Methods for treating pancreatitis

Abstract

Provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or a functional fragment thereof that binds to CA19-9.

Description

Technical Field

[0001] Provided herein are methods for preventing and treating pancreatitis. Specifically, provided herein are methods for treating and / or preventing pancreatitis using molecules that bind to CA19-9.

[0002] Sequence Listing

[0003] This application incorporates by reference a computer readable form (CRF) of a sequence listing in ASCII text format submitted via EFS-Web. The sequence listing text file submitted via EFS-Web is named 12967-046-999_Sequence_Listing.txt, created on September 10, 2018, and is 22,963 bytes in size. Background Art

[0004] Pancreatitis causes 275,000 hospitalizations in the United States each year (Forsmark CE et al. N Engl J Med. 2016; 375(20): 1972-81). Pancreatitis includes chronic pancreatitis and acute pancreatitis and can be accompanied by severe abdominal pain. The mortality rate of patients with chronic pancreatitis is higher than that of the general population, with 10-year survival estimates after diagnosis ranging from 69-80% (Seicean A. et al., J Gastrointestin Liver Dis. 2006; 15(1): 21-6). Due to the complications and morbidity caused by chronic pancreatitis, the life expectancy of patients with chronic pancreatitis is about 8 years shorter than that of the general population (Bang UC. et al., Gastroenterology. 2014; 146(4): 989-94). In severe acute pancreatitis, the mortality rate can be as high as 16.3%, with more than 40% of deaths occurring within 14 days of admission (Fu C. et al., World J Gastroenterol. 2007; 13(13): 1966-1969).

[0005] Despite the increased mortality and significant morbidity associated with pancreatitis, there is little progress in how to treat this debilitating disease. Prior to the disclosure, acute pancreatitis was often treated with general supportive care consisting of intravenous hydration, with different fluid resuscitation rates as the basis of care according to the patient's volume status (Chapter 37-Acute Pancreatitis, GI / Liver Secrets Plus, 4th edition, McNally PR compiles, 2010). The pain associated with severe pancreatitis is managed with intravenous analgesia at the same time (Chapter 37-Acute Pancreatitis, GI / Liver Secrets Plus, 4th edition, McNally PR compiles, 2010). Chronic pancreatitis is often treated only with drugs for the pain or complications of pancreatitis, so that potential chronic pancreatitis itself is not directly treated (Chapter 38-Chronic Pancreatitis, GI / Liver Secrets Plus, 4th edition, McNally PR compiles, 2010).

[0006] To date, there has been a lack of success in developing therapeutic agents that directly treat, prevent, manage or alleviate pancreatitis or target the underlying mechanisms that underlie the development of pancreatitis. Therefore, there is a need to identify therapeutic agents that can treat, prevent, manage or alleviate pancreatitis, including chronic and acute pancreatitis. Summary of the invention

[0007] In one aspect, provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sialyl Lewis a (Sialyl-Lewis a ) binding antibody or its functional fragment (anti-sLe a ).

[0008] In another aspect, provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising:

[0009] (1) administering to the subject a first dose of a sialyl Lewis a The antibody or its functional fragment (anti-sLe a ),

[0010] (2) determining the severity of pancreatitis in the subject, and

[0011] (3) If anti-SLe is administered aIf the severity of pancreatitis in the subject is reduced compared to the severity of pancreatitis in the subject described above, a second dose of the anti-sLe is administered in an amount that is the same as or lower than the first dose. a or if the severity of pancreatitis in the subject has not decreased, administering a second dose of anti-sLe in an amount higher than the first dose; a .

[0012] In certain aspects, provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a therapeutic agent that binds to CA19-9.

[0013] In one aspect, provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising:

[0014] (1) administering to the subject a therapeutic agent that binds to CA19-9 at a first dose,

[0015] (2) determining the severity of pancreatitis in the subject, and

[0016] (3) administering a second dose of the therapeutic agent in an amount that is the same as or lower than the first dose if the severity of pancreatitis in the subject is reduced compared to the severity of pancreatitis in the subject before administration of the dose; or administering a second dose of the therapeutic agent in an amount that is higher than the first dose if the severity of pancreatitis in the subject is not reduced.

[0017] In some embodiments of the methods provided herein, the anti-sLe a The antibody or functional fragment thereof comprises a variable heavy chain (VH) domain comprising an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO:2, residues 20-142 of SEQ ID NO:6, residues 20-142 of SEQ ID NO:10, and residues 20-145 of SEQ ID NO:14.

[0018] In certain embodiments of the methods provided herein, the anti-sLe a The antibody or functional fragment thereof comprises a variable light (VL) domain comprising an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO:4, residues 20-129 of SEQ ID NO:8, residues 20-130 of SEQ ID NO:12, and residues 23-130 of SEQ ID NO:16.

[0019] In other embodiments of the methods provided herein, the anti-sLe a The antibody or its functional fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain and the VL domain respectively comprise an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO:2 and residues 20-130 of SEQ ID NO:4; residues 20-142 of SEQ ID NO:6 and residues 20-129 of SEQ ID NO:8; residues 20-142 of SEQ ID NO:10 and residues 20-130 of SEQ ID NO:12; and residues 20-145 of SEQ ID NO:14 and residues 23-130 of SEQ ID NO:16.

[0020] In some embodiments of the methods provided herein, the anti-sLe a The antibody or its functional fragment comprises a heavy chain variable region comprising three complementarity determining regions (CDRs) having amino acid sequences of three heavy chain variable region CDRs according to the Kabat numbering of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) Accession No. HB-8059; and a light chain variable region comprising three CDRs having amino acid sequences of three light chain variable region CDRs according to the Kabat numbering of an antibody produced by a hybridoma deposited with the ATCC Accession No. HB-8059.

[0021] In certain embodiments of the methods provided herein, the anti-sLe a The antibody or its functional fragment comprises a heavy chain variable region, which consists of the amino acid sequence of the heavy chain variable region of the antibody produced by the hybridoma deposited with the American Type Culture Collection (ATCC) Accession No. HB-8059; and a light chain variable region, which consists of the amino acid sequence of the light chain variable region of the antibody produced by the hybridoma deposited with the ATCC Accession No. HB-8059.

[0022] In other embodiments of the methods provided herein, the anti-sLe a The antibody or its functional fragment comprises a heavy chain consisting of the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited with ATCC Accession No. HB-8059; and a light chain consisting of the light chain amino acid sequence of the antibody produced by the hybridoma deposited with ATCC Accession No. HB-8059.

[0023] In some embodiments of the methods provided herein, the anti-sLe aThe antibody functional fragment is selected from the group consisting of: Fab, Fab', F(ab')2, scFV, diabody, triabody, minibody and single domain antibody (sdAB). a The antibody or its functional fragment is a diabody. In other embodiments, the diabody comprises the amino acid sequence SEQ ID NO: 18 or 20.

[0024] In certain embodiments of the methods provided herein, the anti-sLe a The antibody or its functional fragment is a monoclonal antibody. In some embodiments of the methods provided herein, the anti-sLe a The antibody or its functional fragment is of IgG or IgM isotype. In certain embodiments, the IgG antibody is of IgG1, IgG2, IgG3 or IgG4 subclass.

[0025] In some embodiments of the methods provided herein, the anti-sLe a The antibody or its functional fragment is formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In certain embodiments, the antibody or its functional fragment is administered to the subject via a parenteral administration route. In some embodiments, the parenteral administration is selected from the group consisting of: subcutaneous injection, intramuscular injection, and intravenous injection.

[0026] In some embodiments of the methods provided herein, the antibody or its functional fragment is administered to the subject in a dosage range of about 0.1 mg / kg / dose to about 10 mg / kg / dose. In certain embodiments, the antibody or its functional fragment is administered to the subject weekly, biweekly, monthly, or bimonthly. In some embodiments, the antibody or its functional fragment is administered for about one, two, three, four, five, or six months.

[0027] In certain embodiments of the methods provided herein, the therapeutic agent is a sialyl Lewis a The antibody or its functional fragment (anti-sLe a ).

[0028] In some embodiments of the methods provided herein, the therapeutic agent is a peptide or peptidomimetic molecule. In certain embodiments, the peptide or peptidomimetic has a sialyl Lewis a Specific binding properties.

[0029] In certain embodiments of the methods provided herein, pancreatitis is characterized by one or more markers. In some embodiments of the methods provided herein, the severity of pancreatitis is determined by one or more markers. In certain embodiments of the methods provided herein, the level of one or more markers of pancreatitis in the subject is increased. In some embodiments of the methods provided herein, the administration of the therapeutic agent causes the level of one or more markers of pancreatitis in the subject to be reduced. In certain embodiments, the one or more markers are selected from the group consisting of: physiological markers, histological markers, serological markers and signaling markers. In some embodiments, the histological markers are selected from the group consisting of: immune cell invasion, collagen deposition, fibrotic tissue, fat necrosis, interstitial edema, acinar and vascular destruction, interstitial hemorrhage, ductal dilatation, acinar cell homogenization and CA19-9 expression levels. In certain embodiments, the one or more markers include the expression level of CA19-9. In other embodiments, the one or more markers are determined by immunohistochemistry. In certain embodiments, the serological markers are selected from the group consisting of: serum lipase levels, serum amylase levels and serum C-reactive protein levels. In some embodiments, the one or more markers are serum lipase levels exceeding 200U / L. In certain embodiments, the one or more markers are serum amylase levels exceeding 200U / L. In some embodiments, the one or more markers are serum amylase levels three times higher than the upper limit of normal serum amylase levels in control subjects. In certain embodiments, the one or more markers are serum lipase levels three times higher than the upper limit of normal serum lipase levels in control subjects. In some embodiments, the physiological markers are selected from the group consisting of: weight loss, food malabsorption, pancreatic atrophy, pancreatic enlargement, pancreatic inflammation and bile reflux.

[0030] In one aspect, the present invention provides a method for preventing, treating, alleviating or managing pancreatic sLe a A method for pancreatitis in a subject with elevated sLe comprising administering to the subject a therapeutically effective amount of a Specifically bound therapeutic agent.

[0031] In certain embodiments of the methods provided herein, pancreatitis is pancreatitis selected from the group consisting of chronic pancreatitis and acute pancreatitis. In some embodiments, chronic pancreatitis is selected from the group consisting of: mild chronic pancreatitis, moderate chronic pancreatitis, and severe chronic pancreatitis. In certain embodiments, acute pancreatitis is selected from the group consisting of: mild acute pancreatitis, moderate acute pancreatitis, and severe acute pancreatitis.

