8 expression in the mini-intestines of healthy subjects treated with IGFBP3;

[0027] reduction of β-cell loss in f-IGFBP3-treated β-cells;

[0028] g-IGFBP3-treated β cells increased insulin expression; and

[0029] β-cell apoptosis was reduced in h-IGFBP3-treated β-cells;

[0030] i - Decreased expression of caspase-8 in IGFBP3-treated β cells;

[0031] j-Reduced pancreatitis scores in diabetic animal models;

[0032] k Reduced the incidence of diabetes in diabetic animal models.

[0033] 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%, the decrease in i), j) and k) is at least 50%, and preferably the decrease in k is at least 70%.

[0034] The present invention provides an isolated antibody or antigen-binding fragment thereof, comprising:

[0035] a. A heavy chain variable domain (VH), comprising:

[0036] i. a CDR1 sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 4, 7 or 9;

[0037] ii. a CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 5, 8 or 10; and

[0038] iii. a CDR3 sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 6 or 11; and / or

[0039] b. a light chain variable domain (VL) comprising:

[0040] i. a CDR1 sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 15, 17, 20, 23, 25 or 27;

[0041] ii. a CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 18 or 21; and

[0042] iii. a CDR3 sequence selected from the group consisting of an amino acid sequence of SEQ ID NO: 14, 16, 19, 22, 24, or 26.

[0043] Preferably, the isolated antibody or antigen-binding fragment thereof comprises the CDRs shown in Table 2 and / or Table 3 (including Table 3.1).

[0044] Preferably, it has at least one activity selected from the group consisting of:

[0045] Increased mini-intestinal growth in healthy subjects treated with α-IGFBP3

[0046] b-IBD patients have increased mini-intestinal growth;

[0047] c- Increased mini-intestinal growth in healthy subjects treated with diabetic enteropathy serum;

[0048] The expression of EphB2 and / or LGR5 was increased in the mini intestines of healthy subjects treated with d-IGFBP3;

[0049] e-Reduction of caspase-8 expression in the mini-intestines of healthy subjects treated with IGFBP3;

[0050] reduction of β-cell loss in f-IGFBP3-treated β-cells;

[0051] g-IGFBP3-treated β cells increased insulin expression; and

[0052] β-cell apoptosis was reduced in h-IGFBP3-treated β-cells;

[0053] i-Reduction of caspase 8 expression in IGFBP3-treated β cells.

[0054] 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) are at least 10%.

[0055] Preferably, the isolated antibody or antigen-binding fragment thereof comprises:

[0056] a. a heavy chain variable domain sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO: 36;

[0057] b. a light chain variable domain sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 37 to SEQ ID NO: 45; or

[0058] c. Light chain variable domain of (a) and heavy chain variable domain of (b).

[0059] Preferably, the isolated antibody or antigen-binding fragment thereof comprises:

[0060] - SEQ ID NO: 9 and SEQ ID NO: 10 and SEQ ID NO: 11 and SEQ ID NO: 27 and SEQ ID NO: 18 and SEQ ID NO: 26 or the CDRs of Kabat, IMGT, Chothia, AbM or Contact of M1 or

[0061] - SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 12 and SEQ ID NO: 13 and SEQ ID NO: 14 or the CDRs of Kabat, IMGT, Chothia, AbM or Contact of E08 or

[0062] - the CDRs of Kabat, IMGT, Chothia, AbM or Contact of SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 23 and SEQ ID NO: 18 and SEQ ID NO: 24 or E20.

[0063] Preferably, the isolated antibody or antigen-binding fragment thereof comprises:

[0064] a. A heavy chain variable domain (VH), comprising:

[0065] i. a CDR1 sequence of an amino acid sequence selected from the group consisting of sequences defined by the abysis tool (www.abysis.org);

[0066] ii. a CDR2 sequence of an amino acid sequence selected from the group consisting of a sequence defined by analysis using the abysis tool (www.abysis.org); and

[0067] iii. a CDR3 sequence selected from the group consisting of an amino acid sequence defined by analysis using the abysis tool (www.abysis.org); and / or

[0068] b. a light chain variable domain (VL) comprising:

[0069] i. a CDR1 sequence of an amino acid sequence selected from the group consisting of sequences defined by the abysis tool (www.abysis.org);

[0070] ii. a CDR2 sequence of an amino acid sequence selected from the group consisting of a sequence defined by analysis using the abysis tool (www.abysis.org); and

[0071] iii. A CDR3 sequence selected from the group consisting of amino acid sequences defined by the abysis tool analysis (www.abysis.org).

[0072] Still preferably, as shown in Tables 2-7, the isolated antibody is E01, E02, E08, E14, E19, E20, E23, E24 or M1, or an antigen-binding fragment thereof.

[0073] Still preferably, the isolated antibody is E01, which includes SEQ ID NO: 28 and SEQ ID NO: 37, E02, which includes SEQ ID NO: 29 and SEQ ID NO: 38, E08, which includes SEQ ID NO: 30 and SEQ ID NO: 39, E14, which includes SEQ ID NO: 31 and SEQ ID NO: 40, E19, which includes SEQ ID NO: 32 and SEQ ID NO: 41, E20, which includes SEQ ID NO: 33 and SEQ ID NO: 42, E23, which includes SEQ ID NO: 34 and SEQ ID NO: 43, E24, which includes SEQ ID NO: 35 and SEQ ID NO: 44, M1, which includes SEQ ID NO: 36 and SEQ ID NO: 45.

[0074] The present invention also provides an isolated antibody or antigen-binding fragment thereof, which:

[0075] (a) specifically binds to an epitope on IGFBP3, e.g., an epitope that is the same as or similar to the epitope recognized by monoclonal antibody E01, E02, E08, E14, E19, E20, E23, E24 or M1 comprising a sequence as defined in Tables 2-7; or

[0076] (b) cross-competes for binding with monoclonal antibody E01, E02, E08, E14, E19, E20, E23, E24 or M1 comprising a sequence as defined in Tables 2-7; or

[0077] (c) exhibits the same or similar binding affinity or specificity, or both, as any of E01, E02, E08, E14, E19, E20, E23, E24, or M1 comprising a sequence as defined in Tables 2-7; or

[0078] (d) has one or more biological properties of an antibody molecule described herein, e.g., an antibody molecule selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1 comprising a sequence as defined in, e.g., Tables 2-7; or

[0079] (e) having one or more pharmacokinetic properties of an antibody molecule described herein, e.g., an antibody molecule selected from any one of E01, E02, E08, E14, E19, E20, E23, E24 or M1 comprising a sequence as defined in, e.g., Tables 2-7.

[0080] Preferably, the isolated antibody or antigen-binding fragment thereof of the present invention is a human antibody or a humanized antibody.

[0081] 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.

[0082] The present invention provides an isolated polynucleotide comprising at least one sequence encoding the above-defined antibody or antigen-binding fragment thereof, preferably the polynucleotide is a cDNA.

[0083] The present invention provides a vector comprising the above polynucleotide. Preferably, 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.

[0084] The present invention also provides an isolated cell comprising the polynucleotide as defined above or the 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).

[0085] The present invention further provides an antibody or antigen-binding fragment thereof as defined above or an isolated polynucleotide or vector or an isolated cell as defined above for use as a medicament, preferably for the treatment of: diabetes, intestinal diseases and / or bowel diseases, malabsorption syndromes, cachexia or diabetic bowel disease, preferably diabetes is type I or type II diabetes, preferably the intestinal and / or bowel disease is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction.

[0086] The present invention also provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof or an isolated polynucleotide or a vector or an isolated cell as defined above and a pharmaceutically acceptable carrier, preferably for treating: diabetes, intestinal and / or enteropathy, malabsorption syndrome, cachexia or diabetic enteropathy, preferably the intestinal disease and / or enteropathy is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction.

[0087] The present invention provides a method for inhibiting the binding of IGFBP3 to TMEM219 receptor, comprising contacting IGFBP3 with the antibody or composition defined above.

[0088] The present invention provides a method for treating the following conditions: diabetes, preferably type 1 or type 2 diabetes, intestinal and / or enteropathy, malabsorption syndrome, cachexia or diabetic enteropathy, preferably the intestinal disease and / or enteropathy is inflammatory bowel disease, IBD, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising the above-mentioned isolated antibody or antigen-binding fragment thereof or isolated polynucleotide or vector or isolated cell and a pharmaceutically acceptable carrier, or administering to a subject in need thereof an isolated antibody or antigen-binding fragment thereof or isolated polynucleotide or vector or isolated cell as defined above.

[0089] The present invention also provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising obtaining the cell as defined above and producing the antibody or the antigen-binding fragment thereof.

[0090] In some embodiments, the combination includes an inhibitor of IGFBP3 (e.g., an anti-IGFBP3 antibody molecule as described herein). Thus, disclosed herein are compositions and methods for detecting IGFBP3, as well as methods for treating various diseases including diabetes, as well as intestinal diseases and / or bowel disorders using the anti-IGFBP3 antibody molecules and combinations thereof disclosed herein.

[0091] Thus, in one aspect, the invention features an antibody molecule (e.g., an isolated or recombinant antibody molecule) having one or more of the following properties:

[0092] (i) binds to IGFBP3 (e.g., human IGFBP3) with high affinity, e.g., with a binding affinity of at least about 4×10 6 M -1 , preferably 10 7 M -1 , usually about 10 8 M -1 and more usually about 10 9 M -1 to 10 10 M -1 or a stronger affinity constant;

[0093] (ii) inhibiting or reducing the binding of IGFBP3 to its receptor TMEM;

[0094] (iii) specifically binds to an epitope on IGFBP3, e.g., an epitope that is different from the epitope recognized by the commercially available antibodies LSBIO LS-C45037 or clone 83.8F9;

[0095] (iv) specifically binds to an epitope on IGFBP3, e.g., an epitope that is the same as or similar to the epitope recognized by monoclonal antibody E01, E02, E08, E14, E19, E20, E23, E24 or M1 as defined in Tables 2-7;

[0096] (v) cross-competes for binding with monoclonal antibodies E01, E02, E08, E14, E19, E20, E23, E24 or M1 as defined in Tables 2-7;

[0097] (vi) exhibits the same or similar binding affinity or specificity as any of E01, E02, E08, E14, E19, E20, E23, E24, or M1 as defined in Tables 2-7, or both;

[0098] (vii) exhibits the same or similar binding affinity or specificity, or both, as the antibody molecules (e.g., heavy chain variable region and light chain variable region) described in Tables 2-7;

[0099] (viii) 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 the amino acid sequence shown in Tables 2-7;

[0100] (ix) exhibits the same or similar binding affinity or specificity, or both, as the antibody molecules (e.g., heavy chain variable region and light chain variable region) encoded by the nucleotide sequences shown in Tables 6-7;

[0101] (x) binds to the same or overlapping epitope as a second antibody molecule directed against IGFBP3, wherein the second antibody molecule is an antibody molecule described herein, e.g., an antibody molecule selected from E01, E02, E08, E14, E19, E20, E23, E24 or M1 as defined in Tables 2-7;

[0102] (xi) possesses one or more biological properties of an antibody molecule described herein, e.g., an antibody molecule selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1 as defined in Tables 2-7;

[0103] (xii) has one or more pharmacokinetic properties of an antibody molecule described herein, e.g., an antibody molecule selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1 as defined in Tables 2-7;

[0104] (xiii) inhibiting one or more activities of IGFBP3, e.g., resulting in one or more of: at least a 20% increase in mini-intestine development in an IBD patient-derived tissue sample compared to an untreated sample, and / or at least a 20% increase in mini-intestine growth development in the presence of IGFBP3 compared to an untreated sample, or at least a 20% increase in mini-intestine growth development in the presence of diabetic enteropathy serum compared to an untreated sample;

[0105] (xiv) an increase of at least 50% in inducing EphB2 and LGR5 compared to a sample treated with IGFBP3; or a decrease of at least 50% in the expression level of caspase 8 compared to a sample treated with IGFBP3; or

[0106] (xv) inhibiting 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; a reduction in beta cell loss or an increase in insulin of at least 10% compared to an IGFBP3-treated sample;

[0107] (xvi) inhibiting, reducing or neutralizing one or more activities of IGFBP3, resulting in blocking or reducing IGFBP3-induced cell apoptosis;

[0108] (xvii) Binds to human IGFBP3 and cross-reacts with cynomolgus monkey IGFBP3.

[0109] Also provided are nucleic acid molecules encoding the antibody molecules, expression vectors, host cells, and methods for preparing the antibody molecules. Also provided are immunoconjugates, multispecific antibodies, or bispecific antibody molecules, and pharmaceutical compositions comprising the antibody molecules.

[0110] Without being bound by any theory, it is believed that the IGFBP3 / TMEM219 axis is dysfunctional in inflammatory bowel disease (IBD), leading to loss of ISCs and altered mucosal barrier function, which in turn triggers and maintains microbial invasion to activate immune responses and inflammation. The use of agents that block the IGFBP3-TMEM219 interaction in IBD may protect ISCs and preserve intestinal barrier integrity, thereby preventing the development of local inflammation.

[0111] 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 newly diagnosed and long-standing diabetes, and TMEM219 is expressed in β-cells. TMEM219 expression or overexpression promotes β-cell destruction and impacts β-cell mass, with the resulting hyperglycemia and inflammation playing a role in the development and progression of diabetes. Altered glycemic control and inflammation in prediabetes favor increased hepatic production of IGFBP3, which targets TMEM219 expressed on pancreatic β-cells and triggers a cycle in which TMEM219 overexpression parallels increased IGFBP3 release. TMEM219 can then trigger β-cell death, and targeting the IGFBP3 / TMEM219 axis may prevent this cell death.

[0112] It should be noted that Casp8 is overexpressed in T1D patients compared with the control group.