[0032] In some embodiments of the methods provided herein, the subject is a mammal. In certain embodiments of the methods provided herein, the mammal is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A Shown are the percentage of human chronic pancreatitis patients who exhibit high levels of pancreatic tissue CA19-9 (left panel), and representative immunohistochemistry (IHC) showing that CA19-9 is predominantly in the pancreatic ducts (right two panels, indicated by open arrows). Figure 1B Examples of CA19-9 IHC from excluded (left), CA19-9 high (middle), and CA19-9 low / negative (right) human chronic pancreatitis specimens are shown. Scale bar = 100 μm. Figure 1C Schematic diagram of Lewis antigen generation is shown. FIG1D shows that ectopic FUT3 induces Lewis antigens in mouse pancreatic ductal adenocarcinoma (PDAC) cells as analyzed by flow cytometry. x (Le x ) expression, but not CA19-9 / sialyl Lewis a (SLe a ) expression. Figure 1E shows CA19-9 flow cytometry of mouse PDAC cells stably and constitutively expressing FUT3 and β3GALT5 compared to parental KPC cell controls. Both the expression of FUT3 and β3GALT5 resulted in cell surface expression of CA19-9 reaching the same level observed in a human cancer cell line (Colo205). Figure 1F shows CA19-9 immunofluorescence evaluation of human PDAC cell line Suit2 (upper panel) and mouse PDAC cells stably and constitutively expressing FUT3 and β3GALT5 (lower panel) or PDAC cells expressing an empty vector control. Scale bar = 25 μm. Figure 1G shows circulating CA19-9 levels in nude mice transplanted with CA19-9-negative human PDAC cell line MiaPaCa2, CA19-9-positive human PDAC cell line Suit2, mouse PDAC cell lines transfected with an empty vector control, and mouse PDAC cells stably and constitutively expressing FUT3 and β3GALT5.

[0034] Figure 2 A shows the immunoprecipitation / mass spectrometry (IP / MS) strategy of mouse PDAC cells stably and constitutively expressing FUT3 and β3GALT5 (left panel), immunoblot validation of CA19-9 IP efficiency (middle panel), and overlap of CA19-9 protein loading between three biological replicate lines (FC1199, FC1242, and FC1245) (right panel). Figure 2B shows the IP / MS strategy for human PDAC cells (left), immunoblot validation of CA19-9 IP efficiency (middle), and overlap of CA19-9 protein loading between three biological replicate lines (Capan2, hM1-2D, and Suit2) (right). Figure 2C Overlap between CA19-9 protein carrier identified in 3 of 3 human PDAC cell lines (n=936) and three independent mouse PDAC cell lines expressing FUT3 and β3GALT5 is shown.

[0035] Figure 3 A-3D show the design, validation and generation of the CA19-9 genetically engineered mouse model. Figure 3 A shows a schematic diagram of the inducible CA19-9 mouse model. Figure 3 B shows immunoblot confirmation of CA19-9 production in mES cell clones after Cre transfection and Dox treatment. Figure 3 C shows a southern blot of eGFP in positive control wild-type mES cell gDNA, where the targeting vector was spiked in at 1-5 copies per genome, and in mES cell targeted clones. The correct integration size was 5.548 kb (non-targeted = 4.04 kb). Figure 3 D shows eGFP qPCR copy number assessment of targeted mES cell clones relative to a previously validated targeted mES cell clone carrying a single and correctly integrated ColA1 targeting vector (P12).

[0036] Figure 4 Shown is the whole body CA19-9 mouse model, whole body histology in haematoxylin and eosin (H&E) histological evaluation of mouse tissues from untreated R;F mice. Scale bar = 100 μm.

[0037] Figure 5 Shown is the whole body CA19-9 mouse model, whole body histology in H&E histological evaluation of mouse tissues from R;F mice treated with Dox for 7 days. Scale bar = 100 μm.

[0038] Figure 6 Shown is the whole body CA19-9 mouse model, whole body histology in H&E histological evaluation of mouse tissues from R;F mice treated with Dox for 28 days. Scale bar = 100 μm.

[0039] Figure 7Shown are whole body CA19-9 mouse model genetic negative control littermates, whole body histology in H&E histology evaluation of mouse tissues from untreated R;F genetic negative control littermates. Scale bar = 100 μm.

[0040] Figure 8 Shown are whole body CA19-9 mouse model genetic negative control littermates, whole body histology in H&E histology evaluation of mouse tissues from R;F genetic negative control littermates treated with Dox for 7 days. Scale bar = 100 μm.

[0041] Fig. 9 Shown are whole body CA19-9 mouse model genetic negative control littermates, whole body histology in H&E histology evaluation of mouse tissues from R;F genetic negative control littermates treated with Dox for 28 days. Scale bar = 100 μm.

[0042] Fig.10 Shown in untreated C PDX ; R LSL ; F LSL Focal pancreas, PDX1-Cre driven CA19-9 mouse model, whole body histology in H&E histological assessment of mouse tissues in mice. Scale bar = 100 μm.

[0043] Fig.11 The results show that the C PDX ; R LSL ; F LSL Focal pancreas, PDX1-Cre driven CA19-9 mouse model, whole body histology in H&E histological assessment of mouse tissues in mice. Scale bar = 100 μm.

[0044] Fig.12 The results show that the C PDX ; R LSL ; F LSL Focal pancreas, PDX1-Cre driven CA19-9 mouse model, whole body histology in H&E histological assessment of mouse tissues in mice. Scale bar = 100 μm.

[0045] Fig.13 Shown in untreated C PDX / p48 ; R LSL ; F LSL Focal pancreas, genetic negative control littermates, whole body histology in H&E histological evaluation of mouse tissues from genetic negative control littermates in the CA19-9 mouse model. Scale bar = 100 μm.

[0046] Fig.14The results show that the C PDX / p48 ; R LSL ; F LSL Focal pancreas, CA19-9 mouse model genetic negative control littermates, whole body histology in H&E histological evaluation of mouse tissues from untreated genetic negative control littermates. Scale bar = 100 μm.

[0047] Fig.15 The results show that the C PDX / p48 ; R LSL ; F LSL Focal pancreas, genetic negative control littermates, whole body histology in H&E histological evaluation of mouse tissues from genetic negative control littermates in the CA19-9 mouse model. Scale bar = 100 μm.

[0048] Fig.16 Shown in untreated C p48 ; R LSL ; F LSL Focal pancreas, p48-Cre driven CA19-9 mouse model, whole body histology in H&E histological assessment of mouse tissues in mice. Scale bar = 100 μm.

[0049] Fig.17 The results show that the C p48 ; R LSL ; F LSL Focal pancreas, p48-Cre driven CA19-9 mouse model, whole body histology in H&E histological assessment of mouse tissues in mice. Scale bar = 100 μm.

[0050] Fig.18 The results show that the C p48 ; R LSL ; F LSL Focal pancreas, p48-Cre driven CA19-9 mouse model, whole body histology in H&E histological assessment of mouse tissues in mice. Scale bar = 100 μm.

[0051] Fig.19 Shown in untreated C PDX ; R LSL ; F H&E histological evaluation of whole pancreas CA19-9 mouse model, whole body histology of mouse tissues in mice. Scale bar = 100 μm.

[0052] Fig. 20 The results show that the C PDX ; R LSL; F H&E histological evaluation of whole pancreas CA19-9 mouse model, whole body histology of mouse tissues in mice. Scale bar = 100 μm.

[0053] Fig.21 The results show that the C PDX ; R LSL ; F H&E histological evaluation of whole pancreas CA19-9 mouse model, whole body histology of mouse tissues in mice. Scale bar = 100 μm.

[0054] Fig. 22 Shown in untreated C PDX ; R LSL ; F H&E histological evaluation of whole pancreas tissues from genetic negative control littermates CA19-9 mouse model genetic negative control littermates, whole body histology. Scale bar = 100 μm.

[0055] Fig.23 The results show that the difference between untreated C and Dox treated C for 7 days is 1. PDX ; R LSL ; F H&E histological evaluation of whole pancreas tissues from genetic negative control littermates CA19-9 mouse model genetic negative control littermates, whole body histology. Scale bar = 100 μm.

[0056] Fig.24 The results show that the difference between untreated C and Dox treated C for 28 days is not statistically significant. PDX ; R LSL ; F H&E histological evaluation of whole pancreas tissues from genetic negative control littermates CA19-9 mouse model genetic negative control littermates, whole body histology. Scale bar = 100 μm.

[0057] Fig.25 A shows the expression of CA19-9 by H&E staining, Masson's trichrome staining (MT, blue indicates collagen deposition) and by IHC (open arrows, CA19-9+ ducts; solid arrows, CA19-9+ islets) after treatment with Dox. PDX ; R LSL ; F Histological evaluation of pancreas. Scale bar = 50 μm. Fig.25 B shows the treatment of C with Dox PDX ; R LSL Quantification of the percentage of pancreatic area showing histological signs of pancreatitis in ; F mice, n=5, 4, 5, 3, 9, respectively (F(4,21)=22.46, p<0.0001). Fig.25 C shows the treatment of C with Dox PDX ; R LSL; Circulating levels (U / L) of amylase (F(5,61)=74.44, p<0.0001) and lipase (F(5,61)=27.59, P<0.0001) after F mice. The dotted line indicates the threshold elevation required for diagnosing pancreatitis. The short dash indicates the maximum detection level, n=14, 12, 19, 8 and 9, respectively. The horizontal red line in the middle represents the mean, and the error bars represent the standard deviation; each data point represents the measurement from a single mouse. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for multiple comparisons using the Holm-Sidak method after one-way ANOVA.

[0058] Fig.26 R from a genetic negative control littermate treated with Dox evaluated using H&E histology; F, C PDX ; R LSL ; F LSL or C p48 ; R LSL ; F LSL Comparison of the severity of pancreatitis in mouse models of mouse pancreas. Scale bar = 50 μm.

[0059] Fig. 27 Shown are the results of Masson's trichrome staining (blue, collagen deposition) from genetic negative control littermates R treated with Dox; F or C PDX ; R LSL ; F LSL Comparison of stromal abundance in the mouse pancreas between mouse and mouse models. Scale bar = 50 μm.