[0113] The anti-IGFBP3 antibody molecules disclosed herein can be used (alone or in combination with other agents or treatment modalities) to treat, prevent, and / or diagnose diseases such as diabetes, as well as intestinal diseases and / or enteropathy, malabsorption syndrome, inflammatory bowel disease, cachexia, IBD, celiac disease, diabetic enteropathy. In addition, the methods and compositions disclosed herein comprise a combination of two, three, or more therapeutic agents selected from one, two, or all of the following categories (i)-(iii): (i) an agent for treating diabetes; (ii) an anti-inflammatory agent; or (iii) an immunotherapeutic agent.

[0114] Other therapeutic agents may be selected from agents for treating diabetes, including: 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), agents for preventing diabetes, aspirin, anticoagulants and platelet antiaggregants (e.g., enoxaparin, eparin, sulodexide); cholesterol-lowering drugs (e.g., statins, bile ducts); acid chelators, ezetimibe, fibrates as detailed in Marsha et al. 2011 (21, incorporated by reference)); other antihypertensive drugs (such as thiazides, ACE inhibitors, beta and alpha blockers); anti-apoptotic agents, anti-inflammatory agents, corticosteroids and immunosuppressants (22, incorporated by reference), adjuvant therapy in organ transplantation, protective agents in cell therapy approaches, analgesics, antibiotics, probiotics, TNF-alpha blockers (23, incorporated by reference), SGLT2 inhibitors (such as gliflozin derivatives), integrin inhibitors (24, incorporated by reference).

[0115] Methods of measuring increased minigut growth compared to minigut growth in the presence of IGFBP3 and / or in the presence of diabetic enteropathy serum are known in the art and described in several publications (4, 18, 27, 28) or as described in the Methods section below.

[0116] 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 quantitative RT-PCR, real-time RT-PCR, microarrays, northern blots, RNASeq (29, 30), or as described in the Methods section below.

[0117] Methods for measuring reduced β cell loss compared to β cell loss in the presence of IGFBP3 are known in the art and include 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 as described in the Methods section below.

[0118] Methods for measuring increased insulin levels compared to insulin levels in the presence of IGFBP3 are known in the art and include Western blot, ELISA, mass spectrometry (31-33).

[0119] 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, microcatheter membrane permeabilization, Annexin V binding (34) or as described in the Methods section below.

[0120] In some embodiments, the antibody molecule binds to IGFBP3 with high affinity, e.g., having 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 anti-IGFBP3 antibody molecule, a chimeric anti-IGFBP3 antibody molecule, or a commercially available anti-IGFBP3 antibody molecule. In some embodiments, the KD of a murine or chimeric anti-IGFBP3 antibody molecule is less than about 0.4, 0.3, 0.2, 0.1, or 0.05 nM, e.g., as measured by Biacore or KinExA kinetic exclusion assay. In some embodiments, the KD of a murine or chimeric anti-IGFBP3 antibody molecule is less than about 0.2 nM. In other embodiments, the murine or chimeric anti-IGFBP3 antibody molecule has a KD of 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 murine or chimeric anti-IGFBP3 antibody molecule has a KD of less than about 1 nM.

[0121] Methods for measuring binding to IGFBP3 are known in the art as protein-protein interaction assays and include ELISA, co-immunoprecipitation, surface plasmon resonance, FRET-Förster resonance energy transfer (35) or as described in the Methods section below.

[0122] In some embodiments, the antibody molecule is expressed at a level greater than the expression level of a murine or chimeric antibody molecule (e.g., a murine, commercially available, or chimeric anti-IGFBP3 antibody molecule, such as LSBIO LS-C45037, clone 83.8F9, or Novus NBP2-12364), for example, at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold. In some embodiments, the antibody molecule is expressed in HEK293 cells, CHO cells, or any suitable mammalian cell line known in the art.

[0123] In some embodiments, the anti-IGFBP3 antibody molecule reduces one or more IGFBP3-associated activities with an IC50 (concentration for 50% inhibition) that is about equal to or less than the IC50 of a murine, commercially available or chimeric anti-IGFBP3 antibody molecule, e.g., a murine commercially available or chimeric anti-IGFBP3 antibody molecule described herein, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0124] In some embodiments, the anti-IGFBP3 antibody molecule has improved stability, e.g., at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold greater stability in vivo or in vitro than a murine, commercially available, or chimeric anti-IGFBP3 antibody molecule (e.g., a murine, commercially available, or chimeric anti-IGFBP3 antibody molecule, e.g., LSBIO LS-C45037, clone 83.8F9, or Novus NBP2-12364).

[0125] In one embodiment, the anti-IGFBP3 antibody molecule is a humanized antibody molecule.

[0126] In another embodiment, the anti-IGFBP3 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 one of E01, E02, E08, E14, E19, E20, E23, E24, or M1), as defined in Tables 2-5, or encoded by a nucleotide sequence in Tables 6-7; or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0127] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, three or four variable regions from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24 or M1, as defined in Tables 2-5, or encoded by the nucleotide sequence in Tables 6-7; 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.

[0128] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5, or encoded by the nucleotide sequence in Tables 6-7; 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.

[0129] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5, or encoded by the nucleotide sequence in Tables 6-7; 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.

[0130] In another embodiment, the anti-IGFBP3 antibody molecule comprises a heavy chain constant region of 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 another embodiment, the anti-IGFBP3 antibody molecule comprises a heavy chain constant region of 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 comprises a substitution at position 234, a substitution at position 235, or both (e.g., a Leu to Ala substitution at position 234 and / or a Leu to Ala substitution at position 235). In one embodiment, the heavy 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).

[0131] In another embodiment, the anti-IGFBP3 antibody molecule comprises a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the light chain constant region comprises an amino acid sequence as 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).

[0132] In another embodiment, the anti-IGFBP3 antibody molecule comprises an IgG4 (e.g., human IgG4) heavy chain constant region and a kappa light chain constant region (e.g., a human kappa light chain constant region), e.g., a heavy chain and light chain constant region comprising 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). In another embodiment, the anti-IGFBP3 antibody molecule comprises an IgG1 (e.g., human IgG1) heavy chain constant region and a kappa light chain constant region (e.g., a human kappa light chain constant region), e.g., a heavy chain and light chain constant region comprising 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). In one embodiment, the human IgG1 or IgG4 includes substitutions in the variable regions to reduce aggregation, reduce charge heterogeneity, increase affinity, and modulate antigen binding; instability hotspots in the CDRs are removed by mutation, and putative N-glycosylation sites in the variable regions are as described in (26), incorporated by reference.

[0133] In another embodiment, the anti-IGFBP3 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 one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5, or encoded by the nucleotide sequence in Tables 6-7; 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-IGFBP3 antibody molecule optionally comprises a leader sequence from the heavy chain, the light chain, or both.

[0134] In another embodiment, the anti-IGFBP3 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 one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-7, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any one of the sequences present in Tables 2-7.

[0135] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs (or the entire set of CDRs) from a heavy chain variable region comprising an amino acid sequence shown in Tables 2-5 or encoded by a nucleotide sequence shown in Tables 6-7. In one embodiment, one or more CDRs (or the entire set of CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Tables 2-5 or encoded by the nucleotide sequence shown in Tables 6-7.

[0136] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs from a light chain variable region from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 and 3.1, or encoded by the nucleotide sequence in Tables 6-7; 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.

[0137] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs (or the entire set of CDRs) from a light chain variable region comprising an amino acid sequence shown in Tables 2-5 or encoded by a nucleotide sequence shown in Tables 6-7. In one embodiment, one or more CDRs (or the entire set of CDRs) have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequences shown in Tables 2-5 and 3.1 or encoded by the nucleotide sequences shown in Tables 6-7. In certain embodiments, the anti-IGFBP3 antibody molecule comprises substitutions in light chain CDRs, e.g., one or more substitutions in CDR1, CDR2, and / or CDR3 of the light chain.

[0138] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, three, four, five, or six CDRs (or the entire set of CDRs) from the heavy and light chain variable regions comprising the amino acid sequences shown in Tables 2-5, or encoded by the nucleotide sequences shown in Tables 6-7. In one embodiment, one or more CDRs (or the entire set of CDRs) have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequences shown in Tables 2-5 and 3.1, or encoded by the nucleotide sequences shown in Tables 6-7.

[0139] In one embodiment, the anti-IGFBP3 antibody molecule comprises all six CDRs from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 and 3.1, or encoded by the nucleotide sequences in Tables 6-7, or closely related CDRs, e.g., CDRs 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). In one embodiment, the anti-IGFBP3 antibody molecule comprises any of the CDRs described herein. In certain embodiments, the anti-IGFBP3 antibody molecule comprises substitutions in the light chain CDRs, e.g., one or more substitutions in CDR1, CDR2, and / or CDR3 of the light chain. In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition or other definitions as shown in Tables 2-5 and 3.1) of the heavy chain variable region from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 and 3.1, or encoded by the nucleotide sequence in Tables 6-7; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the above sequences; or a sequence having at least one amino acid alteration, but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. or other definitions as shown in Tables 2-5 and 3.1.

[0140] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to Kabat or other definitions as shown in Tables 2-3 and 3.1) of the light chain variable region from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5, or encoded by the nucleotide sequence in Tables 6-7; or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the above sequences; or a sequence that has at least one amino acid alteration, but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. or other definitions as shown in Tables 2-5 and 3.1.

[0141] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., at least one, two, three, four, five, or six CDRs according to Kabat et al., or as defined in Tables 2-3 and 3.1 ) of the heavy and light chain variable regions from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 . , or encoded by the nucleotide sequence in Tables 6-7; or a sequence substantially identical to any of the above sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity); or a sequence having at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to one, two, three, four, five or six CDRs as defined by Kabat et al. or others as shown in Tables 2-5 and 3.1.

[0142] In another embodiment, the anti-IGFBP3 antibody molecule comprises all six CDRs according to Kabat et al. (e.g., all six CDRs according to the Kabat definition or other definitions shown in Tables 2-5 and 3.1) of the heavy and light chain variable regions from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, or as described in Tables 2-5, or encoded by the nucleotide sequences in Tables 6-7; or a sequence substantially identical to any of the above sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical); or a sequence having at least one amino acid alteration, but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Kabat et al. shown in Tables 2-5 or 3.1. In one embodiment, the anti-IGFBP3 antibody molecule can comprise any CDR described herein.

[0143] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three Chothia or Kabat hypervariable loops of the heavy chain variable region (e.g., at least one, two, or three hypervariable loops according to the Chothia or Kabat definitions listed in Tables 2-5) from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5, or encoded by the nucleotide sequences in Tables 6-7; or at least amino acids from those hypervariable loops that contact IGFBP3; or having at least one amino acid alteration, but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to one, two, or three hypervariable loops according to Chothia et al., as shown in Tables 2-5.

[0144] In another embodiment, the anti-IGFBP3 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 definition listed in Tables 2-5) of the light chain variable region from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 (including 3.1), or encoded by the nucleotide sequences in Tables 6-7; or at least amino acids from those hypervariable loops that contact IGFBP3; or having at least one amino acid alteration, but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), relative to one, two, or three hypervariable loops according to Chothia et al., as shown in Tables 2-5 (including 3.1).

[0145] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, three, four, five, or six hypervariable loops (e.g., at least one, two, three, four, five, or six hypervariable loops according to the Chothia definition listed in Tables 2-5) of the heavy and light chain variable regions from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or MI, or as defined in Tables 2-5 (including 3.1); or encoded by the nucleotide sequence in Tables 6-7; or at least amino acids from those hypervariable loops that contact IGFBP3; or having at least one amino acid alteration, but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six hypervariable loops according to Chothia et al. as shown in Tables 2-5 (including 3.1).

[0146] In one embodiment, the anti-IGFBP3 antibody molecule comprises all six hypervariable loops (e.g., all six hypervariable loops according to the Chothia definition as described in Tables 2-5) from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 (including 3.1), or closely related hypervariable loops, e.g., 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); or hypervariable loops that 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) relative to all six hypervariable loops according to Chothia et al. as shown in Tables 2-5. In one embodiment, the anti-IGFBP3 antibody molecule can include any hypervariable loop described herein.

[0147] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three hypervariable loops having the same canonical structure as corresponding hypervariable loops of an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 (including 3.1), e.g., the same canonical structure as at least loop 1 and / or loop 2 of the heavy and / or light chain variable domains of an antibody described herein. For descriptions of canonical hypervariable loop structures, 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 determined by consulting the tables described in these references.

[0148] In certain embodiments, the anti-IGFBP3 antibody molecule comprises a combination of CDRs or hypervariable loops defined according to Kabat et al. and Chothia et al., or other definitions known in the art.

[0149] In one embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs or hypervariable loops of a heavy chain variable region from an antibody described herein, e.g., an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 (including 3.1), at least one, two, or three CDRs or hypervariable loops according to Kabat and Chothia or other definitions (e.g., as shown in Tables 2-5 (including 3.1), R or hypervariable loop); or encoded by the nucleotide sequence in Tables 6-7; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the above sequences; or a sequence having at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to one, two or three CDRs or hypervariable loops as defined by Kabat and / or Chothia or others as shown in Tables 2-5 (including 3.1).

[0150] For example, an anti-IGFBP3 antibody molecule can include a VH CDR1 according to Kabat et al. or a VH hypervariable loop 1 according to Chothia et al., or a combination thereof, for example, as shown in Tables 2-5 (including 3.1). The anti-IGFBP3 antibody molecule can further include, for example, VH CDRs 2-3 according to Kabat et al. and VL CDRs 1-3 according to Kabat et al., for example, or as otherwise defined in Tables 2-5 (including 3.1). 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-IGFBP3 antibody molecule can include a VH FR1 defined based on VH hypervariable loop 1 according to Chothia et al. and a VH FR2 defined based on VH CDRs 1-2 according to Kabat et al., for example, or as otherwise defined in Tables 2-5 (including 3.1). The anti-IGFBP3 antibody molecule can further comprise, for example, VH FRs 3-4 defined based on VH CDRs 2-3 according to Kabat et al. or others, and VL FRs 1-4 defined based on VL CDRs 1-3 according to Kabat et al. or others.