[0060] Fig.28 A- Fig.28 C shows a mouse model comparison of pancreatitis severity in genetically engineered mice. Including genetic negative control littermates R treated with Dox (days); F, C PDX ; R LSL ; F LSL or C p48 ; R LSL ; F LSL Mouse, from Fig.25 B-25C C; R LSL ; Data from the F model are used for relative comparison. Fig.28 A shows the percentage of pancreatic area affected by histological signs of pancreatitis (F(4,22)=0.5589; (4,12)=16.02; (4,21)=22.46; (3,19)=2.705). Fig.28B-28C shows circulating levels (U / L) of pancreatic enzymes amylase (B) (F(4,71) = 8.828; (4,22) = 25.07; (5,61) = 74.44; (4,58) = 6.343) and lipase (C) (F(4,71) = 2.967; (4,22) = 7.108; (5,61) = 27.59; (4,58) = 5.637) after treatment with Dox (days). The dotted line indicates the threshold elevation required for diagnosis of pancreatitis. The short dash line indicates the maximum possible detection level of amylase. Lines represent mean values; each data point represents measurements from a single mouse. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, multiple comparisons were performed using one-way ANOVA.

[0061] Figures 29A-29B IHC of eGFP and CA19-9 in the pancreas of each CA19-9 mouse model is shown. Fig.29A R from treatment with Dox for 0 or 7 days is shown; F, C PDX ; R LSL ; F, C PDX ; R LSL ; F LSL or C p48 ; R LSL ; F LSL eGFP IHC of mouse pancreas from mice. Open arrows indicate positive islet cells, solid arrows indicate positive ducts, * indicates negative blood vessels, and "a" indicates positive acinar cells. Scale bar = 50 μm. Fig.29B Shown are R from a genetic negative control littermate treated with Dox; F, C PDX ; R LSL ; F LSL or C P48 ; R LSL ; F LSL CA19-9 IHC of mouse pancreas from mouse. Open arrows indicate positive islet cells, solid arrows indicate positive ducts, and orange arrows indicate positive acinar cells. Inset shows an enlarged positive duct indicated by solid arrows. Scale bar = 50 μm.

[0062] Figures 30A-30B Shown are R; F whole body pancreatic lineage immunofluorescence (IF) of the CA19-9 mouse model. Fig. 30A Shown are IFs of the ductal marker CK19 as well as eGFP (a Dox-dependent recombination marker), DAPI, and CA19-9 in mouse pancreas from R;F mice treated with Dox for 0, 7, or 28 days. Fig. 30B Shown are annotated IF images from A. Scale bar = 50 μm.

[0063] Fig.31 Whole body pancreatic lineage immunofluorescence (IF) of the R;F CA19-9 mouse model is shown. IF of the fibroblast marker Podoplanin and eGFP (Dox-dependent recombinant marker), DAPI, and CA19-9 in mouse pancreas from R;F mice treated with Dox for 28 days. CA19-9+, Podoplanin+, eGFP- cells are indicated by hollow pink arrows. CA19-9-, Podoplanin+, eGFP- cells are indicated by solid red arrows. Scale bar = 50 μm.

[0064] Fig.32 Systemic CA19-9 mouse model: eGFP expression in non-pancreatic tissues is shown. eGFP IHC from untreated R; F mouse tissues is shown. Scale bar = 100 μm.

[0065] Fig.33 Systemic CA19-9 mouse model: eGFP expression in non-pancreatic tissues is shown. eGFP IHC of mouse tissues from R;F mice treated with Dox is shown. Scale bar = 100 μm.

[0066] Fig.34 Whole pancreas CA19-9 mouse model: eGFP expression in non-pancreatic tissues is shown. PDX ; R LSL ; eGFP IHC of mouse tissues from F mice. Scale bar = 100 μm.

[0067] Fig.35 Whole pancreas CA19-9 mouse model: eGFP expression in non-pancreatic tissues is shown. PDX ; R LSL ; eGFP IHC of mouse tissues from F mice. Scale bar = 100 μm.

[0068] Fig.36 Focal pancreatic, PDX1-Cre driven CA19-9 mouse model: eGFP expression in non-pancreatic tissues is shown. PDX ; R LSL ; F LSL eGFP IHC of mouse tissues from mice. Scale bar = 100 μm.

[0069] Fig.37 Focal pancreatic, PDX1-Cre driven CA19-9 mouse model: eGFP expression in non-pancreatic tissues. PDX ; R LSL ; FLSL eGFP IHC of mouse tissues from mice. Scale bar = 100 μm.

[0070] Fig.38 Focal pancreatic, p48-Cre driven CA19-9 mouse model: eGFP expression in non-pancreatic tissues is shown. P48 ; R LSL ; F LSL eGFP IHC of mouse tissues from mice. Scale bar = 100 μm.

[0071] Fig.39 Focal pancreatic, p48-Cre driven CA19-9 mouse model: eGFP expression in non-pancreatic tissues. P48 ; R LSL ; F LSL eGFP IHC of mouse tissues from mice. Scale bar = 100 μm.

[0072] Fig.40 Systemic CA19-9 mouse model: CA19-9 expression in non-pancreatic tissues is shown. CA19-9 IHC of mouse tissues from untreated R;F mice is shown. Scale bar = 100 μm.

[0073] Fig.41 Shown is a systemic CA19-9 mouse model: CA19-9 expression in non-pancreatic tissues. Shown is CA19-9 IHC of mouse tissues from R;F mice treated with Dox for 7 days. Scale bar = 100 μm.

[0074] Fig.42 Shown is a systemic CA19-9 mouse model: CA19-9 expression in non-pancreatic tissues. Shown is CA19-9 IHC of mouse tissues from R;F mice treated with Dox for 28 days. Scale bar = 100 μm.

[0075] Fig.43 Systemic CA19-9 mouse model: CA19-9 expression in non-pancreatic tissues is shown. CA19-9 IHC of mouse tissues from untreated R;F genetic negative control littermates is shown. Scale bar = 100 μm (except for heart, where the scale bar = 500 μm).

[0076] Fig.44 Focal pancreatic CA19-9 mouse model: CA19-9 expression in non-pancreatic tissues. PDX ; R LSL ; F LSL and C P48 ; RLSL ; F LSL CA19-9 IHC of mouse tissues from mice. Scale bar = 100 μm.

[0077] Fig.45 Focal pancreatic CA19-9 mouse model: CA19-9 expression in non-pancreatic tissues; C from Dox treated for 7 days is shown. PDX ; R LSL ; F LSL and C P48 ; R LSL ; F LSL CA19-9 IHC of mouse tissues from mice. Scale bar = 100 μm.

[0078] Fig.46 Focal pancreatic CA19-9 mouse model: CA19-9 expression in non-pancreatic tissues; C from 28-day Dox treatment is shown. PDX ; R LSL ; F LSL and C P48 ; R LSL ; F LSL CA19-9 IHC of mouse tissues from mice. Scale bar = 100 μm.

[0079] Fig.47 Focal pancreatic CA19-9 mouse model: CA19-9 expression in non-pancreatic tissues. PDX ; R LSL ; F LSL and C P48 ; R LSL ; F LSL CA19-9 IHC of mouse tissues from genetic negative control littermates. Scale bar = 100 μm.

[0080] Fig.48 A-48B shows a mouse model comparison of body and pancreas weights. Fig.48 A shows genetic negative control littermate R treated with Dox (weeks); F, C PDX ; R LSL ; F or C PDX ; R LSL ; F LSL Percent change in mouse body weight. Each data point line represents the same individual mouse over time, and p values ​​were determined by multiple t-test (Holm-Sidak) without assuming a consistent SD. Fig.48 B shows the genetic negative control littermate R; F, C PDX ; R LSL ; F or C PDX ; RLSL ; F LSL Percentage of pancreas weight normalized to body weight after 12 weeks of Dox treatment in mice, p values ​​determined by unpaired, parametric, two-tailed t-test (t = 1.225, 5.874, 0.7731, 1.059; df = 29, 4, 10, 4. Lines represent mean values; each data point represents measurement from a single mouse. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0081] Fig.49 A-49D shows assessment of indicators of autoimmunity, exocrine pancreatic insufficiency, glucose homeostasis, and CA19-9 secretion. Fig.49 A shows serological measurements of CA19-9-directed antibodies in mouse plasma from the R;F systemic CA19-9 mouse model as well as mice immunized directly with CA19-9. Fig.49 B shows fecal levels of chymotrypsin and elastase measured in R;F mice after 0 or 7 days of Dox treatment. Fig.49 C shows the changes in the expression of proteins in R; F and C after treatment with sucrose vehicle water or Dox sucrose water for 7 days followed by switching to Dox or normal food. PDX ; R LSL ; Blood glucose levels measured in random-fed mice of the F model. Fig.49 D shows treatment of genetic negative control littermates R with Dox (days); F, C; R LSL ; F or C; R LSL ; F LSL Circulating levels of CA19-9 (U / ml) after 12-week follow-up in mice, p values ​​determined by one-way ANOVA with multiple comparisons (F(3,30)=2.305; (3,14)=13.46, (3,24)=35.36; (3,35)=4.667). Lines represent mean values; each data point represents measurements from a single mouse. Values ​​above 37 U / ml (dashed line) were considered elevated. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0082] Fig.50 A shows the treatment of C with Dox PDX ; R LSL ; F Circulating levels of CA19-9 (U / ml) in mice after 4-hectare lipopolysaccharide infusion. Values ​​above 37 U / ml (dashed line) are elevated (F(3,24)=35.36, p<0.0001), n=5, 5, 8 and 10, respectively. Fig.50B shows the expression of cerulein-treated mice (C57B1 / 6j, n=5, 5, and 5, respectively) and Dox-treated mice (C57B1 / 6j, n=5, 5, and 5, respectively) compared to genetic negative controls (GN, n=3 and 3, respectively). PDX ; R LSL ; Immune cell infiltration assessed by flow cytometry in F mice (n=4, 4, and 6, respectively) (F(7,26 / 27 / 26)=41.06, 14.38, 41.23, all p<0.0001). Fig.50 A-50B, the horizontal red line in the middle represents the mean, and the error bars represent the standard deviation; each data point represents measurement from a single mouse; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for multiple comparisons using the Holm-Sidak method after one-way ANOVA.