[0151] According to the Kabat and Chothia definitions, the anti-IGFBP3 antibody molecule can comprise any combination of CDRs or hypervariable loops. In one embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs from a light chain variable region from an antibody described herein, such as an antibody selected from any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 (including 3.1), according to Kabat and Chothia, or other definitions (e.g., at least one, two, or three CDRs as shown in Tables 2-5 according to the Kabat and Chothia definitions). Preferred anti-IGFBP3 antibodies are E01, E02, E08, E14, E19, E20, E23, E24, or M1 as defined in Tables 2-5 (including 3.1).

[0152] In one embodiment, e.g., an embodiment comprising a variable region, CDR (e.g., Chothia CDR or Kabat CDR), or other sequences mentioned herein (e.g., in Tables 2-5 (including 3.1)), 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 an embodiment, the antibody molecule is a bispecific antibody molecule having a first binding specificity for IGFBP3 and a second binding specificity for TNFα, integrin, IL1, IL12 and IL23, CD3, CD20, CD80, CD86.

[0153] In one embodiment, the anti-IGFBP3 antibody molecule comprises:

[0154] (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 7 or 9; a VHCDR2 amino acid sequence selected from any one of SEQ ID NOs: 2, 5, 8 or 10; and a VHCDR3 amino acid sequence selected from any one of SEQ ID NOs: 3, 6 or 11; and / or

[0155] (i) a light chain variable region (VL) comprising a VLCDR1 amino acid sequence selected from any one of SEQ ID NOs: 12, 15, 17, 20, 23, 25, or 27, a VLCDR2 amino acid sequence selected from any one of SEQ ID NOs: 13, 18, or 21, and a VLCDR3 amino acid sequence selected from SEQ ID NOs: 14, 16, 19, 22, 24, or 26.

[0156] In another embodiment, the anti-IGFBP3 antibody molecule comprises:

[0157] (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 9; a VHCDR2 amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 10; and a VHCDR3 amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 11;

[0158] (ii) a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, a VLCDR2 amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 18 or SEQ ID NO: 21, and a VLCDR3 amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 26.

[0159] In one embodiment, the light chain or heavy chain variable framework (e.g., a region comprising at least FR1, FR2, FR3, and optionally FR4) of the anti-IGFBP3 antibody molecule can be selected from: (a) a light chain or heavy chain variable framework comprising at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% of the amino acid residues from a human light chain or heavy chain variable framework, such as a light chain or heavy chain variable framework from a human mature antibody, a human germline sequence, or a human consensus sequence; (b) a light chain or heavy chain variable framework comprising 20% ​​to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of amino acid residues from a human light chain or heavy chain variable framework, such as a light chain or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (c) a non-human framework (e.g., a rodent framework); or (d) a modified non-human framework, e.g., modified to remove antigenic or cytotoxic determinants, e.g., deimmunization or partial humanization. In one embodiment, the light chain or heavy chain variable framework region (particularly FRI, FR2, and / or FR3) comprises a light chain 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.

[0160] In certain embodiments, the anti-IGFBP3 antibody molecule comprises a heavy chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more changes, such as amino acid substitutions or deletions.

[0161] In one embodiment, the heavy chain or light chain variable region, or both, of the anti-IGFBP3 antibody molecule comprises an amino acid sequence encoded by a nucleic acid sequence described herein, or a nucleic acid that hybridizes (e.g., under low stringency, medium stringency, or high stringency, or other hybridization conditions described herein) to a nucleic acid sequence described herein (e.g., a nucleic acid sequence as shown in Tables 6 and 7), or its complement.

[0162] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, three, or four antigen-binding regions, e.g., variable regions, having an amino acid sequence as described in Tables 2-5, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical, or a sequence that differs by no more than 1, 2, 5, 10, or 15 amino acid residues from a sequence as described in Tables 2-5). In another embodiment, the anti-IGFBP3 antibody molecule comprises a VH and / or VL domain encoded by a nucleic acid having a nucleotide sequence as described in Tables 6-7, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical, or a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotide residues from a sequence as described in Tables 6-7).

[0163] In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs from a heavy chain variable region having an amino acid sequence as described in Tables 2-5 (including 3.1), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three, or more substitutions, insertions, or deletions, such as conservative substitutions). In another embodiment, the anti-IGFBP3 antibody molecule comprises at least one, two, or three CDRs from a light chain variable region having an amino acid sequence as described in Tables 2-5 (including 3.1), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three, or more substitutions, insertions, or deletions, such as conservative substitutions). In another embodiment, the anti-IGFBP3 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 as shown in Tables 2-5 (including 3.1), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three or more substitutions, insertions or deletions, e.g., conservative substitutions).

[0164] In other embodiments, the anti-IGFBP3 antibody molecule comprises a heavy chain constant region (Fc) selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; specifically, a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, and IgG4, more specifically, an IgG1 or IgG4 heavy chain constant region (e.g., human IgG1, IgG2, or IgG4). In one embodiment, the heavy chain constant region is human IgG1. In another embodiment, the anti-IGFBP3 antibody molecule comprises a light chain constant region selected from, for example, a kappa or lambda light chain constant region. In one embodiment, the constant region is altered (e.g., mutated) to modify the properties of the anti-IGFBP3 antibody molecule (e.g., to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, complement function, half-life, aggregation, and stability). In certain embodiments, the anti-IGFBP3 antibody molecule comprises a mutated human IgG4.

[0165] In one embodiment, the anti-IGFBP3 antibody molecule is isolated or recombinant.

[0166] In one embodiment, the anti-IGFBP3 antibody molecule is a humanized or human antibody molecule.

[0167] The present invention features nucleic acid molecules comprising one or two nucleotide sequences encoding the heavy and light chain variable regions, CDRs, hypervariable loops, and framework regions of the anti-IGFBP3 antibody molecules described herein. In certain embodiments, the nucleotide sequences encoding the anti-IGFBP3 antibody molecules are codon-optimized. For example, the present invention features first and second nucleic acids encoding the heavy and light chain variable regions, respectively, of the anti-IGFBP3 antibody molecules selected from, for example, any one or more of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 (including 3.1), or sequences substantially identical thereto. For example, the nucleic acid can comprise a nucleotide sequence as set forth in Tables 6-7, 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 by no more than 3, 6, 15, 30, or 45 nucleotides from a sequence shown in Tables 6-7).

[0168] In other embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable domain and / or a heavy chain constant region, wherein the heavy chain variable domain and / or the heavy chain constant region comprises the amino acid sequence of any one of E01, E02, E08, E14, E19, E20, E23, E24 or M1, as defined in Tables 2-5 (including 3.1); or the nucleotide sequence in Tables 6-7; or a sequence substantially identical to any of the above sequences (e.g., a sequence with at least about 85%, 90%, 95%, 99% or more identity).

[0169] In other embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain variable domain and / or a light chain constant region, and the heavy chain variable domain and / or heavy chain constant region comprises the amino acid sequence of any one of E01, E02, E08, E14, E19, E20, E23, E24 or M1, as defined in Tables 2-5 (including 3.1); or the nucleotide sequence in Tables 6-7; or a sequence substantially identical to any of the foregoing sequences (e.g., a sequence with at least about 85%, 90%, 95%, 99% or more identity).

[0170] The nucleotide sequences encoding the variable domains and constant regions of the heavy and light chains of anti-IGFBP3 may be present in separate nucleic acid molecules or in the same nucleic acid molecule. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a leader sequence.

[0171] 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 listed in Tables 2-5, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three or more substitutions, insertions or deletions, such as conservative substitutions).

[0172] 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 listed in Tables 6-7, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three or more substitutions, insertions or deletions, such as conservative substitutions).

[0173] In 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 the heavy and light chain variable regions having an amino acid sequence listed in Tables 2-5 (including 3.1), or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or having one, two, three or more substitutions, insertions or deletions, e.g., conservative substitutions).

[0174] In another embodiment, the nucleic acid molecule comprises one or more heavy chain framework regions (e.g., any of VHFW1(a-type), VHFW1(b-type), VHFW1(c-type), VHFW1(d-type), VHFW2(a-type), VHFW2(a′-type), VHFW2(b-type), VHFW2(c-type), VHFW2(d-type), VHFW2(e-type), VHFW3(a-type), VHFW3(b-type), VHFW3(c-type), VHFW3(d-type), VHFW3(e-type), or VHFW4, or any combination thereof, such as the framework combinations described herein) for any of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 (including 3.1), or a sequence substantially identical thereto. For example, the nucleic acid molecule may comprise a nucleotide sequence as described in Tables 2-5 (including 3.1), 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 by no more than 3, 6, 15, 30 or 45 nucleotides from a sequence shown in Tables 2-5 (including 3.1)).

[0175] In another embodiment, the nucleic acid molecule includes one or more light chain framework regions (e.g., any one of VLFW1(a-type), VLFW1(b-type), VLFW1(c-type), VLFW1(d-type), VLFW1(e-type), VLFW1(f-type), VLFW2(a-type), VLFW2(c-type), VLFW3(a-type), VLFW3(b-type), VLFW3(c-type), VLFW3(d-type), VLFW3(e-type), VLFW3(f-type), VLFW3(g-type), or VLFW4, or any combination thereof, such as the framework combinations described herein) for any one of E01, E02, E08, E14, E19, E20, E23, E24, or M1, as defined in Tables 2-5 (including 3.1), or a sequence substantially identical thereto. For example, the nucleic acid molecule can comprise a nucleotide sequence as set forth in Tables 6-7, 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 by no more than 3, 6, 15, 30, or 45 nucleotides from a sequence shown in Tables 6-7).

[0176] In another embodiment, the nucleic acid molecule comprises one or more heavy chain framework regions and one or more light chain framework regions as described herein.The heavy chain and light chain framework regions can be present in the same vector or in separate vectors.

[0177] On the other hand, the application is characterized in that host cells and vectors comprise nucleic acids described herein or modified for codon optimization according to known methods. The nucleic acid can be present in a single vector in the same host cell or non-same host cell or in separate multiple vectors. 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 Escherichia coli. For example, the mammalian cell can be a cultured cell or a cell line. Exemplary mammalian cells include lymphocyte lines (such as NSO), Chinese hamster ovary cells (CHO), COS cells, oocytes, and cells from transgenic animals, such as mammary epithelial cells.

[0178] In one aspect, the invention features a method of providing an antibody molecule described herein. The method includes providing an IGFBP3 antigen (e.g., an antigen comprising at least a portion of an IGFBP3 epitope); obtaining an antibody molecule that specifically binds to an IGFBP3 polypeptide; and evaluating whether the antibody molecule specifically binds to the IGFBP3 polypeptide, or evaluating the efficacy of the antibody molecule in modulating (e.g., inhibiting) the activity of IGFBP3. The method can further include administering the antibody molecule to a subject, e.g., a human or non-human animal.

[0179] 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-IGFBP3 antibody molecules described herein. In one embodiment, the composition, e.g., pharmaceutical composition, comprises a combination of an antibody molecule and one or more agents (e.g., therapeutic agents or other antibody molecules), as described herein. In one embodiment, the antibody molecule is conjugated to a label or therapeutic agent.

[0180] The anti-IGFBP3 antibody molecules disclosed herein can inhibit, reduce, or neutralize one or more activities of IGFBP3 as described above. Therefore, such antibody molecules can be used to treat or prevent conditions in which it is desirable to inhibit, reduce, or neutralize IGFBP3-induced activity in a subject.

[0181] Uses of anti-IGFBP3 antibody molecules

[0182] The antibodies of the invention are useful in methods of treating various diseases or conditions, such as diabetes, as well as intestinal diseases, malabsorption syndromes, inflammatory bowel disease, cachexia, Crohn's disease, ulcerative colitis, celiac disease, and diabetic bowel disease.

[0183] Thus, in another aspect, a method for modulating the IGFBP3 / TMEM219 axis in a subject is provided. The method comprises administering an anti-IGFBP3 antibody molecule disclosed herein (e.g., a therapeutically effective amount of an anti-IGFBP3 antibody molecule), alone or in combination with one or more agents or procedures, to a subject, such that the IGFBP3 / TMEM219 axis in the subject is modulated. In one embodiment, the antibody molecule inhibits, reduces, neutralizes, or blocks IGFBP3 / TMEM219 axis activity in the subject. The subject can be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject is in need of inhibition, reduction, neutralization, or blocking of the IGFBP3 / TMEM219 axis. In one embodiment, the subject has or is at risk of suffering from a disease described herein, such as diabetes or inflammatory bowel disease (IBD), malabsorption syndrome, irritable bowel syndrome, cachexia, celiac disease, diabetic bowel disease, as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0184] Figure 1IGFBP3-ecto-TMEM219 binding was tested using a competitive ELISA screening assay in the presence of the newly generated anti-IGFBP3 mAb alone (10 μg / mL) or ecto-TMEM219 (10 μg / mL). In this assay, microplates were coated with rhIGFBP3 and labeled ecto-TMEM219 was added. Monoclonal antibody M1 was added, and its ability to displace ecto-TMEM219 was assessed by measuring absorbance after washing the plates. The newly generated anti-IGFBP3 antibody M1 significantly reduced the ecto-TMEM219 signal (one-way ANOVA, ****p<0.0001).

[0185] Figure 2 Rescue effect of anti-IGFBP3 mAbs on human miniintestine growth after IGFBP3 exposure (50 ng / mL). Miniintestines were generated from crypts obtained from human healthy controls. Newly generated anti-IGFBP3 mAb M1 (10 μg / mL) and exo-TMEM219 (130 ng / mL) were tested on miniintestines treated with IGFBP3. Self-renewal properties were assessed by morphological evaluation. The development of large crypt organoids with at least one crypt domain was considered the primary criterion. The tested anti-IGFBP3 mAbs rescued miniintestine development. ****p<0.001 compared to hIGFBP3.