[0083] Fig.51 A shows immune cell representation during pancreatitis induction in mice. Flow cytometry was used to determine the expression of cerulein in mice (C57B1 / 6j) induced with acute pancreatitis and Dox in mice (C57B1 / 6j) treated for 0, 1, or 3 days compared with genetically negative littermate controls (GN). PDX ; R LSL ; Representative immune cell types in spleen, peripheral lymph nodes, mesenteric lymph nodes, and pancreas of F mice. Data are expressed as the percentage of CD45 positive cells. Fig.51 B shows mice (C57B1 / 6j) treated with bombesin to induce acute pancreatitis and treated with Dox for 0, 1 or 3 days as determined by flow cytometry, compared to genetically negative littermate controls (GN). PDX ; R LSL ; Representative immune cell types in the pancreas of F mice. Lines represent mean values; each data point represents measurements from a single mouse. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, multiple comparisons were performed using one-way ANOVA (F(8,30)=2.709; 2.034; 3.049; 3.784; 4.739; 6.46; 3.986; 4.688).

[0084] Fig.52 A shows that CA19-9 expression induces ductal proliferation. C After treatment with Dox, flow cytometry was used to evaluate PDX ; R LSL; EdU incorporation in F mice (n=3, 3, and 4, respectively) and genetically negative littermate controls (GN, n=3 and 2, respectively). The percentage of EdU positive cells in the whole pancreas (F(4,10)=14.48, p<0.0004), in the infiltrating immune compartment (CD45, F(4,10)=14.82, p<0.0003), or in the ductal epithelial lineage (CK19, F(4,9)=48.35, p<0.0001) is shown. Lines represent mean values; each data point represents measurements from a single mouse. Fig.52 B-52C shows the assessment of proteotoxic stress markers in primary acinar cultures after Dox treatment. Fig.52 B shows the results from C treated with vehicle (PBS), Dox or bombesin for 12 hours. PDX ; R LSL ; F Amylase and lipase levels in the conditioned medium of acinar cell explants. **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA for multiple comparisons (F(2,3)=92.37; 37.17); Fig.52 C shows the levels of Atf4 and Bip relative to the loading control cofilin and untreated time points by immunoblotting of C from cells treated with vehicle (PBS), Dox or bombesin for 12 h. PDX ; R LSL ; F Whole-cell lysates of acinar cell explants were quantified.

[0085] Fig.53 A-53B shows the proliferation pattern during pancreatitis induction in mice. Fig.53 A shows the genetic negative control littermates R treated with Dox (days); F, C PDX ; R LSL ; F or C PDX ; R LSL ; F LSL Ki67 IHC in mice. Fig.53 B shows quantification of Ki67 IHC from at least three mice, with five high-power fields counted per animal. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, multiple comparisons using one-way ANOVA (F(3,91)=5.097; (4,75)=57.28; (7,197)=113.1; (4,86)=12.16), scale bar=50 μm.

[0086] Fig.54 A-54F shows C PDX ; R LSL ; F RNAseq analysis of organoids. Fig.54 A shows a MA plot (Bland-Altman, mean expression versus fold change). Fig.54 B shows a volcano plot (fold change vs. q value). Fig.54 In A and 54B, significantly differentially expressed genes are annotated in blue, and the most differentially expressed genes are further annotated. Fig.54 C shows transcripts per million of the transgene introduced into the genetically engineered mouse model. Fig.54 D shows the expression levels of Fut3 and β3GalT5, normalized to HPRT1, in organoids isolated from normal or malignant human pancreas after Dox treatment compared with C. PDX ; R LSL ; F was compared with normal mouse organoids. Fig.54 E shows display C PDX ; R LSL ; F is an inverted image of the overlap between the organoid and the genetic negative control. PDX ; R LSL ; F The list of differentially expressed genes in organoids was subtracted from genes that were differentially expressed in response to Dox in the absence of CA19-9. Fig.54 F shows the difference between 0 and 24 h treatment in C after subtracting Dox-induced gene expression changes. PDX ; R LSL ; Heat map of differentially expressed genes in F organoids.

[0087] Fig.55 A shows the enrichment of C in ERBB signaling. PDX ; R LSL ; F GSEA of organoids. Fig.55 B and 55C show the PDX ; R LSL ; F GSEA of differentially expressed genes from RNAseq analysis of organoids. Depicted are bar graphs of normalized enrichment scores (NES) of pathways with p < 0.05 and FDR < 0.15, as well as the enrichment scores of the pathways from Dox-treated C PDX ; R LSL ; F Organoid post-enrichment ( Fig.55 B) or depletion ( Fig.55 C) GSEA plot of selected cancer gene signature sets.

[0088] Fig.56 A-56B shows the PDX ; R LSL ; F GSEA of differentially expressed genes from RNAseq analysis of organoids. Depicted are bar graphs of normalized enrichment scores (NES) of pathways with p < 0.05 and FDR < 0.15, as well as the enrichment scores of the pathways from Dox-treated C PDX ; RLSL ; F Organoid post-enrichment ( Fig.56 A) or depletion ( Fig.56 B) GSEA plot of selected KEGG pathway gene sets.

[0089] Fig.57 A shows the activation of signaling pathways as assessed by immunoblotting after treatment with Dox. PDX ; R LSL ; F organoids (n=3 biological replicates) and genetically negative organoids (GN, n=2 biological replicates). Blots represent three technical replicates, C PDX ; R LSL ; Three biological replicates for F organoids and two biological replicates for GN organoids. Quantification was performed relative to the untreated time point after normalization to cofilin. Fig.57 B-57C shows organoid pathway activation following induction of CA19-9 expression. Fig.57 B shows the chromatin from C as assessed by immunoblotting after treatment with Dox (hours). PDX ; R LSL ; Normal ductal organoids from F and genetically negative littermate controls (GN). Fig.57 C shows the expression of mitochondria from C as assessed by immunoblotting after treatment with Dox (hours) in organoid culture medium lacking EGF and NAC. PDX ; R LSL ; Normal ductal organoids of F (n=2) and genetically negative littermate controls (GN, n=2). Fig.57 D shows the C treated with Dox for 0 (n=4) or 3 (n=5) days PDX ; R LSL ; Representative phospho-EGFR immunohistochemistry (IHC) in F mice. Inset is a higher magnification of the pancreatic duct. Scale bar = 50 μm.

[0090] Fig.58 A-58D shows the proliferation status of organoids after induction of CA19-9 expression. Depicted are C cells grown for several days as assessed daily by cell titer Glo PDX ; R LSL ; F( Fig.58 A), R; F( Fig.58 B) and C PDX ; R LSL ; F LSL ( Fig.58 C) and normal mouse organoids expressing constitutive CA19-9 ( Fig.58 D) Including C PDX ; R LSL ;F(218,223) and R;F(3384) genetic negative controls for organoid lines.

[0091] Fig.59 A-59C shown in C PDX ; R LSL ; CA19-9 was not detected on EGFR in F organoids. Fig.59 A shows EGFR IP followed by C from 0-8 hours after Dox treatment. PDX ; R LSL ; F and CA19-9 immunoblot of protein lysates of genetically negative (GN) normal ductal organoids. Fig.59 B shows that CA19-9 expression does not affect C; R LSL ; F and sensitivity of genetically negative (GN) normal ductal organoids to ErbB kinase inhibition using erlotinib or neratinib. GN organoids: A218, A223. C PDX ; R LSL ; F organoids: A217, A220, A225.

[0092] Fig.60 Shown is the C after treatment with Dox (hours) in the presence of isotype control (MOPC21) or CA19-9 blocking mAb (NS19-9) as assessed by immunoblotting. PDX ; R LSL ; F Signaling in organoids (n=3 biological replicates). Quantification was relative to untreated time points after normalization to cofilin. Blots are representative of two technical replicates and three biological replicates.

[0093] Fig.61 A-61B shows that CA19-9-mediated pancreatitis is reversible. Fig.61 A shows C pulsed with Dox for 3 days and chased for 4 days PDX ; R LSL Serological levels of amylase and lipase in F mice. Serological levels of amylase and lipase are shown, compared with those from Fig.25 C data for comparison. Fig.61 B shows C pulsed with Dox for 3 days and chased for 4 days PDX ; R LSL ; F H&E staining of mice. Scale bar = 100 μm.

[0094] Fig.62 A shows the 8-day treatment with human isotype control (n=5) or 5B1 (n=5) and the final 7 days of Dox treatment. PDX ; R LSL; Serum amylase and lipase levels in F mice and percentage of pancreatic tissue area showing histological signs of pancreatitis. The horizontal red line in the middle represents the mean value, and the error bars represent the standard deviation; Each data point represents the measurement from a single mouse, and the p value is determined using an unpaired, parametric, two-tailed t-test for amylase and lipase comparisons (t=4.611, 2.981; df=8) and an unpaired, parametric t-test for the percentage of affected pancreas with Welch correction (t=2.935, df=4,065); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Fig.62 B shows H&E staining of representative fields from mice treated with isotype or 5B1 as described above. Scale bar = 100 μm. Fig.62 C-62D shows the effect of isotype ( Fig.62 C) or 5B1( Fig.62 D) pEGFR IHC of a representative treated mouse. Scale bar = 100 μm.

[0095] Fig.63 A-63C shows that CA19-9-mediated pancreatitis can be attenuated by CA19-9 antibodies in vivo. Depicted are C PDX ; R LSL ; H&E staining in F mice ( Fig.63 A), serum amylase and lipase levels ( Fig.63 B) and pEGFR IHC ( Fig.63 C). Scale bar = 100 μm, inset scale bar = 50 μm. Fig.63 In B, sample size is MOPC21 (n=5) or NS19-9 (n=5). Lines represent mean values; each data point represents measurement from a single mouse, and p values ​​were determined using an unpaired, parametric, two-tailed t-test (t=4.108, 2.961, 3.216; df=8, 8, 7).

[0096] Fig.64 A-64C shows that CA19-9-mediated pancreatitis can be attenuated in vivo by CA19-9 antibodies. Fig.64 A shows the results of 96 h of MOPC21 (n=5) or NS19-9 (n=5) treatment and the last 24 h of Dox treatment. PDX ; R LSL ; Percentage of pancreatic tissue area showing histological signs of pancreatitis in F mice. Fig.64B shows the expression of 444 cells from normal mice or C mice treated with Dox in the presence of isotype control MOPC21 or CA19-9 antibody NS19-9. PDX ; R LSL ; Representative whole pancreas histology (H&E) of F mouse. Orange inset represents higher magnification of pancreatitis area. Black inset represents higher magnification of normal pancreatic area containing ductal structures. Scale bar = 100 μm. Fig.64 C shows C PDX ; R LSL ; Amylase and lipase levels in F mice after treatment with a lower dose of Dox (0.5 g / L) in their drinking water. Lines represent mean values; each data point represents measurements from a single mouse, and p values ​​were determined using an unpaired, parametric, two-tailed t-test.

[0097] Fig.65 A shows the C after treatment with Dox and erlotinib or vehicle. PDX ; R LSL ; F H&E staining of mouse pancreas. Scale bars are 200 μm, 200 μm, 100 μm and 50 μm from left to right, respectively. Fig.65 B shows the C after treatment with Dox and erlotinib or vehicle PDX ; R LSL ; Serological levels of amylase and lipase in F mice. Fig.65 C shows H&E staining of C57B1 / 6J mouse pancreas after treatment with sucrose or Dox sucrose and vehicle or erlotinib. Scale bar = 100 μm. Fig.65 D shows serum levels of amylase and lipase in the pancreas of C57B1 / 6J mice after treatment with sucrose or Dox-sucrose and vehicle or erlotinib.

[0098] Fig.66 A-66B shows that secretion of CA19-9 ligand activates EGFR. Fig.66 A shows immunoblot evaluation of organoids from genetically negative (GN) littermates compared with those from C previously treated with Dox or vehicle (24 h). PDX ; R LSL ; F organoids were incubated with conditioned medium. Quantification was performed relative to the untreated time point after normalization to cofilin. Fig.66 B shows the expression of Fc-control or EGFR-conjugated Fc from C cells previously treated with Dox (24 h). PDX ; R LSL Immunoblot evaluation of organoids from GN littermates incubated with conditioned media from F organoids. Quantification was performed relative to the untreated time point after normalization to cofilin.

[0099] Fig.67 A-67D shows an exemplary gating strategy for flow cytometry experiments. Fig.67 A shows flow cytometry reverse gating of Figure 1D. Fig.67 B shows flow cytometry reverse gating of Figure 1E. Fig.67 C shows Fig.50 B Flow cytometry reverse gating. Fig.67 D shows Fig.52 A. Flow cytometry reverse gating. DETAILED DESCRIPTION

[0100] 6.1 Definitions

[0101] As used herein, the term "sialyl Lewis a ”(sLe a ), also known or referred to as sialyl Le a , sialyl Lewis A, sialyl Lewis a, intended to mean a compound of formula C 31 H 52 N2O 23 A tetrasaccharide compound with a molar mass of 820.74 g / mol. a The structure of sLe may include Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ. a It is also a known ligand for E-selectin, also known as endothelial leukocyte adhesion molecule (ELAM). a This includes all possible conformations, including biologically active and inactive conformations.

[0102] As used herein, the term "CA19-9", "CA 19-9", "CA-19.9" or "CA 19.9" refers to a sialyl Lewis a CA19-9 encompasses any antigen that has been modified with one or more sialyl Lewis a Partially modified proteins, such as glycoproteins. For example, CA19-9 includes a sialyl Lewis aGlycoproteins such as C2, CD59, F5, SPARC, MUC1, MUC5AC, MUC16, apolipoprotein, prokinase, A2M, ADAM9, AFP, B3GNT5, CD44, CSF3, CTBS, CTSL2, EFNB3, EPHA3, FGFBP1, IGFBP5, IL18RAP, INHBC, MSLN, NPY, PDGFA, PROM1, RAF1, S100A7, TFF3, TGFB2, TNFRSF10A, WISP1 and WNT9B. CA19-9 also includes sialyl Lewis a Sialyl Lewis a The lipid of epitope, such as glycolipid. Exemplary glycolipid CA19-9 includes glycolipids associated with bile globular membrane (membrane vesicles secreted by normal bile), as described in Uozumi, N et al., J. Proteome Res. 2010, 9, 6345-6353, which is incorporated herein by reference in its entirety. Additional examples of CA19-9 are described in Rho J et al., J Proteomics. 2014 Jan 16; 96: 291-299 and Trinchera M et al., Biology (Basel). 2017 Feb 23; 6 (1). pii: E16, which are incorporated herein by reference in their entirety.

[0103] The terms "preventing / prevent / prevention", when used with respect to pancreatitis and / or damage (e.g., physical or functional), effects or symptoms of pancreatitis, are intended to mean delaying the onset of, hindering the progression of, hindering the appearance of, protecting from, inhibiting or eliminating the appearance of, or reducing the incidence of pancreatitis or damage, effects or symptoms of pancreatitis. For example, even if some patients in a patient population treated with an antibody or antibody fragment provided herein develop pancreatitis, as long as the patient population treated with the antibody or antibody fragment exhibits a reduction in damage, effects, incidence, rate of occurrence or symptoms of pancreatitis when compared to an untreated population, the antibody or antibody fragment still "prevents" pancreatitis.

[0104] As used herein, the terms "treat" or "treatment" when used in reference to pancreatitis are intended to mean eliminating, reducing, managing or controlling pancreatitis or conditions, damage (including, for example, physical or functional damage) and / or effects or symptoms caused by pancreatitis.

[0105] As used herein, the term "alleviate" when used in relation to pancreatitis is intended to mean to reduce, lessen or alleviate pancreatitis or the conditions, damage (including, for example, physical and functional damage) and / or effects or symptoms caused by pancreatitis. For example, one skilled in the art can alleviate the symptoms of pancreatitis by making it more tolerable, slowing the progression of the disease, or restoring the subject to a state without pancreatitis.

[0106] As used herein, the term "management" when used with respect to pancreatitis is intended to mean achieving one or more beneficial effects on a patient by administering an antibody or antibody fragment as provided herein. The beneficial effect may be, for example, regression of pancreatitis, cessation of progression, or less rapid progression, alleviation, maintenance, or delay of worsening symptoms or effects of pancreatitis, and reversal, maintenance, or delay of the spread of damage associated with pancreatitis. In certain embodiments, an antibody or antibody fragment as provided herein is administered to a patient to limit or control the symptoms of pancreatitis in order to prevent progression or worsening of symptoms of pancreatitis.

[0107] As used herein, the term "subject" is intended to refer to an animal, such as a mammal. Mammals encompassed by the term "subject" include, for example, non-primate mammals such as cows, pigs, horses, cats, dogs, rats, and mice, and primate mammals such as monkeys, apes, and humans. "Patient" refers to a human who is a suitable target for the methods provided herein.

[0108] As used herein, the term "pancreatitis" is intended to mean a disease in which the pancreas is inflamed and damaged. Pancreatitis includes acute pancreatitis and chronic pancreatitis. Acute pancreatitis refers to a sudden inflammation of the pancreas that lasts for a short time. Chronic pancreatitis refers to a long-term inflammation of the pancreas. Chronic pancreatitis can occur after an episode of acute pancreatitis. Chronic pancreatitis also includes mild, moderate and severe chronic pancreatitis.

[0109] As used herein, the phrase "therapeutic agent" is intended to mean an agent that can be used to prevent, treat, manage, or alleviate a disorder related to CA19-9 and sLe aAny agent for pancreatitis and / or symptoms associated with expression. The therapeutic agents provided herein include antibodies (as further defined below), proteins, peptides, nucleic acids, peptidomimetics, peptoids, etc. Proteins include natural proteins, proteins designed from scratch, and proteins produced by animals or cells after stimulation, engineering, or both. Examples of proteins include ligands, soluble parts of receptors, and other biological products mainly formed by amino acids. Nucleic acids as therapeutic agents include aptamers or aptamers with nucleic acid sequences to achieve certain secondary structures with desired functional properties (such as binding or blocking), wherein the sequence can be designed or identified by screening a nucleic acid sequence library. Peptides include peptides with certain sequences to achieve desired functional properties (such as binding or blocking), wherein the sequence can be designed or identified by screening a peptide sequence library. For example, the therapeutic agent can be an antibody or functional fragment of the present disclosure. For another example, the therapeutic agent can be a molecule other than the antibody or functional fragment of the present disclosure. The therapeutic agent can also be a molecule for preventing, treating, managing, or alleviating diseases related to CA19-9 and sLe a Any molecule provided by the present disclosure that expresses a disease of interest and / or one or more symptoms associated therewith.

[0110] As used herein, the term "pancreatitis marker" or "marker for pancreatitis" is intended to mean any ascertainable and / or observable indicator of pancreatitis. Pancreatitis markers may include physical changes or damage to the pancreas; functional or physiological changes in the pancreas; functional measurements of the pancreas; histological changes in the pancreas; pancreatic abnormalities in imaging examinations; expression or structural abnormalities of one or more proteins (including glycoproteins in the pancreas); expression or structural abnormalities of one or more carbohydrates in the pancreas; DNA, RNA, microRNA sequence or structural abnormalities; serum indicators of pancreatic inflammation and any measurement or indicator abnormality of pancreatic inflammation and / or pancreatic function. Examples of pancreatitis markers include histological markers such as immune cell invasion, collagen deposition, the presence and level of fibrotic tissue, fat necrosis, interstitial edema, acinar and vascular destruction, interstitial hemorrhage, ductal dilatation, acinar cell homogenization, and CA19-9 expression levels. Pancreatitis markers also include physiological markers such as weight loss, food malabsorption, pancreatic atrophy, pancreatic enlargement, pancreatic inflammation, and bile reflux. Pancreatitis markers may also include serum lipase levels, serum amylase levels, and serum C-reactive protein (CRP) levels.

[0111] As used herein, the term "signaling marker" when used in relation to the pancreas or pancreatitis is intended to refer to a molecule in a signaling cascade in the pancreas or pancreatic cells (e.g., a chain of interconnected biochemical reactions, biophysical reactions, quantitative changes (such as changes in expression levels), and / or positional changes of components of the cascade), wherein activation, stimulation, blocking, inhibition, or alteration of the signaling cascade is the basis of the disease mechanism of pancreatitis. Such signaling markers include, for example, elevated Akt phosphorylation in the pancreas, elevated EGFR phosphorylation in the pancreas, elevated Erk1 / 2 phosphorylation in the pancreas, and elevated CA19-9 expression.