[0186] Figure 3 Newly generated anti-IGFBP3 mAbs reconstitute ISC (intestinal stem cell) marker expression in IGFBP3-treated miniguts. Normalized mRNA expression of the ISC markers EphB2 (A) and LGR5 (B) was analyzed by RT-PCR in miniguts co-cultured with IGFBP3 and selected anti-IGFBP3 mAbs / exo-TMEM219. *p < 0.05 compared to IGFBP3.

[0187] Figure 4 Newly generated anti-IGFBP3 mAbs downregulated caspase-8 expression in IGFBP3-treated miniguts. Normalized caspase-8 mRNA expression in miniguts incubated with IGFBP3 (50 ng / mL) and selected anti-IGFBP3 mAbs (10 μg / mL) was analyzed by RT-PCR. ****p<0.001 compared to IGFBP3.

[0188] Figure 5Rescue effect of anti-IGFBP3 mAbs on mini-intestine growth in IBD re-challenged with IGFBP3 (50 ng / mL). Mini-intestines were generated from crypts obtained from Crohn's disease (CD) patients and re-challenged with / without IGFBP3 (50 ng / mL) and newly generated anti-IGFBP3 mAb M1 (10 μg / mL) or exo-TMEM219 (130 ng / mL). Self-renewal properties were assessed by morphological evaluation. The development of large crypt organoids with at least one crypt domain was considered the primary criterion. The tested anti-IGFBP3 mAbs rescued mini-intestine development. *p<0.05, ***p<0.01 compared to hIGFBP3 or compared to CD.

[0189] Figure 6 Rescue effect of anti-IGFBP3 mAbs on murine miniintestine growth after IGFBP3 exposure (50 ng / mL). Miniintestines were generated from crypts obtained from control C57BL6 / J mice. Newly generated anti-IGFBP3 mAb M1 (10 μg / mL) and exo-TMEM219 (130 ng / mL) were tested on miniintestines treated with IGFBP3. Self-renewal properties were assessed by morphological evaluation. The development of large crypt organoids with at least one crypt domain was considered the primary criterion. The tested anti-IGFBP3 mAbs rescued miniintestine development. ****p<0.01 compared to mIGFBP3.

[0190] Figure 7 Newly generated anti-IGFBP3 mAbs downregulated caspase-8 expression in human β-cell lines treated with IGFBP3. Normalized caspase-8 mRNA expression in β-cells cultured with IGFBP3 (50 ng / mL) and selected anti-IGFBP3 mAbs (10 μg / mL) was analyzed by RT-PCR. **p<0.01 compared to IGFBP3.

[0191] Figure 8 Rescue effect of anti-IGFBP3 mAbs on human minigut growth after IGFBP3 exposure (50 ng / mL). Miniguts were generated from crypts obtained from human healthy controls. Newly generated anti-IGFBP3 mAbs (10 μg / mL) and exo-TMEM219 (130 ng / mL) were tested on miniguts treated with IGFBP3. Self-renewal properties were assessed by morphological evaluation. The development of large crypt organoids with at least one crypt domain was considered the primary criterion. The tested anti-IGFBP3 mAbs rescued minigut development. ***p < 0.01, ****p < 0.001 compared to hIGFBP3.

[0192] Figure 9 Newly generated anti-IGFBP3 mAbs reconstitute ISC (intestinal stem cell) marker expression in IGFBP3-treated miniguts. Normalized mRNA expression of the ISC markers EphB2 (A) and LGR5 (B) was analyzed by RT-PCR in miniguts co-cultured with IGFBP3 and selected anti-IGFBP3 mAbs / exo-TMEM219. *p < 0.05 compared to IGFBP3.

[0193] Figure 10 Newly generated anti-IGFBP3 mAbs downregulated caspase-8 expression in IGFBP3-treated miniguts. Normalized caspase-8 mRNA expression in miniguts incubated with IGFBP3 (50 ng / mL) and selected anti-IGFBP3 mAbs (10 μg / mL) was analyzed by RT-PCR. ****p<0.001 compared to IGFBP3.

[0194] Figure 11 Rescue effect of anti-IGFBP3 mAb on murine minigut growth after IGFBP3 exposure (50 ng / mL). Miniguts were generated from crypts obtained from control C57BL6 / J mice. Newly generated anti-IGFBP3 mAb (10 μg / mL) and exo-TMEM219 (130 ng / mL) were tested on miniguts treated with IGFBP3. Self-renewal properties were assessed by morphological evaluation. The development of large crypt organoids with at least one crypt domain was considered the primary criterion. The tested anti-IGFBP3 mAb rescued minigut development. **p<0.01, ***p<0.01 compared to mIGFBP3.

[0195] Figure 12 Newly generated anti-IGFBP3 mAbs downregulated caspase-8 expression in human β-cell lines exposed to pooled T1D serum. RT-PCR was used to analyze normalized caspase-8 mRNA expression in β-cells cultured with pooled T1D serum and selected anti-IGFBP3 mAbs (10 μg / mL). ***p < 0.001 compared to IGFBP3.

[0196] Figure 13 Experimental timeline

[0197] Figure 14 Effects of the newly generated anti-IGFBP3 mAb on the development of diabetes in a T1D mouse model. (A) Effect of anti-IGFBP3 mAb on preventing the onset of diabetes in NOD mice at 24 weeks of age and (B) effect on protecting blood glucose levels. Anti-IGFBP3 mAb prevented the development of diabetes in 80% of mice. Non-diabetic mice are defined as mice with normal blood glucose levels.

[0198] Diabetes was defined as the onset of diabetes when blood glucose was >250 mg / dl for three consecutive measurements. Non-diabetic mice did not have blood glucose >250 mg / dl for three consecutive measurements.

[0199] Figure 15 Serial paraffin sections of pancreatic tissue obtained at the time of euthanasia were prepared and stained with H&E, and islet morphology was analyzed microscopically. (Ai) shows representative images; original magnification 20X. (A-ii) shows representative images of insulin staining (brown); original magnification 20X. (B) shows pancreatitis scoring. In (B), the degree of cellular infiltration is scored from 0 to 4. Pancreatitis scoring was performed by examining at least 30 islets per animal. Detailed Description of the Invention

[0201] The antibodies of the present invention specifically bind to human IGFBP3. As discussed herein, the antibodies of the present invention are collectively referred to as "anti-IGFBP3 antibodies." All such antibodies are encompassed by the discussion herein. In the methods of the present invention, the respective antibodies can be used alone or in combination.

[0202] By "the antibody specifically binds" to IGFBP3 is meant that the antibody does not substantially cross-react with another, non-homologous, human polypeptide. By "does not substantially cross-react" is meant that the antibody or fragment has less than 10%, more preferably less than 5%, and even more preferably less than 1% of its binding affinity for IGFBP3 as compared to the binding affinity for the non-homologous protein.

[0203] In various embodiments, as used herein, an antibody that "specifically binds" IGFBP3 includes antibodies that bind to human IGFBP3 with a KD of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or about 0.5 nM, as measured using an Octet biolayer interferometer or surface plasmon resonance assay, e.g., using BIAcore TM System (Biacore Life Sciences, GE Healthcare, Piscataway, NJ) or kinetic size exclusion assay or any method known in the art.

[0204] As used herein, the term "antibody" is used in the broadest sense as understood in the art and includes all polypeptides described as antibodies in (25), which are incorporated herein by reference.

[0205] For example, the term "antibody" as used herein includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (such as bispecific antibodies), and antibody fragments, so long as the fragment exhibits the desired antigen-binding activity (antigen-binding fragment). The term has its broadest art-recognized meaning and includes all known forms, including but not limited to: bivalent monospecific monoclonal antibodies, bivalent bispecific antibodies, trivalent trispecific antibodies, F(ab) fragments, F(ab)'2 fragments, scFv fragments, diabodies, single domain antibodies, including camelid VHH single domain antibodies, Tandabs, and flexobodies.

[0206] As used herein, the term "antigen-binding fragment" of an antibody or equivalently "antigen-binding portion" of an antibody, etc., includes any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein, which includes a portion of an antibody and can specifically bind to an antigen to form a complex. Antigen-binding fragments of an antibody can be derived from intact antibody molecules using any suitable standard techniques, such as proteolysis or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable domains and (optionally) constant domains. Such DNA is known and / or easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0207] Like whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies generally include at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen.

[0208] In particular embodiments, the 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 can be found within an antigen-binding fragment of an antibody include: (i) VH-CH1; (ii) VH-CH2; (iii) 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 can be directly linked to each other, or can be linked by a full or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids in various embodiments, which form a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of the antibody may comprise, in various embodiments, homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, which are non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via one or more disulfide bonds).

[0209] The term "antigen-binding fragment" of an antibody further includes single domain antibodies.

[0210] Single-domain antibodies are antibody fragments composed of a single monomeric variable antibody domain. In some embodiments, single-domain antibodies are derived from the variable domain of the antibody heavy chain of camelids (also known as nanobodies, or VHH fragments). In some embodiments, single-domain antibodies are autonomous human heavy chain variable domains (aVH) or VNAR fragments from sharks.

[0211] 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 the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), such as a CDR3 peptide), or a restricted 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.

[0212] The antigen-binding fragment of an antibody will generally comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR adjacent to or within one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains can be arranged relative to each other in any suitable arrangement. For example, the variable domain can be a dimer, including a VH-VH, VH-VL, or VL-VL dimer. Alternatively, the antigen-binding fragment of an antibody can comprise a monomeric VH or VL domain.

[0213] The antibodies or binding molecules of the present invention may be further linked to an active substance, preferably a nanoparticle or a radionucleotide.

[0214] As used herein, the term "antigen binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are antibodies, including antigen binding fragments and scaffold antigen binding proteins.

[0215] The term "antigen binding portion" refers to the portion of an antigen binding molecule that specifically binds to an antigenic determinant. Antigen binding portions include antibodies and antigen-binding fragments thereof, such as scFv, that are capable of specifically binding to an antigen on a target cell. In certain aspects, an antigen binding portion is capable of directing an entity (e.g., a cell) to which it is attached to the target site.

[0216] In addition, antigen binding moieties capable of specific binding to a target cell antigen include scaffold antigen binding proteins as defined below, e.g., binding domains based on designed repeat proteins or designed repeat domains, such as designed ankyrin repeat proteins (DARPins) (see, e.g., WO 2002 / 020565) or lipocalins (Anticalins).

[0217] Designed ankyrin repeat proteins (DARPins) are derived from ankyrin, 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 the residues in each repeated β-turn and the first α-helix. Their binding contact surface can be increased by increasing the number of modules (affinity maturation method). For more details, see J.Mol.Biol.332, 489-503 (2003), PNAS 100 (4), 1700-1705 (2003) and J.Mol.Biol.369, 1015-1028 (2007) and US200401320028.

[0218] In certain embodiments, the antibodies and antigen binding molecules provided herein are changed to increase or decrease the degree of glycosylation of the antigen-binding moiety. One or more glycosylation sites can be created or removed by changing the amino acid sequence, and the glycosylation variants of the molecule can be easily obtained. When the antigen binding molecules include the Fc region, the carbohydrate attached thereto can be changed. On the one hand, there is provided a variant of the antigen binding molecules, whose carbohydrate structure lacks (directly or indirectly) fucose attached to the Fc region. Such fucosylated variants can have improved ADCC function, see, for example, U.S. Patent Publication No. US 2003 / 0157108 (Presta, L.) or US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Other variants of the antigen binding molecules of the present invention include those with bisected oligosaccharides (bisectedoligosaccharide) molecules, for example, wherein the biantennary oligosaccharide (biantennaryoligosaccharide) attached to the Fc region is bisected by GlcNAc. This variant can reduce fucosylation and / or improve ADCC function, see, for example, WO 2003 / 011878 (Jean-Mairet etc.); U.S. Patent number 6,602,684 (Umana etc.); and US2005 / 0123546 (Umana etc.). There is also provided a variant having at least one galactose residue attached to the Fc region in oligosaccharides. Such antibody variants can have improved CDC function and are described in, for example, WO 1997 / 30087 (Patel etc.), WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0219] In certain embodiments, it may be desirable to create a cysteine ​​engineered variant of an antibody or antigen-binding molecule of the invention, such as a "thioMAb," in which one or more residues of the molecule are replaced by cysteine ​​residues. In a specific embodiment, the substituted residues appear at accessible sites of the molecule. By replacing these residues with cysteine, the active sulfhydryl groups are thereby located at accessible sites of the antibody and can be used to couple the antibody to other moieties (e.g., a drug moiety or a linker-drug moiety) to produce an immunoconjugate. In certain embodiments, any one or more of the following residues can be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine-engineered antigen-binding molecules can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0220] In some aspects, the antibody or antigen-binding molecules provided herein can be further modified to include other non-protein moieties known in the art and easily available. Suitable for the part of the antibody or antigen-binding molecules being derivatized include but are not limited to water-soluble polymers. The limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamic acid (homopolymer or random copolymer), dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol and mixtures thereof. Due to the stability of polyethylene glycol propionaldehyde in water, there may be advantages in manufacturing. The polymer can have any molecular weight and can be branched or non-branched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on factors including, but not limited to, the specific properties or functions of the antibody to be modified, whether the antibody derivative is to be used therapeutically under a prescribed condition, and the like.

[0221] On the other hand, there is provided an antibody and a conjugate of a non-protein portion that can be selectively heated by exposure to radiation. In one embodiment, the non-protein portion is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, and includes but is not limited to not harming ordinary cells, but heating the non-protein portion to a wavelength at a temperature at which cells close to the non-protein portion of the antibody are killed. On the other hand, an immunoconjugate of an antigen binding molecule provided herein can be obtained. "Immunoconjugate" is an antibody coupled to one or more heterologous molecules (including but not limited to cytotoxic agents).

[0222] The antibody constant region is important for the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Therefore, the isotype of the antibody can be selected based on whether the antibody needs to mediate cytotoxicity. In certain embodiments, the constant region is an IgG1, IgG2, IgG3 or IgG4 constant region.