[0112] As used herein, the term "amylase" refers to a group of enzymes that hydrolyze starch and glycogen. Exemplary chemical bonds hydrolyzed by amylases include glycosidic bonds present in starch. Amylases include, for example, α-amylases (EC 3.2.1.1 according to the numerical classification scheme of enzymes called Enzyme Commission numbers; the following EC numbers similarly indicate the enzyme classification scheme according to the Enzyme Commission numbers; alternative names: 1,4-α-D-glucan glucanohydrolase; glycogenase), which catalyzes the internal hydrolysis of (1->4)-α-D-glycosidic bonds in polysaccharides containing three or more (1->4)-α-connected D-glucose units. Amylases also include β-amylases (EC 3.2.1.2; alternative names: 1,4-α-D-glucan maltohydrolase; glycogenase; glycogen amylase), which catalyzes the hydrolysis of (1->4)-α-D-glycosidic bonds in polysaccharides to remove continuous maltose units from the non-reducing end of the chain. Amylases also include, for example, γ-amylase (EC 3.2.1.3; alternative names: glucan 1,4-α-glucosidase; amyloglucosidase; Exo-1,4-α-glucosidase; glucoamylase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase), which catalyzes the continuous hydrolysis of terminal (l->4)-linked α-D-glucose residues from the non-reducing end of the chain and releases β-D-glucose. "Serum amylase" or "serological amylase" refers to the total amylase or total amylase in serum. About 35%-45% of normal serum amylase is of pancreatic origin. Elevated serum amylase levels can be used as a marker for pancreatitis because when the pancreas is inflamed, high levels of amylase will be released into the blood. Normal serum levels of amylase in healthy human subjects are 0-137 U / L, which may be method- and population-related. Thus, in some embodiments, normal serum amylase levels may vary from laboratory to laboratory and from human population to human population.

[0113] As used herein, the term "lipase" refers to a subclass of esterases that catalyze the hydrolysis of fat (lipid). The term lipase can include triacylglycerol lipases (EC 3.1.1.3) that catalyze the hydrolysis of triacylglycerols into glycerol, diacylglycerols, monoglycerols and free fatty acids. The term lipase also includes acylglycerol lipases (EC 3.1.1.23) that catalyze the hydrolysis of monoglycerides of long-chain fatty acids. "Serum lipase" or "serological lipase" refers to the total lipase or total lipase in serum. The lipase of pancreatic origin accounts for a significant and important part of serum lipase. The increase in serum lipase levels can be used as a marker for pancreatitis, because when the pancreas is inflamed, high levels of lipase will be released into the blood. The normal serum level of lipase in healthy human subjects is 12-70U / L, which is related to method and population. Therefore, in some embodiments, normal serum lipase levels can vary due to laboratories and due to human populations.

[0114] As used herein, the term "elevated level" in reference to a marker is intended to mean that the level of the marker is above the normal reference range or the level of a corresponding control subject with normal pancreatic histology, physiology and / or function. Such elevated levels can be 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times or more of the levels of a control subject or a normal reference range with normal pancreatic histology, physiology and / or function. Such reference ranges are further described below.

[0115] As used herein, the term "antibody" is intended to mean a polypeptide product of a B cell within the immunoglobulin class of polypeptides, or a synthetic or recombinantly produced equivalent thereof, which is capable of binding to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), and each amino-terminal portion of each chain contains a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain contains a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering , 2nd edition, Oxford University Press.; Kuby (1997) Immunology, 3rd edition, WH Freeman and Company, New York). In the context of the present disclosure, specific molecular antigens that can be bound by the antibodies of the present disclosure include target carbohydrates sLe a The term antibody encompasses, for example, an anti-sLe alone as described below a Monoclonal antibodies and anti-CA-19-9 monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), anti-sLe with multi-epitope or mono-epitope specificity a Antibodies and anti-CA-19-9 antibody compositions, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity). Antibodies can be human, humanized, chimeric and / or affinity matured, as well as antibodies from other species, such as mice and rabbits, etc. The antibodies provided herein can be any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibodies provided herein include those produced biologically, recombinantly, or synthetically.

[0116] The term "human" when used in reference to an antibody or a functional fragment thereof refers to an antibody or a functional fragment thereof having a human variable region and / or a human constant region or a portion thereof corresponding to a human germline immunoglobulin sequence. Such human germline immunoglobulin sequences were described by Kabat et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. In the context of the present disclosure, human antibodies may include antibodies with sLe a Antibodies that bind and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. Exemplary methods for producing human antibodies are provided in Example 1, but any method well known to those skilled in the art may be used.

[0117] The term "monoclonal antibody" refers to an antibody that is the product of a single cell clone or hybridoma or a population of cells derived from a single cell. Monoclonal antibodies are also intended to refer to antibodies produced by recombinant methods from heavy and light chain encoding immunoglobulin genes to produce a single molecule immunoglobulin species. The amino acid sequence of the antibodies within a monoclonal antibody preparation is essentially homogeneous, and the binding activity of the antibodies within such a preparation exhibits essentially the same antigen binding activity. In contrast, polyclonal antibodies are obtained from different B cells within a population, and the polyclonal antibodies are a combination of immunoglobulin molecules that bind to a specific antigen. Each immunoglobulin of a polyclonal antibody can bind to a different epitope of the same antigen. Methods for producing monoclonal and polyclonal antibodies are well known in the art (Harlow and Lane., Antibodies: ALaboratory Manual , Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (ed.), Antibody Engineering:A Practical Guide , WH Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

[0118] As used herein, the term "functional fragment" when used in relation to an antibody is intended to refer to a portion of an antibody comprising a heavy or light chain polypeptide, which portion retains some or all of the same binding activity as the antibody from which the fragment was derived. Such functional fragments may include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, and small molecule antibodies. Other functional fragments may include, for example, heavy or light chain polypeptides, variable region polypeptides, or complementary determining region (CDR) polypeptides or portions thereof, as long as such functional fragments retain binding activity. Such antibody binding fragments may be described, for example, in Harlow and Lane, Antibodies:A Laboratory Manual , Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec.Biology and Biotechnology:A Comprehensive Desk Reference , New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22: 189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178: 497-515 (1989) and Day, ED, Advanced Immunochemistry , 2nd ed., Wiley-Liss, Inc., New York, NY (1990).

[0119] The term "heavy chain", when used with respect to an antibody, refers to a polypeptide chain of about 50-70 kDa, wherein the amino terminal portion includes a variable region of about 120 to 130 or more amino acids and the carboxyl terminal portion includes a constant region. The constant region can be one of five different types (called α, δ, ε, γ and μ) based on the amino acid sequence of the heavy chain constant region. Different heavy chains are of different sizes: α, δ and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, these different types of heavy chains produce five well-known antibody classes, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, i.e. IgG1, IgG2, IgG3 and IgG4. The heavy chain can be a human heavy chain.

[0120] The term "light chain" when used in relation to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids and the carboxyl terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa or lambda, based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. The light chain can be a human light chain.

[0121] The term "variable domain" or "variable region" refers to a portion of the light or heavy chain of an antibody that is usually located at the amino terminus of the light or heavy chain and is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain, and is used for the binding and specificity of each particular antibody to its particular antigen. The variable domains vary widely in sequence between different antibodies. The variability of the sequence is concentrated in the CDRs, while the smaller variable portions of the variable domains are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. The numbering of the amino acid positions used herein is according to the EU index, as in Kabat et al. (1991) Sequences of Proteins of Immunological interest . (USDepartment of Health and Human Services, Washington, DC.) 5th Edition. The variable region may be a human variable region.

[0122] CDR refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of the immunoglobulin (Ig or antibody) VHβ fold (β-sheet) framework, or one of the three hypervariable regions (L1, L2 or L3) in the non-framework region of the antibody VLβ fold framework. Therefore, CDR is a variable region sequence interspersed within the framework region sequence. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the most highly variable region within the antibody variable (V) domain (Kabat et al., J.Biol.Chem.252:6609-6616 (1977); Kabat, Adv.Prot.Chem.32:1-75 (1978)). CDR region sequences have also been structurally defined by Chothia as those residues that are not part of the conserved β-fold framework and are therefore able to adapt to different conformations (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). Both terms are recognized in the art. The position of CDR in the variable domain of a canonical antibody has been determined by comparing numerous structures (Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); Morea et al., Methods 20: 267-279 (2000)). Because the number of residues in the hypervariable region varies in different antibodies, the additional residues relative to the canonical position are conventionally numbered with a, b, c, etc. next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

[0123] For example, CDRs defined according to the Kabat (hypervariable) or Chothia (structural) designations are listed in Table 1 below.

[0124] Table 1: CDR Definition

[0125]

[0126] 1 The residue numbering follows the nomenclature of Kabat et al., supra.

[0127] 2 The residue numbering follows the nomenclature of Chothia et al., supra.

[0128] One or more CDRs may also be covalently or non-covalently incorporated into a molecule to make the molecule an immunoadhesin. An immunoadhesin may incorporate a CDR as part of a larger polypeptide chain, may covalently link the CDR to another polypeptide chain, or may incorporate the CDR non-covalently. The CDR allows the immunoadhesin to bind to a particular antigen of interest.

[0129] Separation refers to that the molecule mentioned does not contain at least one component found in nature. The term includes antibodies or antibody functional fragments found in their natural environment that are removed from some or all of the other components. The components of the natural environment of the antibody include, for example, red blood cells, white blood cells, platelets, plasma, proteins, nucleic acids, salts and nutrients. The components of the natural environment of the antibody functional fragment include, for example, lipid membranes, organelles, proteins, nucleic acids, salts and nutrients. The antibodies or antibody functional fragments provided herein may also be free of or until substantially free of all these components or any other components of the cell from which they are separated or recombinantly produced.

[0130] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region gene. The heavy chain of a given antibody or functional fragment determines the class of the antibody or functional fragment: IgM, IgG, IgA, IgD or IgE. Each class can have a kappa or lambda light chain. The term "subclass" refers to the slight differences in the heavy chain amino acid sequence that distinguish subclasses. In humans, there are two IgA subclasses (IgA1 and IgA2 subclasses), and there are four IgG subclasses (IgG1, IgG2, IgG3 and IgG4 subclasses). Such classes and subclasses are well known to those skilled in the art.

[0131] As used herein, the term "binds" or "binding" refers to interactions between molecules that form a complex. The interactions can be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules that are bound together by covalent or non-covalent bonds, interactions, or forces. The binding of an antibody or its functional fragment can be detected using, for example, an enzyme-linked immunosorbent assay (method provided in Section 6.3.1) or any of a number of methods well known to those skilled in the art.

[0132] A single antigen binding site on an antibody or functional fragment binds to a target molecule (such as sLe a The strength of the total non-covalent interaction between the single epitopes of the antibody or functional fragment is the affinity of the antibody or functional fragment for the epitope. The association (k1) and dissociation (k -1 ) ratio (k1 / k -1 ) is the association constant K, which is a measure of affinity. For different complexes of antibody or functional fragment and antigen, the value of K varies and depends on k1 and k -1 The association constant K of the antibodies or functional fragments of the present disclosure can be determined using any of the methods provided herein or any other method well known to those skilled in the art.

[0133] The affinity at one binding site does not always reflect the true strength of the interaction between the antibody or functional fragment and the antigen. a )When contacting an antibody containing multiple binding sites, the interaction of the antibody or functional fragment with the antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity. The avidity of an antibody or functional fragment can better measure its binding ability than the affinity of its single binding site. For example, high avidity can compensate for the low affinity of pentamer IgM antibodies that are sometimes found, which may have a lower affinity than IgG, but due to the multivalency of IgM, its high avidity enables it to effectively bind antigens.

[0134] The specificity of an antibody or its functional fragment refers to the ability of a single antibody or its functional fragment to react with only one antigen. An antibody or functional fragment is considered specific when it can distinguish differences in the primary, secondary or tertiary structure of an antigen or in isomeric forms of an antigen.

[0135] As used herein, the term "therapeutically effective amount" is intended to mean an amount of a therapeutic agent (e.g., an antibody or functional fragment provided herein or any other therapeutic agent provided herein) sufficient to achieve a beneficial or desired therapeutic outcome, such as treating a disease, reducing and / or alleviating the severity and / or duration of a given disease and / or symptoms associated therewith. A therapeutically effective amount of a therapeutic agent can be regression, reduction, or alleviation of a disease; reduction or alleviation of advancement or progression of a given disease; reduction or alleviation of recurrence, development, or onset of a given disease and / or an amount necessary to improve or enhance the preventive or therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or functional fragment provided herein). The present disclosure provides therapeutically effective amounts of antibodies and antibody fragments provided herein.

[0136] As used herein, when a dose is referred to as a higher amount, such as a dose at a "higher amount", the "amount" is intended to mean that the dose at a "higher amount" contains a higher amount of antibody or antibody fragment to achieve a beneficial or expected effect. Such "higher amounts" include, for example, an increase in the total amount of the antibody or antibody fragment administered in a defined time period compared to a control or basal dose in the same time period or a higher dose. Such "higher amounts" include doses that are administered more frequently in a defined time window. For example, a dose injected 4 times a day is a higher amount of dose compared to a dose injected 3 times a day, where all other conditions are the same. The "higher amount" dose also includes an increase or higher amount of antibody or antibody fragment administered each time, while keeping the same frequency of administration, such as 2 mg of antibody injected 3 times a day compared to 1 mg of antibody injected 3 times a day. The "higher amount" dose also includes a change in the frequency of administration and the amount of each administration that is changed in a given time window, as long as the product of the frequency of administration and the amount of each administration in a given window is higher than the product in the control or basal dose. For example, a dose of 1 mg of an antibody injected 6 times a day is a "higher amount" dose than a dose of 1.5 mg of an antibody injected 3 times a day. Similarly, in continuous administration, a "higher amount" dose includes a dose in which the product of the administration rate and the duration of administration is higher than the product in the control dose. When a dose is referred to as a lower amount (e.g., a dose at a "lower amount"), "amount" is intended to mean the opposite of the dose at a "higher amount" as provided herein.

[0137] As used herein, the term "effector" when used in relation to CA19-9 is intended to mean a molecule in the pancreas or pancreatic cells in a signaling cascade that is the basis of the disease mechanism of pancreatitis, wherein the molecule will respond to the presence or stimulation of CA19-9, e.g., undergo a biochemical, biophysical, expression level or location change. Examples of CA19-9 effectors include molecules in the EGFR signaling pathway such as EGFR, Akt and Erk, as well as molecules that can bind directly or indirectly to CA19-9, and molecules that can be directly or indirectly affected by CA19-9 with an observable biological effect.

[0138] As used herein, the term "peptidomimetic molecule" or "peptidomimetic" is intended to mean a peptide-like molecule having the activity of a peptide on which it is structurally based. Such peptidomimetics include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, and peptoids, and have the activity of the peptide from which the peptidomimetics are derived, such as selective binding activity (see, e.g., Goodman and Ro, Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry and Drug Discovery" Vol. 1 (M E. Wolff, ed.; John Wiley & Sons (1995), pp. 803-861). Peptidomimetic molecules include molecules containing mimetics (amino acid mimetics) of amino acid residues, including but not limited to piperazine core molecules, keto-piperazine core molecules, and diazepine core molecules. Such amino acid mimetics can include both carboxyl and amino groups and groups corresponding to amino acid side chains, or, in the case of glycine mimetics, have no side chains except hydrogen.

[0139] 6.2 Methods of treating pancreatitis

[0140] Provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sialyl Lewis a The antibody or its functional fragment (anti-sLe a ). Also provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising: (1) administering to the subject a first dose of a sialyl Lewis a The antibody or its functional fragment (anti-sLe a ), (2) determining the severity of pancreatitis in the subject, and (3) if associated with administration of anti-sLe a The severity of pancreatitis in the subject is reduced compared to the severity of pancreatitis in the subject before the administration of the antibody, then a second dose of the anti-sLe is administered in an amount that is the same or lower than the first dose. a or if the severity of pancreatitis in the subject has not decreased, administering a second dose of an antibody or a functional fragment thereof that binds to sLe in an amount higher than the first dose; a Antibodies or functional fragments thereof that bind to the sLe a Antibodies are described in Section 6.2.1.1.

[0141] Also provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a therapeutic agent that binds to CA19-9.

[0142] Further provided herein is a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, comprising: (1) administering to the subject a therapeutic agent that binds to CA19-9 at a first dose, (2) determining the severity of pancreatitis in the subject, and (3) if the severity of pancreatitis in the subject is reduced compared to the severity of pancreatitis in the subject before administration of the agent, administering a second dose of the agent in an amount that is the same as or lower than the first dose; or if the severity of pancreatitis in the subject is not reduced, administering the second dose of the agent in an amount that is higher than the first dose.

[0143] In all methods provided herein, particularly in the methods described in paragraphs

[0139] to

[0141] : therapeutic agents that can be used are described in Section 6.2.1, selection of patients for treatment is described in Section 6.2.2, dosing regimens for administering the agents are described in Section 6.2.3 below, biomarkers that can be used to identify the therapeutic agents, select the patients, determine the results of these methods and / or serve as criteria for these methods in any way are described in Section 6.2.2 below, and the treatment results are described in Section 6.2.2 below. Therefore, those skilled in the art will understand that the methods provided herein include all permutations and combinations of patients, therapeutic agents, dosing regimens, biomarkers, and treatment results as described above and below.

[0144] As described in Section 6.2.2 below about patient population, the pancreatitis that can be treated, prevented, managed and alleviated by the methods provided herein include chronic pancreatitis (including various stages of chronic pancreatitis) and acute pancreatitis as described in detail in the definitions and patient population sections above and below. In some embodiments, the methods provided herein are applicable to mild chronic pancreatitis, moderate chronic pancreatitis and / or severe chronic pancreatitis. The methods provided herein can also be used for mild acute pancreatitis, moderate acute pancreatitis and / or severe acute pancreatitis. Because patients with acute pancreatitis can recover but develop into chronic pancreatitis, patients with chronic pancreatitis can develop into acute pancreatitis, and patients can develop from a stage of chronic or acute pancreatitis to another stage of chronic and acute pancreatitis, so the methods provided herein can be used for patients with any permutation or combination of medical records of various stages of chronic or acute pancreatitis as described herein.

[0145] As described below in Section 6.2.1 on therapeutic agents, the agents provided herein include anti-sialylated Lewis a Antibodies, Sialyl Lewis a Bind and / or modulate sialyl Lewis aAny permutation or combination of active peptides or peptidomimetics, or any suitable therapeutic agent as provided by the present disclosure.

[0146] Therapeutic agents provided herein also include anti-CA19-9 antibodies, peptides or peptidomimetics that bind to CA19-9 and / or modulate the activity of CA19-9, or any permutation or combination of any suitable therapeutic agents as provided by the present disclosure.

[0147] As described below in Section 6.2.1 on therapeutic agents, the agents provided herein in the methods for treating pancreatitis can be isolated or purified with various levels of purity and impurities; and the agents provided herein can also be in various pharmaceutical compositions and formulated for various routes of administration. Thus, the methods provided herein include using agents isolated or purified with various levels of purity and impurities, agents in various pharmaceutical compositions, and agents formulated for various routes of administration as provided in the present disclosure.

[0148] The methods provided herein can be used in various subjects as disclosed herein, including mammals. In one embodiment, the methods provided herein can be used in humans.

[0149] As disclosed below in Section 6.2.2 on patient populations and biomarkers, various patients can be subjected to the methods provided herein, and various biomarkers can be used to select patients and evaluate the results of the methods provided herein. In some embodiments, the methods provided herein can be used for selective populations of patients, including patients with mild, moderate or severe chronic pancreatitis, and patients with mild, moderate or severe acute pancreatitis. In other embodiments, the methods provided herein can be used for selective populations of patients, including patients with the following: pancreatic atrophy, increased pancreatic inflammation, increased immune cell invasion in pancreatitis, increased collagen deposition in pancreatitis, increased fibrotic tissue in pancreatitis, increased fat necrosis in pancreatitis, increased interstitial edema in pancreatitis, increased acinar and vascular destruction in pancreatitis, increased interstitial hemorrhage in pancreatitis, increased ductal dilatation in pancreatitis, increased homogenization of acinar cells in pancreatitis, sLe in pancreatitis a The method provided herein can also be used for all permutations and combinations of various patient populations and various biomarkers described herein.

[0150] In other embodiments, the methods provided herein can be used in various patients or in conjunction with various biomarkers for the treatment outcomes of these methods as provided in Section 6.2.2, which can include reduced pancreatic atrophy, reduced pancreatic inflammation, reduced immune cell invasion in pancreatitis, reduced collagen deposition in pancreatitis, reduced fibrotic tissue in pancreatitis, reduced fat necrosis in pancreatitis, reduced interstitial edema in pancreatitis, reduced acinar and vascular destruction in pancreatitis, reduced interstitial hemorrhage in pancreatitis, reduced ductal dilatation in pancreatitis, reduced homogenization of acinar cells in pancreatitis, reduced sLe in pancreatitis a The methods provided herein can further be used for all permutations and combinations of various patient populations, various biomarkers and / or treatment outcomes as provided herein.