[0223] The present invention encompasses, in various embodiments, antibodies having one or more mutations in the hinge, CH2, or CH3 regions, which may be desirable, for example, in production, to improve the yield of a desired antibody form. In some embodiments, for example, the antibodies described herein comprise a human IgG4 constant region. In a specific embodiment, the IgG4 constant region comprises a single amino acid substitution in the hinge region of the human IgG4 hinge, which reduces Fab arm exchange (Angal et al. (1993) Molecular Immunology 30:105) to the levels typically observed using the human IgG1 hinge.

[0224] In certain embodiments, the antibody comprises one or more mutations in the constant region that increase serum half-life, including 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.

[0225] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, the human antibodies described herein 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), such as in CDRs and in some embodiments in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences.

[0226] As used herein, the term "recombinant human antibody" 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., mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295, incorporated herein by reference), or antibodies prepared, expressed, created, or isolated by any other method involving the cleavage of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally occur within the human antibody germline repertoire in vivo.

[0227] As used herein, an "isolated antibody" 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 a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody." In various embodiments, isolated antibodies also include antibodies in situ within recombinant cells. In other embodiments, an isolated antibody is an antibody that has been subjected to at least one purification or separation step. In various embodiments, an isolated antibody can be substantially free of other cellular material and / or chemicals.

[0228] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule, called a paratope. A single antigen may have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and may have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by the spatial juxtaposition of amino acids in different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a carbohydrate, phosphoryl, or sulfonyl moiety on the antigen.

[0229] The anti-IGFBP3 antibodies described herein for use in the methods described herein can, in various embodiments, include 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 compared to the corresponding germline sequences from which the antibody was derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences, for example, available from public antibody sequence databases.

[0230] The present invention, in various embodiments, includes antibodies and methods involving the use of antibodies, as well as antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to one or more corresponding residues in the germline sequence from which the antibody is derived, or one or more corresponding residues in another human germline sequence, or to a conservative amino acid substitution of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations").

[0231] A plurality of antibodies and antigen-binding fragments can be constructed that include one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found within the first 8 amino acids of FRI or the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). In addition, the antibody may comprise any combination of two or more germline mutations within the framework and / or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residues of a certain germline sequence, while certain other residues that are different from the original germline sequence are retained or mutated to the corresponding residues of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing 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 appropriate), reduced immunogenicity, etc. The use of antibodies and antigen-binding fragments obtained in this general manner is encompassed by the present invention.

[0232] The present invention also includes anti-IGFBP3 antibodies and methods involving the use of anti-IGFBP3 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having 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 having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0233] As used herein, the term "bioequivalence" refers to molecules that have similar bioavailability (rate and extent of utilization) after administration at the same molar dose and under similar conditions (e.g., same route of administration), such that they can be expected to be substantially the same as the comparable molecule in terms of efficacy and safety. Two pharmaceutical compositions comprising an anti-IGFBP3 antibody are bioequivalent if they are pharmacologically equivalent, i.e., they contain the same amount of active ingredient (e.g., IGFBP3 antibody), are in the same dosage form, are administered by the same route, and meet the same or comparable criteria. Bioequivalence can be determined by in vivo studies, for example, by comparing the pharmacokinetic parameters of the two compositions. Common parameters used in bioequivalence studies include peak plasma concentration (C 最大值) and the area under the plasma drug concentration-time curve (AUC).

[0234] In certain embodiments, the present invention relates to antibodies comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO: 36 and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 37 to SEQ ID NO: 45, and methods comprising administering to a subject an antibody. The present invention provides pharmaceutical compositions comprising such antibodies, as well as methods of using these compositions.

[0235] In various embodiments, the antibodies are administered to a subject in a formulation comprising suitable carriers, excipients, and other agents to provide improved transfer, delivery, tolerability, etc., and suitable for intravenous or subcutaneous injection.

[0236] Injectable preparations can be prepared by known quick methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or a conventional oily medium for injection. As an aqueous medium for injection, for example, normal saline, an isotonic solution containing glucose and other adjuvants, etc., can be used in combination with a suitable solubilizing agent, for example, alcohol (such as ethanol), polyols (such as propylene glycol, polyethylene glycol), nonionic surfactants [such as polysorbate 20 or 80, HCO-50 (polyoxyethylene (50mol) adducts of hydrogenated castor oil)] etc. As an oily medium, for example sesame oil, soybean oil etc. have been employed, which can be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol etc. The injectable preparation prepared in this way can be filled in a suitable ampoule.

[0237] The antibodies according to the present invention can be administered to a subject using any acceptable device or mechanism. For example, administration can be accomplished using a syringe and needle or using a reusable pen and / or automatic syringe delivery device. The methods of the present invention include administering the antibody (or a pharmaceutical formulation comprising the antibody) using a variety of reusable pens and / or automatic syringe delivery devices. Examples of such devices include, but are not limited to, AUTOPEN®. TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical systems, Burgdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM PEN (Eli Lilly and Company, Indianapolis, IL), NOVOPEN TMI, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPENJUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM PEN (BD Inc., Franklin Lakes, New Jersey), OPTIPEN TM , OPTIPEN PRO TM 、OPTIPEN STARLET TM , and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen and / or autoinjector delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (EliLilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier GmbH, Stuttgart, Germany), EPIPEN (Dey, LP), HUMIRA TM Pen (Abbott Laboratories, Abbott Park, IL), Auto-injector (SHL Group) and any device with PUSHCLICK TM technology auto-injectors (SHL Group), to name a few.

[0238] In one embodiment, the antibody is administered using a pre-filled syringe. In another embodiment, the antibody is administered using a pre-filled syringe that contains a safety system. For example, the safety system can prevent accidental needle stick injuries. In various embodiments, the antibody is administered using a pre-filled syringe that contains a safety system. Antibodies were administered using prefilled syringes from the Safeline® safety system (West Pharmaceutical Services Inc.) See also US Patent Nos. 5,215,534 and 9,248,242, which are incorporated herein by reference in their entireties.

[0239] In another embodiment, the antibody is administered using an automatic injector. TM The antibody is administered using an automatic injector using a device comprising a syringe that allows a dose of a composition and / or antibody to be administered to a subject. See also U.S. Patent Nos. 9,427,531 and 9,566,395, which are incorporated herein by reference in their entirety.

[0240] According to the present invention, a "subject" refers to a human subject or a human patient. Example

[0241] method

[0242] Patients and study design

[0243] Healthy control subjects were individuals without a diagnosis of inflammatory bowel disease (IBD) (CTRL) who were recruited from patients undergoing colonoscopy or intestinal surgery due to diverticular disease, colon cancer, or irritable bowel syndrome.

[0244] CD patients with a long history of Crohn's disease were recruited during surgery due to disease complications (stenosis, fistula) or undergoing preoperative endoscopy. All subjects signed an informed consent before participating in the study.

[0245] Animal studies

[0246] C57BL / 6J (B6) mice were obtained from Charles River Italy Laboratories (Calico, Italy) and their care and use were in compliance with Italian Law No. 116 / 1992 on animal care and European Communities Council Directive EEC / 609 / 86.

[0247] Recombinant proteins and interventional studies

[0248] Recombinant human IGFBP3 was obtained from Life Technologies (IGFBP3, Life Technologies, 10430H07H5). Ecto-TMEM219, the extracellular domain of the TMEM219 receptor, was used as a positive control. In relevant disease models, ecto-TMEM219 has been shown to successfully prevent IGFBP3-mediated damage in vitro and in vivo. See WO 2016 / 193496 and WO 2016 / 193497. Ecto-TMEM was obtained through Genescript Custom Protein Services. The protein (produced in E. coli) has the following amino acid sequence:

[0249] Human exo-TMEM amino acid sequence:

[0250]

[0251] Murine extracellular-TMEM amino acid sequence:

[0252]

[0253] 50 ng / ml of IGFBP3 and 130 ng / ml of exo-TMEM219 were added to the culture medium on day +1 of minigut culture (see below).

[0254] Newly generated anti-IGFBP3 monoclonal antibodies were added at a molecular ratio of 1:1 compared to a final concentration of 10 μg / ml IGFBP3.

[0255] Crypt isolation and mini-intestine development

[0256] people

[0257] Crypts were extracted from samples of intestinal mucosa and submucosa from healthy subjects (healthy controls) or from patients with established Crohn's disease who underwent surgery for complications of the disease (strictures, fistulas). The mucosa was incubated with a cocktail of antibiotics (Normocin [Invivogen, San Diego, CA 92121, USA; Catalog No. ant-nr], Gentamycin [Invivogen, Carlsbad, CA, USA; Catalog No. ant-gn], and Fungizone [Invitrogen 15290018]) at room temperature for 15 minutes. The tissue was then cut into small pieces and incubated two to three times with 10 mM dithiothreitol (DTT) (Sigma) in PBS for several minutes. The samples were then transferred to 8 mM EDTA in PBS and incubated at 37°C for 30 minutes. Following this step, the samples were shaken vigorously to produce a supernatant enriched in colonic crypts. Fetal bovine serum (FBS, Sigma 12103C-500ML) was added to a final concentration of 5%, and single cells were removed by centrifugation at 40×g for 2 minutes. The crypts were mixed with 50 μl of Matrigel (BD Biosciences 354234) and plated on a pre-warmed culture dish. After solidification, crypts were covered with complete crypt medium: Wnt3a-conditioned medium and advanced DMEM / F12 (Life Technologies 1263010) 50:50, supplemented with Glutamax 10 mM (Life Technologies 35050038), HEPES (Life Technologies 15630080), N-2 [1×] (Life Technologies 17502048), retinoic acid-free B-27 [1×] (Life Technologies 12587010), 10 mM nicotinamide (Sigma N0636), 1 mM N-acetyl-L-cysteine ​​(Sigma A965), 50 ng / ml human EGF (Life Technologies PHG0311), 1 μg / ml RSPO1 (Sino Biological). Biological) 11083-H08H), 100 ng / ml human Noggin (Peprotech 12010C), 1 μg / ml Gastrin (Sigma-Aldrich SCP0152), 500 nM LY2157299 (AxonMedChem 1491), 10 μM SB202190 (Sigma S7067) and 0.01 μM PGE2 (Sigma P6532).

[0258] The culture medium was changed every 3 days. Purified crypts were cultured for 8 days with or without recombinant proteins / antibodies as described in the Recombinant Protein and Interventional Studies section. After 8 days, crypts were collected and morphology, mini-intestine growth, expression of intestinal markers (EphB2, LGR5, h-TERT), and caspase 8 (Life Technologies) were examined using RT-PCR. The percentage of mini-intestines with at least one crypt domain was assessed as described (4, 18).

[0259] mouse

[0260] Crypts were obtained from C57BL / 6J mice. Briefly, the colon was cut into 2-4 mm pieces with scissors, the fragments were washed with 30 ml of ice-cold PBS, and then incubated in 20 mM EDTA-PBS at 37°C. Finally, the fragments were treated with a trypsin / DNase solution to obtain crypts. Following this step, the sample was vigorously shaken to produce a supernatant enriched in colonic crypts. The crypts were mixed with Matrigel and plated on prewarmed culture dishes. After Matrigel solidification (37°C, 10-15 min), crypts were covered with culture medium: (ADF, 10 mM HEPES, N-2, B27 without retinoic acid, 10 μM Y-27632, 1 μM JAG1 peptide (Anaspec, Fremont, CA, USA), 1 μg / ml R-Spondin 1, 50 ng / ml EGF (Invitrogen), and 100 ng / ml Noggin (Peprotech, Rocky Mount, NJ, USA), and the culture medium was changed every other day until day 8. After 8 days, the percentage of developed mini-guts was assessed.

[0261] qRT-PCR analysis

[0262] RNA from purified intestinal crypts was extracted using Trizol reagent (Invitrogen), and qRT-PCR analysis was performed using TaqMan assays (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. Normalized expression values ​​were determined using the ΔΔCt or ΔCt method. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data were normalized using the expression of ACTB. Statistical analysis of comparative gene expression across all cell populations for each patient was performed by one-way ANOVA, followed by a Bonferroni post hoc test for multiple comparisons between the population of interest and all other populations. Analyses were performed in technical and biological triplicates.

[0263] The following is a list of genes whose expression has been quantified by qRT-PCR.

[0264]

[0265]

[0266] Competitive ELISA binding assay

[0267] The following reagents were used to screen newly generated anti-IGFBP3 antibodies: recombinant human IGFBP3 (0.223 mg / ml R&D System 8874-B3-025), exo-TMEM219 (0.5 mg / ml GenScript), newly generated anti-IGFBP3 mAb (Trianni), anti-human IgG HRP (Life Technologies A24470), bovine serum albumin (BSA), Tween 20 (Tween 20), ELISA colorimetric TMB reagent (HRP substrate, Item H Sigma, RABTMB3), ELISA STOP solution (Item I Sigma, RABSTOP3). We also used blocking reagent solution (3% BSA in PBS) and dilution solution (0.5% BSA, 0.05% Tween 20 in PBS).

[0268] Microplates (Thermo Fisher Scientific, Thermo Electron Corporation, 2801) were coated with 50 μl / well of 4 μg / ml rhIGFBP3 dissolved in PBS or PBS alone (no coating). The plates were incubated at 37°C for 90 minutes, washed with PBS (300 μl / well), and incubated with blocking reagent (200 μl / well) for 2 hours at room temperature. Samples were then diluted in dilution solution (50 μl / well) and added to the plates as follows: anti-IGFBP3 mAb 10 μg / ml alone, dilution solution (none), exo-TMEM219 10 μg / ml, exo-TMEM219 10 μg / ml + anti-IGFBP3 mAb 10 μg / ml. After a wash step, the plates were then incubated with anti-6X His-tag HRP (50 μl / well) diluted 1:2000 in dilution solution for 1 hour at room temperature. After adding the colorimetric solution to the ELISA plate reader, read the ELISA plate and measure the absorbance.

[0269] β-cells

[0270] Betalox-5 cells, a human β-cell line (36), were grown in culture flasks containing DMEM (glucose 1 g / L), BSA fraction V (0.02% weight / volume), nonessential amino acids (1X), penicillin (100 units / mL), and streptomycin (100 μg / mL). Cells were cultured in a humidified incubator at 37°C in a 5% CO2 atmosphere. Cells were passaged every second week.

[0271] Beta cells were cultured with or without IGFBP3, exo-TMEM219, or a newly generated monoclonal antibody (see Recombinant Proteins and Interventional Studies) and harvested for immunofluorescence studies, RNA extraction, apoptosis detection, and protein analysis. Supernatants were collected for insulin assessment. Insulin levels were measured using a microparticle enzyme immunoassay (Mercodia Iso-insulin ELISA, 10-1113-01).

[0272] Statistical analysis

[0273] Data are presented as mean and standard error of the mean (SEM) and were tested for normal distribution using the Kolmogorov-Smimov test and for homoscedasticity using the Levene's test. Statistically significant differences were tested using a two-tailed t-test. Significance between the two groups was determined by a two-sided unpaired Student's t-test. For multiple comparisons, a Bonferroni-corrected ANOVA test was used. GraphPad Prism version 6.0 (GraphPad Software, La Jolla, California) was used to generate charts and data. All statistical tests were performed at the 5% significance level.

[0274] Efficacy of anti-IGFBP3 mAb in T1D mouse model after intraperitoneal (IP) administration

[0275] animal

[0276] Female non-obese diabetic (NOD) mice (10 weeks old) were obtained from Charles River Laboratories, Calco, Varese, Italy (stock number 613). All mice were cared for and used in accordance with Italian law No. 116 / 1992 on animal care and European Community Council Directive EEC / 609 / 86.

[0277] Diabetes monitoring and treatment

[0278] Overt diabetes (the most advanced stage, characterized by elevated fasting blood glucose concentrations and typical symptoms) is defined as three consecutive blood glucose levels above 250 mg / dL. Monitor blood glucose twice a week.

[0279] The inventors set up the following treatment groups:

[0280] 1) Unprocessed

[0281] 2) External-TMEM219 0.1 mg / day (ip) for 10 days

[0282] 3) Anti-IGFBP3 M1 0.5 mg / day (ip) for 10 days

[0283] Exo-TMEM and antibodies were dissolved in PBS.

[0284] Each treatment group included N = 10 mice. Treatment began on day 0 when mice were 10 weeks old. Mice were followed up until 23 weeks of age. Mice were harvested when diabetes was assessed or at week 23. Plasma samples and pancreas were collected for ex vivo analysis. The experimental timeline is described in Figure 13 .

[0285] Pancreatitis score and islet histopathological examination

[0286] Pancreatitis scoring was performed on 5-μm-thick formalin-fixed, paraffin-embedded, hematoxylin and eosin (H&E)-stained pancreatic sections as previously described (Vergani A et al. Diabetes 2010; Ben Nasr M et al. Sci Transl Med 2017). Pancreatitis scoring was performed on H&E- and insulin-stained pancreatic sections. An experienced pathologist assigned a score of 0 to 4 based on islet infiltration. Pancreatitis scores were graded as follows: grade 0, normal islets; grade 1, mild mononuclear infiltration (25%) at the periphery; grade 2, infiltration of 25-50% of islets; grade 3, infiltration of 50% of islets; and grade 4, complete infiltration of islets with no remaining parenchymal remnants. At least 30 islets per group were analyzed and pooled from sections obtained from different mice.

[0287] Statistical analysis

[0288] Unless otherwise reported, data are presented as mean and standard error of the mean (SEM). The incidence of diabetes among different groups was analyzed using the log-rank (Mantel-Cox) test. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA). All statistical tests were performed at the 5% significance level.

[0289] Example 1: Monoclonal Antibody Development

[0290] Monoclonal anti-IGFBP3 antibodies were discovered using transgenic mice in which the relevant human immunoglobulin sequences had been genetically engineered (Trianni Mouse TM (Trianni)) were introduced into the animal genome.

[0291] By this technique, chimeric monoclonal antibodies are produced that contain a repertoire of fully human heavy and light chain variable domains while retaining the mouse constant domains.

[0292] Basically, two cohorts of Trianni Mouse TM Immunizations were performed with a purified preparation of the IGFBP3 antigen (lot AB08BP1210) (cohort 1: ALD / MDP adjuvant, cohort 2: SAS / Ribi adjuvant) twice weekly for 4 weeks, followed by a 2-week extension. Antibody-expressing lymphocytes (e.g., B cells) were then recovered from the mice and fused with myeloid cells to generate immortalized hybridoma cell lines. These hybridoma cell lines were screened and selected to identify hybridoma cell lines that produce antibodies specific for human IGFBP3 (lot AB08BP1210) using ELISA. Hybridoma cell lines reactive to the antigen of interest were expanded. Sequencing was performed by RNA isolation, followed by Sanger sequencing of human VH and VK cDNA.

[0293] Antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding antibodies can be used to transform appropriate mammalian host cells. In fact, monoclonal antibody M1 derived from cohort 1 was expressed in a transient gene expression system in mammalian cells.

[0294] Methods for expressing recombinant proteins in CHO cells

[0295] The corresponding M1 cDNA was cloned into the evitria vector system using conventional (non-PCR-based) cloning techniques to generate a complete human IgG4 mAb. The evitria vector plasmids were gene synthesized. Plasmid DNA was prepared using anion exchange chromatography under low-endotoxin conditions. Sequence accuracy was verified by Sanger sequencing (up to two sequencing reactions per plasmid, depending on the size of the cDNA).

[0296] Suspension-adapted CHO K1 cells (evitria) were used for production. The inoculum was grown in eviGrow medium, a chemically defined, animal component-free, serum-free medium. Cells were transfected with eviFect, a proprietary transfection reagent custom-made by evitria, and after transfection, cells were grown in eviMake, an animal component-free, serum-free medium, at 37°C in 5% CO2 for 7 days. The supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter).

[0297] Using MabSelect TM SuRe TMThe antibodies were purified using Dulbecco's PBS (Lonza BE17-512Q) as wash buffer and 0.1 M glycine pH 3.5 as elution buffer, followed by size exclusion chromatography on a HiLoad Superdex 200 pg column using the final buffer as running buffer.

[0298] Monomericity was determined by analytical size exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7 μm 7.8 x 300 mm) and DPBS as running buffer at 0.8 ml / min. Notably, the monomericity of M1 was >95% and only exhibited <5% aggregation. The high monomericity of a protein is an excellent property that should facilitate its manufacture.

[0299] Affinity measurement

[0300] Octet BLI-based analysis

[0301] The antibodies possess high affinity for their targets. Binding affinity measurements were performed using the Octet instrument (Octet BMIA), a biolayer interferometry (BLI) platform for biomolecular interaction analysis. To set up the assay, target monoclonal antibodies (30 μg / ml in PBS) were immobilized via the Fc region on anti-mouse IgG Fc capture (AMC) or anti-human IgG Fc capture (AMC) biosensors, and their interactions with the antigen, human IGFBP3 (R&D, catalog number 675B3), were measured at 150 nM.

[0302] Affinity measurements of anti-IGFBP3 mAbs for target human IGFBP3 are reported in Table 1 .

[0303]

[0304] Tables 2-5 below report the sequences of nine novel anti-IGFBP3 antibodies.

[0305]

[0306]

[0307]

[0308]

[0309] CDR definitions were provided using the annotation tool from http: / / www.abysis.org / , based on the complete VH and VL amino acid sequences defined in Tables 4 and 5.

[0310] For example, the VH amino acid sequence of any antibody disclosed herein is inserted into the annotation tool and provides the CDR sequences defined by Kabat, or IMGT, or Chothia, or AbM, or Contact. Using the "all in parallel" feature, the defined CDR sequences are provided. The following examples are based on SEQ ID Nos. 36 and 45.

[0311] Table 3.1: All, side by side, defined VH CDR sequences (SEQ ID No. 36) and VL CDR sequences (SEQ ID No. 45):

[0312] Region Definition - All Parallel

[0313]

[0314]

[0315] All area definitions are parallel

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334] Example 2:

[0335] Inhibition of IGFBP3-TMEM219 binding by hybridoma-produced anti-IGFBP3 mAb

[0336] A competitive ELISA binding assay was used to screen novel anti-IGFBP3 monoclonal antibodies produced by hybridomas for their ability to compete with exo-TMEM219 for the interaction with IGFBP3. IGFBP3, exo-TMEM219, and available antibodies were all used at a 1:1 ratio. Anti-IGFBP3 mAbs were able to inhibit the IGFBP3-exo-TMEM219 ( Figure 1 ). This demonstrates that the anti-IGFBP3 mAb of the present invention can inhibit the binding of IGFBP3 to the native TMEM219 receptor and may mimic the neutralizing activity of exo-TMEM219 protein.

[0337] Newly generated monoclonal anti-IGFBP3 antibodies rescue IGFBP3-induced damage in human mini-intestine assays

[0338] This newly discovered monoclonal antibody was also tested in a mini-gut assay. Briefly, mini-guts were generated from crypts obtained from healthy controls (n=3), cultured for 8 days in the presence of IGFBP3, and treated with exo-TMEM219 or the newly generated anti-IGFBP3 mAb at a 1:1 ratio (mAb / exo-TMEM219:IGFBP3).

[0339] We observed that anti-IGFBP3 mAb was comparable to exo-TMEM219 in rescuing the negative effects of IGFBP3 on the self-renewal capacity (% development) and morphology (lack of crypt domains, production of small spheroids) of large crypt organoids. Figure 2 This demonstrates the ability of the anti-IGFBP3 mAb of the present invention to rescue mini-intestine growth under intestinal stem cell (ISC) damage disease conditions by preventing IGFBP3 from binding to TMEM219 and mimicking exo-TMEM219.

[0340] Newly generated monoclonal anti-IGFBP3 antibodies rescue IGFBP3-induced damage in ISC markers

[0341] A newly generated anti-IGFBP3 mAb was shown to effectively promote mini-intestine development after IGFBP3 exposure and also restore the expression of ISC markers EphB2 and LGR5 ( Figure 3The deleterious effects of IGFBP3 on ISCs are mediated by caspase-8. Anti-IGFBP3 mAb was able to inhibit the upregulation of caspase-8 induced by IGFBP3 treatment, further suggesting that it exerts a protective effect on the ISC pool by blocking IGFBP3 / TMEM219 caspase-8-mediated apoptotic damage ( Figure 4 ).

[0342] Newly discovered anti-IGFBP3 antibodies rescue mini-intestine growth in disease models.

[0343] To confirm that the newly discovered anti-IGFBP3 monoclonal antibodies could prevent the deleterious effects of IGFBP3 on TMEM219-expressing intestinal stem cells, the inventors further tested them in vitro in mini-guts obtained from IBD patients.

[0344] The novel anti-IGFBP3 mAb significantly improved the development of minibowels from IBD patients by at least 20%, similar to exo-TMEM219 treatment ( Figure 5 ).

[0345] This highlights that the anti-IGFBP3 mAbs of the invention, selected for their ability to competitively inhibit exo-TMEM binding to IGFBP3, are able to rescue ISC function and protect ISC sets from the deleterious effects of IGFBP3.

[0346] Newly generated monoclonal anti-IGFBP3 antibodies rescue IGFBP3-induced damage in mouse mini-gut

[0347] Crypt isolation and murine mini-intestine development

[0348] To confirm that the antibodies of the invention have a similar tissue cross-reactivity profile in murine tissues relative to human tissues, the inventors further tested the monoclonal anti-IGFBP3 antibodies of the invention in an in vitro mini-intestine assay in murine crypts. Crypts were obtained from control mice (n=3) (632C57BL / 6J Charles River Laboratories, Lyon, France).

[0349] Isolated crypts were cultured for 8 days in the presence or absence of exo-TMEM219 in the presence of IGFBP3 to generate large crypt organoids, or mini-guts. Freshly generated anti-IGFBP3 mAb was added at a 1:1 ratio (mAb / exo-TMEM219:IGFBP3) on day 0. Mini-gut development was calculated as the percentage of organoid growth compared to plated isolated crypts after 8 days (D'Addio F et al., Cell Stem Cell 2015 Oct 1;17(4):486-498).

[0350] like Figure 6As shown, anti-IGFBP3 mAb rescued the negative effects of IGFBP3 on the self-renewal capacity (% development) and morphology of macrocrypt organoids (lack of crypt domains, generation of small spheroids) of murine minigut, similar to what was observed with exo-TMEM219.

[0351] Newly generated monoclonal anti-IGFBP3 antibodies inhibit IGFBP3-mediated caspase-8 pro-expression in human β cells Overexpression

[0352] The deleterious effects of IGFBP3 on human β cells are mediated by caspase-8. Interestingly, the newly discovered anti-IGFBP3 mAb was able to inhibit the upregulation of caspase-8 induced by IGFBP3 treatment by at least 50% compared with samples treated with IGFBP3 alone ( Figure 7 ).

[0353] These results suggest that the discovered anti-IGFBP3 mAb exerts a protective effect on human β cells by blocking IGFBP3 / TMEM219 caspase-8-mediated apoptotic injury.

[0354] Example 3:

[0355] Newly generated monoclonal anti-IGFBP3 antibodies rescue IGFBP3-induced damage in human mini-intestine assays

[0356] Anti-IGFBP3 monoclonal antibodies were tested in a mini-intestine assay. Briefly, mini-intestines were generated from crypts obtained from healthy controls (n=3), cultured for 8 days after IGFBP3 exposure, and treated with anti-IGFBP3 mAb at a 1:1 ratio (mAb:IGFBP3). The inventors observed that among the eight mAbs, E08 and E20 were comparable to exo-TMEM219 in rescuing the self-renewal capacity of large crypt organoids in the presence of IGFBP3, thus supporting a relevant effect on local stem cells to prevent IGFBP3-mediated damage. Figure 8 ).

[0357] Newly generated monoclonal anti-IGFBP3 antibodies rescue IGFBP3-induced damage in ISC markers

[0358] Anti-IGFBP3 mAb, which was shown to effectively promote mini-intestine development, was also able to restore the expression of ISC markers EphB2 and LGR5 ( Figure 9 This effect was caspase 8-mediated, as caspase 8 expression was downregulated after exposure to E08 and E20, further supporting that these anti-IGFBP3 mAbs exerted a protective effect on ISC assembly by blocking IGFBP3 / TMEM219 caspase-8-mediated apoptotic damage ( Figure 10 ).

[0359] Newly generated monoclonal anti-IGFBP3 antibodies rescue IGFBP3-induced damage in mouse mini-gut

[0360] Crypt isolation and murine mini-intestine development

[0361] To confirm that the antibodies of the invention have a similar tissue cross-reactivity profile in murine tissues relative to human tissues, the inventors further tested the monoclonal anti-IGFBP3 antibodies of the invention in an in vitro mini-intestine assay in murine crypts. Crypts were obtained from control mice (n=3) (632C57BL / 6J Charles River Laboratories, Lyon, France).

[0362] Isolated crypts were cultured for 8 days in the presence or absence of exo-TMEM219 in the presence of IGFBP3 to generate large crypt organoids, or mini-guts. Freshly generated anti-IGFBP3 mAb was added at a 1:1 ratio (mAb / exo-TMEM219:IGFBP3) on day 0. Mini-gut development was calculated as the percentage of organoid growth compared to plated isolated crypts after 8 days (D'Addio F et al., Cell Stem Cell 2015 Oct 1;17(4):486-498).

[0363] like Figure 11 As shown, antibody E08 rescues mini-gut growth in the presence of IGFBP3 and is a relevant candidate for further testing.

[0364] Anti-IGFBP3 mAb protects β-cell lines from apoptosis in vitro

[0365] To confirm that anti-IGFBP3 mAbs prevented the pro-apoptotic effects of IGFBP3 on TMEM219-expressing cells in the pancreas, the inventors further tested them in vitro in the human β-cell line Betalox-5. Exposure of β-cells to pooled T1D serum increased CASP8 expression, and anti-IGFBP3 mAb E08 was able to counteract this effect, supporting a beneficial role for these newly generated monoclonal anti-TMEM219 antibodies in preventing pancreatic β-cell apoptosis. Figure 12 ).

[0366] Example 3: T1D mouse model

[0367] like Figure 14 As shown, the inventors evaluated whether 10 days of administration of a newly generated anti-IGFBP3 mAb could prevent the onset of clinical diabetes in NOD mice, a mouse model of choice for studying autoimmune type 1 diabetes (T1D). Intraperitoneal administration of the anti-IGFBP3 mAb maintained long-term control of blood glucose levels and delayed the onset of diabetes in the NOD mouse model of T1D, with 80% of treated mice free of diabetes at week 24 (compared to 50% of untreated controls).

[0368] Next, we analyzed 24-week-old pancreatic tissue sections from untreated, M1S, and exo-TMEM-treated NOD mice and demonstrated a decrease in islet infiltration and a slightly increased detection of insulin-positive cells compared to untreated controls ( Figure 15 ).

[0369] Incorporation by Reference

[0370] All publications, patents, patent applications, and / or other references cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, patent application, and / or other reference cited in this application were individually indicated to be incorporated by reference for all purposes.

[0371] Equivalent form

[0372] Although various specific embodiments have been shown and described, the above description is not intended to be limiting. It should be understood that various modifications may be made without departing from the spirit and scope of the present invention. After reading this description, many changes will become apparent to those skilled in the art.

[0373] References

[0374] 1. Baxter RC. J Cell Commun Signal. 2013;7(3):179-89.

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[0377] 4. D'Addio F, et al. Cell stem cell. 2015; 17(4): 486-98.

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[0380] 7. Ben Nasr M, et al., Pharmacological res.: the official journal of the Italian Pharmacological Society. 2015;98:31-8.

[0381] 8. Keenan HA, et al. Diabetes. 2010;59(11):2846-53.

[0382] 9. Meier JJ, et al. Diabetologia. 2005;48(11):2221-8.

[0383] 10. Atkinson MA, et al. Diabetes care. 2015;38(6):979-88.

[0384] 11. Nguyen KH, et al., Endocrinology. 2011; 152(6): 2184-96.

[0385] 12. Yakar S, et al. FASEB J. 2009;23(3):709-19.

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[0388] 15. Kaplan GG. Nat Rev Gastroenterol Hepatol. 2015; 12(12): 720-7.

[0389] 16. Cui S, and Chang PY. World J Gastroenterol. 2016;22(31):7099-110.

[0390] 17. Yancu D, et al., J Gastroenterol Hepatol. 2017; 32(1): 146-53.

[0391] 18. Jung P, et al., Nature Medecine 2011;17:1225-1227.

[0392] 20. George MJ, et al., Curr Diab Rep 2013;13(1):72-80.

[0393] 21. Marsha JD. Am Health Drug Benefits 2011;4(5):312-322.

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[0395] 23. Pithadia AB, Sunita J. Pharmacoligal rep 2011;63:629-642.

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[0397] 25.Sumit G, Wei W, Tsutomu and Satoshi O, Antibodies 2013;2:452-500.

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Sequence Listing <110> Enthera Srl <120> IGFBP3 antibodies and their therapeutic uses <130> PCT 145204 <150> EP19210646.6 <151> 2019-11-21 <150> EP20167464.5 <151> 2020-04-01 <160> 110 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 1 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 2 Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 <210> 3 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 3 Ala Arg Gly Gly Glu Tyr Phe Tyr Tyr Tyr Gly Leu Asp Val 1 5 10 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 4 Gly Tyr Thr Phe Ser Asn Tyr Gly 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 5 Ile Asn Thr Tyr Asn Gly Asn Thr 1 5 <210> 6 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 6 Ala Arg Asp Arg Gly Tyr Ser Ser Ser Pro Tyr Tyr Tyr Tyr Tyr Gly 1 5 10 15 Met Asp Val <210> 7 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 7 Gly Tyr Thr Phe Thr Asn Tyr Gly 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 8 Ile Asn Ala Tyr Asn Gly Asn Thr 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 9 Gly Gly Ser Ile Ser Thr Tyr Tyr 1 5 <210> 10 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 10 Ile Tyr Tyr Ser Gly Ser Thr 1 5 <210> 11 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 11 Ala Arg Tyr Asp Ile Val Thr Gly Tyr Pro His Tyr Tyr Tyr Tyr Val 1 5 10 15 Met Asp Val <210> 12 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 12 Gln Ser Val Ser Ser Ser Ser 1 5 <210> 13 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 13 Gly Ala Ser 1 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 14 Gln Gln Asp Tyr Asn Leu Pro Leu Thr 1 5 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 15 Gln Ser Val Ser Ser Ser His 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 16 Gln Gln Asp Tyr Asn Leu Thr Ile Thr 1 5 <210> 17 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 17 Gln Gly Ile Ser Asn Tyr 1 5 <210> 18 <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 18 Ala Ala Ser 1 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 19 Gln Gln Tyr Asn Ser Tyr Pro Phe Thr 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 20 Gln Gly Ile Ser Ser Ala 1 5 <210> twenty one <211> 3 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty one Asp Ala Ser 1 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty two Gln Gln Phe Asn Asn Tyr Pro Ser Thr 1 5 <210> twenty three <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty three Gln Gly Ile Arg Asn Asp 1 5 <210> twenty four <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty four Leu Gln His Asn Ser Tyr Pro Tyr Thr 1 5 <210> 25 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 25 Gln Gly Ile Arg Asn Ala 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 26 Leu Gln Asp Tyr Asn Tyr Pro Leu Thr 1 5 <210> 27 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 27 Arg Gly Ile Arg Asn Ala 1 5 <210> 28 <211> 121 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 28 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Asn Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Glu Tyr Phe Tyr Tyr Tyr Gly Leu Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 29 <211> 126 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 29 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Ala Leu Arg Gly Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Tyr Ser Ser Ser Pro Tyr Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 30 <211> 126 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Ala Leu Arg Gly Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Tyr Ser Ser Ser Pro Tyr Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 31 <211> 126 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 31 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Ala Leu Arg Gly Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Tyr Ser Ser Ser Pro Tyr Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 32 <211> 121 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 32 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Asn Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Glu Tyr Phe Tyr Tyr Tyr Gly Leu Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 33 <211> 126 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 33 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Ala Leu Arg Gly Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Tyr Ser Ser Ser Pro Tyr Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 34 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 34 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Asn Tyr Val Val Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Glu Tyr Phe Tyr Tyr Tyr Gly Leu Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 35 <211> 126 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Val Thr Tyr Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Tyr Ser Ser Ser Pro Tyr Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 36 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Asp Ile Val Thr Gly Tyr Pro His Tyr Tyr Tyr Tyr Tyr Val Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 37 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 37 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Ser Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Leu Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 38 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 38 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 His Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Leu Thr 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 39 <211> 108 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 39 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Ser Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Leu Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 40 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 41 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 41 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ile Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Asn Tyr Pro Ser 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 42 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 43 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 43 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 44 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 44 Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Ala 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 45 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 45 Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Gly Ile Arg Asn Ala 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 46 <211> 363 <212> DNA <213> Artificial Sequence <220>[[ID=2​​​​​​​​​​​​​​​​​​​​​​<212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 47 caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta caccttttcc aattatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcaacactt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatg accactgaca catccacgag cacagcctac 240 atggcgctga ggggcctgag atctgacgac acggccgtgt attattgtgc gagagatagg 300 gggtatagca gcagccctta ctactactac tacggaatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctca 378 <210> 48 <211> 378 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 48 caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta caccttttcc aattatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcaacactt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatg accactgaca catccacgag cacagcctac 240 atggcgctga ggggcctgag atctgacgac acggccgtgt attattgtgc gagagatagg 300 gggtatagca gcagccctta ctactactac tacggaatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctca 378 <210> 49 <211> 378 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 49 caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta caccttttcc aattatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcaacactt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatg accactgaca catccacgag cacagcctac 240 atggcgctga ggggcctgag atctgacgac acggccgtgt attattgtgc gagagatagg 300 gggtatagca gcagccctta ctactactac tacggaatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctca 378 <210> 50 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 50 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaaaactat 180 gtagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagaggaggg 300 gagtacttct actattacgg tttggacgtc tggggccaag ggaccacggt caccgtctcc 360 tca 363 <210> 51 <211> 378 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 51 caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta caccttttcc aattatggta tcagctgggt gcgacaggcc 120 tcctgcaagg cttctggtta caccttttcc aattatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcaacactt acaatggtaa cacaaactat 180 cctggacaag ggcttgagtg gatgggatgg atcaacactt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatg accactgaca catccacgag cacagcctac 240 gcacagaagc tccagggcag agtcaccatg accactgaca catccacgag cacagcctac 240 atggcgctga ggggcctgag atctgacgac acggccgtgt attattgtgc gagagatagg 300 atggcgctga ggggcctgag atctgacgac acggccgtgt attattgtgc gagagatagg 300 gggtatagca gcagccctta ctactactac tacggaatgg acgtctgggg ccaagggacc 360 gggtatagca gcagccctta ctactactac tacggaatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctca 378 acggtcaccg tctcctca 378 <210> 52<210> 52 <211> 363<211> 363 <212> DNA<212> DNA <213> 人工序列<213> Artificial sequence <220> <220> <223> 合成的 <223> Synthetic <400> 52 <400> 52 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaaaactat 180 ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaaaactat 180 gtagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 gtagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagaggaggg 300 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagaggaggg 300 gagtacttct actattacgg tttggacgtc tggggccaag ggaccacggt caccgtctcc 360 tca 363 <210> 53 <211> 378 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 53 caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggagcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc aactatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcaacgctt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatg accacagtca catacacgag tacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagatagg 300 gggtatagca gcagccctta ttactactac tacggtatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctca 378 <210> 54 <211> 375 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 54 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt acttactact ggagctggat ccggcagccc 120 acctgcactg tctctggtgg ctccatcagt acttactact ggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240 aagctgagct ctgtgaccgc tgcggacacg gccgtttatt actgtgcgag gtacgatatt 300 aagctgagct ctgtgaccgc tgcggacacg gccgtttatt actgtgcgag gtacgatatt 300 gtgactggtt atcctcacta ctactactac gttatggacg tctggggcca agggaccacg 360 gtgactggtt atcctcacta ctactactac gttatggacg tctggggcca agggaccacg 360 gtcaccgtct cctca 375 gtcaccgtct cctca 375 <210> 55<210> 55 <211> 324<211> 324 <212> DNA<212> DNA <213> 人工序列<213> Artificial Sequence <220> <220> <223> 合成的 <223> Synthetic <400> 55 <400> 55 gaaattgtaa tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 gaaattgtaa tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctcct tatcctggta ccagcagaaa 120 ctctcctgca gggccagtca gagtgttagc agcagctcct tatcctggta ccagcagaaa 120 cctgggcagg ctcccaggct cctcatctat ggtgcatcca ccagggccac tggcatccca 180 cctgggcagg ctcccaggct cctcatctat ggtgcatcca ccagggccac tggcatccca 180 gccaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagcctgcag 240 gccaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagcctgcag 240 cctgaagatt ttgcagttta ttactgtcag caggattata acttaccgct cactttcggc 300 ggagggacca aggtggagat caaa 324 <210> 56 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 56 gaaattgtaa tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagccatt tatcctggta ccagcagaaa 120 cctgggcagg ctcccaggct cctcatctat ggtgcatcca ccagggccac tggcatccca 180 gccaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagcctgcag 240 cctgaagatt ttgcagttta ttattgtcag caggattata atttaacgat caccttcggc 300 caagggacac gactggagat taaa 324 <210> 57 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 57 gaaattgtaa tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctcct tatcctggta ccagcagaaa 120 cctgggcagg ctcccaggct cctcatctat ggtgcatcca ccagggccac tggcatccca 180 gccaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagcctgcag 240 cctgaagatt ttgcagttta ttactgtcag caggattata acttaccgct cactttcggc 300 ggagggacca aggtggagat caaa 324 [[ID=~10]]<210> 58 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 58 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctataggaga cagagtcacc 60 atcacttgtc gggcgagtca gggcattagc aattatttag cctggtttca gcagaaacca 120 gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgccaacag tataatagtt acccattcac tttcggccct 300 gggaccaaag tggatatcaa a 321 <210> 59 <211> 321 Note: The tag <210> 58 and <210> 59 are likely some kind of identifiers within the context of the sequence data. Without further context, it's not clear exactly how they should be interpreted in a more meaningful way. The translation attempts to be as literal as possible while maintaining the integrity of the tags and the sequence data. <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 59 gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaggtca gggcattagc agtgctttag cctggtatca gcagaaacca 120 gggaaagctc ctaagatcct gatctatgat gcctccagtt tggaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc ggggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag tttaataatt accctagcac ttttggccag 300 gggaccaagc tggagatcaa a 321 <210> 60 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 60 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtctacag cataatagtt acccgtacac ttttggccag 300 gggaccaagc tggagatcaa a 321 <210> 61 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 61 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctataggaga cagagtcacc 60 atcacttgtc gggcgagtca gggcattagc aattatttag cctggtttca gcagaaacca 120 gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgccaacag tataatagtt acccattcac tttcggccct 300 gggaccaaag tggatatcaa a 321 <210> 62 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 62 gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga caaagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgctttag gctggtatca gcagaaacca 120 ggaacagccc ctaaactcct gatctatgct gcatccagtt tacagagtgg ggtcccatca 180 aggttcagcg gcagtggatc tggcacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattctg caacttatta ctgtctacaa gattacaatt acccgctcac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 63 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 63 gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtcg gggcattaga aatgctttag gctggtatca gcagaaacca 120 ggaacagccc ctaaactcct gatctatgct gcatccagtt tacagagtgg ggtcccatca 180 aggttcagcg gcagtggatc tggcacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattctg caacttatta ctgtctacaa gattacaatt acccgctcac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 64 <211> 327 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 64 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 65 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 65 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg [[ID=2​​​​​​​​​​​​​​​​​​​​ Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 66 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 66 Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 67 <211> 106 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 67 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 68 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 68 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 69 <211> 162 <212> PRT <213> artificial sequence <220> <223> Synthetic <400> 69 Thr His Arg Thr Gly Leu Arg Ser Pro Asp Ile Pro Gln Asp Trp Val 1 5 10 15 Ser Phe Leu Arg Ser Phe Gly Gln Leu Thr Leu Cys Pro Arg Asn Gly 20 25 30 Thr Val Thr Gly Lys Trp Arg Gly Ser His Val Val Gly Leu Leu Thr 35 40 45 Thr Leu Asn Phe Gly Asp Gly Pro Asp Arg Asn Lys Thr Arg Thr Phe 50 55 60 Gln Ala Thr Val Leu Gly Ser Gln Met Gly Leu Lys Gly Ser Ser Ala 65 70 75 80 Gly Gln Leu Val Leu Ile Thr Ala Arg Val Thr Thr Glu Arg Thr Ala 85 90 95 Gly Thr Cys Leu Tyr Phe Ser Ala Val Pro Gly Ile Leu Pro Ser Ser 100 105 110 Gln Pro Pro Ile Ser Cys Ser Glu Glu Gly Ala Gly Asn Ala Thr Leu 115 120 125 Ser Pro Arg Met Gly Glu Glu Cys Val Ser Val Trp Ser His Glu Gly 130 135 140 Leu Val Leu Thr Lys Leu Leu Thr Ser Glu Glu Leu Ala Leu Cys Gly 145 150 155 160 Ser Arg <210> 70 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 70 Thr His Thr Thr Gly Leu Arg Ser Pro Asp Ile Pro Gln Asp Trp Val 1 5 10 15 Ser Phe Leu Arg Ser Phe Gly Gln Leu Ser Leu Cys Pro Met Asn Glu 20 25 30 Thr Val Thr Gly Thr Trp Gln Gly Pro His Val Val Gly Leu Leu Thr 35 40 45 Thr Leu Asn Phe Gly Asp Gly Pro Asp Arg Asn Lys Thr Gln Thr Phe 50 55 60 Gln Ala Lys Ile His Gly Ser Gln Ile Gly Leu Thr Gly Ser Ser Ala 65 70 75 8​​​​​​​​​Gln Pro Pro Ile Ser Cys Ser Glu Glu Gly Val Gly Asn Ala Thr Leu 115 120 125 Ser Pro Val Met Gly Glu Glu Cys Val Arg Val Trp Ser His Glu Arg 130 135 140 Leu Val Leu Thr Glu Leu Leu Thr Ser Glu Glu Leu Ala Leu Cys Gly 145 150 155 160 Ser <210> 71 <211> 25 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 71 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 72 <211> 30 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 72 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser 20 25 30 <210> 73 <211> 29 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 73 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 20 25 <210> 74 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 74 Gly Gly Ser Ile Ser Thr Tyr 1 5 <210> 75 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 75 Gly Gly Ser Ile Ser Thr Tyr Tyr Trp Ser 1 5 10 <210> 76 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 76 Thr Tyr Tyr Trp Ser 1 5 <210> 77 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 77 Ser Thr Tyr Tyr Trp Ser 1 5 <210> 78 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 78 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 1 5 10 15 Gly Tyr Ile <210> 79 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 79 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> 80 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 80 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 1 5 10 <210> 81 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 81 Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 15 Tyr <210> 82 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 82 Tyr Tyr Ser Gly Ser 1 5 <210> 83 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 83 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn 1 5 <210> 84 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 84 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 85 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 85 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn 1 5 10 <210> 86 <211> 41 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 86 Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp 1 5 10 15 Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala 20 25 30 Asp Thr Ala Val Tyr Tyr Cys Ala Arg 35 40 <210> 87 <211> 39 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 87 Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser 1 5 10 15 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 20 25 30 Ala Val Tyr Tyr Cys Ala Arg 35 <210> 88 <211> 32 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 88 Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 89 <211> 37 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 89 Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser 1 5 10 15 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 20 25 30 Ala Val Tyr Tyr Cys 35 <210> 90 <211> 38 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 90 Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr 1 5 10 15 Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 91 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 91 Tyr Asp Ile Val Thr Gly Tyr Pro His Tyr Tyr Tyr Tyr Val Met Asp 1 5 10 15 Val <210> 92 <211> 18 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 92 Ala Arg Tyr Asp Ile Val Thr Gly Tyr Pro His Tyr Tyr Tyr Tyr Val 1 5 10 15 Met Asp <210> 93 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 93 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 94 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 94 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 95 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 95 Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 96 <211> 29 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 96 Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Gly Ile 20 25 <210> 97 <211> 26 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 97 Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 98 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 98 Arg Ala Ser Arg Gly Ile Arg Asn Ala Leu Gly 1 5 10 <210> 99 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 99 Arg Asn Ala Leu Gly Trp Tyr 1 5 <210> 100 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 100 Trp Tyr Gln Gln Lys Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 101 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 101 Gln Gln Lys Pro Gly Thr Ala Pro Lys 1 5 <210> 102 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 102 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Thr Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 103 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 103 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 104 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 104 Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln 1 5 10 <210> 105 <211> 32 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 105 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys 20 25 30 <210> 106 <211> 33 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 106 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 1 5 10 15 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Tyr 20 25 30 Cys <210> 107 <211> 37 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 107 Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 1 5 10 15 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ser 20 25 30 Ala Thr Tyr Tyr Cys 35 <210> 108 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 108 Leu Gln Asp Tyr Asn Tyr Pro Leu 1 5 <210> 109 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 109 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 110 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 110 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to human IGFBP3 with an affinity constant less than or equal to 1.1 x 10 -9 M, which inhibits or reduces the binding of IGFBP3 to TMEM219, comprising a heavy chain variable domain and a light chain variable domain, comprising: SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 as CDRs 1-3 of the heavy chain variable domain, and SEQ ID NO: 27, SEQ ID NO: 18, and SEQ ID NO: 26 as CDRs 1-3 of the light chain variable domain or - SEQ ID NO: 74 and SEQ ID NO: 82 and SEQ ID NO: 91 as CDR1-3 of the heavy chain variable domain, and SEQ ID NO: 98 and SEQ ID NO: 103 and SEQ ID NO: 26 as CDR1-3 of the light chain variable domain or - SEQ ID NO: 75 and SEQ ID NO: 83 and SEQ ID NO: 91 as CDR1-3 of the heavy chain variable domain, and SEQ ID NO: 98 and SEQ ID NO: 103 and SEQ ID NO: 26 as CDR1-3 of the light chain variable domain or - SEQ ID NO: 76 and SEQ ID NO: 84 and SEQ ID NO: 91 as CDR1-3 of the heavy chain variable domain, and SEQ ID NO: 98 and SEQ ID NO: 103 and SEQ ID NO: 26 as CDR1-3 of the light chain variable domain or - SEQ ID NO: 77, SEQ ID NO: 85 and SEQ ID NO: 92 as CDR1-3 of the heavy chain variable domain, and SEQ ID NO: 99, SEQ ID NO: 104 and SEQ ID NO: 108 as CDR1-3 of the light chain variable domain or - SEQ ID NO: 9 and SEQ ID NO: 10 and SEQ ID NO: 11 as CDRs 1-3 of the heavy chain variable domain, and SEQ ID NO: 27 and AA and SEQ ID NO: 26 as CDRs 1-3 of the light chain variable domain.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, which inhibits, reduces or neutralizes the activation of the TMEM219 receptor induced by IGFBP3.

3. The isolated antibody or antigen-binding fragment thereof of claim 1, which effectively controls blood glucose levels in an in vivo model.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1, which has at least one activity selected from the group consisting of: Mini-intestine growth was increased in healthy subjects treated with a-IGFBP3; b-IBD patients have increased mini-intestinal growth; c-increased mini-intestine growth in healthy subjects treated with diabetic enteropathy serum; The expression of EphB2 and / or LGR5 was increased in the mini intestines of healthy subjects treated with d-IGFBP3; e-Reduction of caspase-8 expression in the mini-intestines of healthy subjects treated with IGFBP3; reduction in β-cell loss in f-IGFBP3-treated β-cells; Increased insulin expression in g-IGFBP3-treated β cells; and β-cell apoptosis was reduced in h-IGFBP3-treated β-cells; i - Decreased expression of caspase-8 in IGFBP3-treated β cells; j-Reduced pancreatitis scores in diabetic animal models; k Reduced the incidence of diabetes in diabetic animal models.

5. The isolated antibody or antigen-binding fragment thereof of claim 4, wherein 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) are at least 10%.

6. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising: a. a heavy chain variable domain sequence of the amino acid sequence shown in SEQ ID NO: 36; and b. The light chain variable domain sequence of the amino acid sequence shown in SEQ ID NO:

45.

7. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising 8. The isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a human or humanized antibody.

9. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is an IgG2 or IgG4 antibody. 10 . The isolated antibody or antigen-binding fragment thereof according to claim 9 , which is an IgG2κ antibody, an IgG2λ antibody, an IgG4κ antibody or an IgG4λ antibody.

11. The isolated antibody or antigen-binding fragment thereof according to claim 9, which is human IgG2 or human IgG4.

12. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 7, comprising: -SEQ ID NO: 64 or 65 and / or -SEQ ID NO: 66, 67 or 68.

13. The isolated antibody or antigen-binding fragment thereof of claim 9, comprising human IgG4 with a Ser to Pro substitution at position 228 and / or a Leu to Glu substitution at position 235.

14. The isolated antibody or antigen-binding fragment thereof of claim 13, wherein the substitution at position 228 is a Ser to Pro substitution.

15. The isolated antibody or antigen-binding fragment thereof of claim 13, wherein the substitution at position 235 is a Leu to Glu substitution.

16. An isolated polynucleotide comprising at least one sequence encoding the antibody or antigen-binding fragment thereof of any one of the preceding claims.

17. The isolated polynucleotide of claim 16, which is a cDNA. A vector comprising the polynucleotide according to claim 16 or 17.

19. The vector of claim 18 is selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, and an expression vector.

20. The vector according to claim 18, which is a recombinant expression vector.

21. An isolated cell comprising the polynucleotide of claim 16 or 17 or the vector of claim 18 or 19.

22. The isolated cell of claim 21, which is a hybridoma or a Chinese hamster ovary (CHO) cell or a human embryonic kidney cell (HEK293).

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the polynucleotide according to any one of claims 16 to 17, or the vector according to any one of claims 18 to 19, or the cell according to any one of claims 21 to 22, in the preparation of a medicament for treating: diabetes, intestinal diseases and / or enteropathy, malabsorption syndrome, cachexia, or diabetic enteropathy.

24. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the polynucleotide according to any one of claims 16 to 17, the vector according to any one of claims 18 to 19, or the cell according to any one of claims 21 to 22 in the preparation of a medicament for treating type I or type II diabetes.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the polynucleotide according to any one of claims 16 to 17, the vector according to any one of claims 18 to 19, or the cell according to any one of claims 21 to 22 in the preparation of a medicament for treating inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease, or intestinal obstruction.

26. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof, the isolated polynucleotide, the vector, or the isolated cell according to any one of the preceding claims and a pharmaceutically acceptable carrier.

27. Use of the pharmaceutical composition according to claim 26 in the preparation of a medicament for treating diabetes, intestinal diseases and / or enteropathy, malabsorption syndrome, cachexia or diabetic enteropathy.

28. Use of the pharmaceutical composition according to claim 26 in the preparation of a medicament for treating inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction.

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