[0151] 6.2.1 Therapeutic Agents for Use with Methods of Treating Pancreatitis

[0152] Various molecules with such properties can be used in the methods provided herein. The present disclosure provides that elevated expression of CA19-9 in the pancreas triggers signal transduction and / or pathological pathways, which are the basis for the initiation, development and exacerbation of pancreatitis. Therefore, the present disclosure provides molecules that can inhibit, block, reverse, reduce or attenuate elevated expression of CA19-9, sialyl Lewis a Therapeutic agents that increase the level of or a combination thereof can be used in the methods provided herein for preventing, treating, alleviating or managing pancreatitis. The present disclosure further provides that elevated CA19-9 expression leads to activation of the EGFR signaling pathway. In some specific embodiments, the therapeutic agents that can be used in the methods provided herein include agents that have the activity of reducing one or more signaling markers in the pancreas, wherein the signaling markers are selected from the group consisting of: Akt phosphorylation in the pancreas, EGFR phosphorylation in the pancreas, Erk1 / 2 phosphorylation in the pancreas, CA19-9 levels in the pancreas, and sialyl Lewis in the pancreas. a level.

[0153] As discussed above, CA19-9 encompasses a sialyl Lewis a The present disclosure provides a list of CA19-9 identified by immunoprecipitation assays with anti-CA19-9 antibodies (such as 5B1 and NS19-9 in Examples 1, 2, and 3). The present disclosure also provides CA19-9, which includes sialyl Lewis antigens known to those skilled in the art. aProteins such as glycoproteins and lipids such as glycolipids, such as those described in the definition of CA19-9 above.

[0154] As noted above, therapeutic agents useful in the methods provided herein include various classes of agents, eg, antibody agents (such as anti-CA19-9 as described in Section 6.2.1.1) and peptides or peptidomimetics as described in Section 6.2.1.2.

[0155] The therapeutic agent can regulate the activity of CA19-9. "Regulating" the activity of a molecule refers to changing the activity of a molecule. For example, regulating CA19-9 activity includes causing an increase or decrease in the size of a certain activity of CA19-9 compared to the size of the CA19-9 activity in the absence of such regulation. In some instances, regulating CA19-9 activity includes reducing the size of one or more activities of CA19-9. In some instances, regulating CA19-9 activity includes completely preventing one or more activities of CA19-9. In other instances, regulating CA19-9 activity includes increasing the size of at least one activity of CA19-9. In another instance, regulating CA19-9 activity includes inducing CA19-9 activity that would not normally occur in the absence of such regulation. A "regulator" relating to a target molecule being regulated refers to a molecule that can "regulate" the activity of the target molecule described in this paragraph.

[0156] The activity of CA19-9 refers to the activity of CA19-9 in cells, tissues, organs, organisms or any biological system, such as in the pancreas or pancreatic cells. The effect of CA19-9 refers to the result of CA19-9 activity in cells, tissues, organs, organisms or any biological system, such as in the pancreas or pancreatic cells. Such activity of CA19-9 includes the immediate activity of CA19-9, such as inducing biochemical, biophysical, positional or quantitative changes in another molecule that directly binds to CA19-9 or otherwise interacts with CA19-9. Then, the corresponding effect of CA19-9 will include the results induced by CA19-9 of other molecules that directly bind to CA19-9 or otherwise interact with CA19-9, such as biochemical, biophysical, positional or quantitative changes. Such activity also includes activities such as inducing a biochemical, biophysical, positional or quantitative change on another molecule that does not directly bind to or otherwise interact with CA19-9 but is further downstream in a cascade of activity (e.g., a signaling cascade) induced by CA19-9 in response to the presence or stimulation of CA19-9. The corresponding effect of CA19-9 will then include consequences such as a biochemical, biophysical, positional or quantitative change induced by CA19-9 of another molecule that does not directly bind to or otherwise interact with CA19-9 but is further downstream in a cascade of activity (e.g., a signaling cascade) induced by CA19-9 in response to the presence or stimulation of CA19-9. Examples of CA19-9 activity include activation of the EGFR signaling pathway, resulting in increased phosphorylation of molecules such as EGFR, Akt, and Erk, binding to molecules such as selectins (including E-selectin) that can interact directly or indirectly with CA19-9, and changes in the levels of molecules that can be directly or indirectly affected by CA19-9 in the form of observable biological effects.

[0157] In some embodiments, the therapeutic agents provided herein can antagonize CA19-9 or CA19-9 activity. This therapeutic CA19-9 antagonist can inhibit, reduce or completely block one or more biological activities of CA19-9. Antagonists of CA19-9 activity include molecules that can block, inhibit or reduce CA19-9-mediated or CA19-9-dependent signal transduction in cells expressing CA19-9 or expressing CA19-9 ligands (such as CA19-9 receptors). Antagonists of CA19-9 activity also include molecules that can block, inhibit or reduce CA19-9 signal transduction. In some embodiments, antagonists of CA19-9 activity also include molecules that can block, inhibit or reduce CA19-9 and natural CA19-9 binding molecules. In other embodiments, antagonists of CA19-9 activity additionally include molecules that can block, inhibit or reduce CA19-9 and CA19-9 ligands such as CA19-9 receptors. In another embodiment, antagonists of CA19-9 activity also include molecules that can block, inhibit or reduce remodeling, processing, conformational changes or modifications of CA19-9 binding molecules or CA19-9 effectors.An "antagonist" of a target molecule is "antagonistic" to the target molecule.

[0158] In some embodiments, the therapeutic agents provided herein can be blocking antibodies, neutralizing antibodies, or antagonist antibodies to CA19-9 or CA19-9 activity, wherein the antibody binds to CA19-9 and acts as an antagonist of CA19-9 or CA19-9 activity as defined in paragraph

[0156] above.

[0159] 6.2.1.1. The antibodies in the methods serve as therapeutic agents.

[0160] In some embodiments, the therapeutic agent used in the methods provided herein is an antibody that has the ability to inhibit, block, reverse, reduce or attenuate elevated expression of CA19-9 in the pancreas, sialyl Lewis a Exemplary antibodies suitable for use in the method include anti-CA19-9 antibodies and anti-sialyl Lewis a Antibodies. In one embodiment, exemplary antibodies suitable for use in the methods are blocking or neutralizing antibodies.

[0161] In some specific embodiments, the therapeutic agent used in the methods provided herein is an anti-CA19-9 antibody or an anti-sialyl Lewis antibody. a In other specific embodiments, the therapeutic agent used in the methods provided herein is a blocking anti-CA19-9 antibody or a blocking anti-sialylated Lewis a Antibody.

[0162] As will be appreciated by those of ordinary skill in the art, anti-sialylated Lewis a The antibody may be a subset of an anti-CA19-9 antibody, and both antibodies may be used in the methods provided herein. In some embodiments, the antibodies useful in the methods provided herein may be antibodies that bind to CA19-9 and / or sialyl Lewis a A binding antibody or a functional fragment thereof, wherein the antibody comprises a VH domain having an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO:2, residues 20-142 of SEQ ID NO:6, residues 20-142 of SEQ ID NO:10, and residues 20-145 of SEQ ID NO:14.

[0163] In another embodiment, the antibodies useful in the methods provided herein can be antibodies that bind to CA19-9 and / or sialyl Lewis a A binding antibody or a functional fragment thereof, wherein the antibody comprises a VL domain, and the VH domain has an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO:4, residues 20-129 of SEQ ID NO:8, residues 20-130 of SEQ ID NO:12, and residues 23-130 of SEQ ID NO:16.

[0164] In another embodiment, the anti-CA19-9 antibody in the method provided herein can be an antibody or a functional fragment thereof, wherein the antibody heavy chain or light chain or a functional fragment thereof has the Figure 8 One or more complementary determining regions (CDRs) depicted in or listed in Table 2. In another aspect, the anti-CA19-9 antibodies in the methods provided herein may include complement dependent cytotoxicity (CDC) activity and / or antibody dependent cell-mediated cytotoxicity (ADCC) activity of any of the clonal isolates 5B1, 9H3, 5H11 or 7E3 described in U.S. Pat. No. 9,475,874, which is incorporated by reference in its entirety. Methods for evaluating the specificity, affinity and / or avidity of antibodies or functional fragments thereof are well known in the art, and exemplary methods are provided herein.

[0165] Table 2: CDRs of cloned isolates

[0166]

[0167] In some embodiments, the anti-CA19-9 antibody in the methods provided herein may comprise an antibody or a functional fragment thereof having less than six CDRs. In some embodiments, the therapeutic anti-CA19-9 antibody or a functional fragment thereof comprises one, two, three, four or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3. In a specific embodiment, the therapeutic anti-CA19-9 antibody or a functional fragment thereof comprises one, two, three, four or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and / or VL CDR3 of the clonal isolates 5B1, 9H3, 5H11 or 7E3 described in U.S. Patent No. 9,475,874, which is incorporated by reference in its entirety.

[0168] In some embodiments, the present disclosure provides therapeutic anti-CA19-9 antibodies or functional fragments thereof, wherein the antibodies or functional fragments comprise a variable heavy (VH) chain domain having the CDR1, CDR2 and CDR3 amino acid sequences of clonal isolates 5B1, 9H3, 5H11 or 7E3 as described in U.S. Patent No. 9,475,874. Such VH domains may comprise amino acid residues 55-62, 70-77 and 116-131 of SEQ ID NO: 2, or alternatively amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 6, or alternatively amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 10, or alternatively amino acid residues 45-52, 70-77 and 116-134 of SEQ ID NO: 14.

[0169] In another embodiment, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof, wherein the antibody comprises a variable light (VL) chain domain having the CDR1, CDR2 and CDR3 amino acid sequences of clonal isolates 5B1, 9H3, 5H11 or 7E3 as described in U.S. Patent No. 9,475,874. Such a VL domain may comprise amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 4, or alternatively amino acid residues 45-52, 70-72 and 109-119 of SEQ ID NO: 8, or alternatively amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 12, or alternatively amino acid residues 49-53, 72-74 and 111-120 of SEQ ID NO: 16.

[0170] In some embodiments, the present disclosure provides therapeutic anti-CA19-9 antibodies or functional fragments thereof, wherein the antibodies are combined with sLe a Therefore, in some aspects, the present disclosure provides a method for combining with sLe a A bound isolated antibody or a functional fragment thereof, wherein the antibody or a functional fragment thereof comprises a VH domain having an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO:2, residues 20-142 of SEQ ID NO:6, residues 20-142 of SEQ ID NO:10, and residues 20-145 of SEQ ID NO:14.

[0171] In some embodiments, the present disclosure provides a A conjugated therapeutic anti-CA19-9 antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a VL domain having an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO:4, residues 20-129 of SEQ ID NO:8, residues 20-130 of SEQ ID NO:12, and residues 23-130 of SEQ ID NO:16.

[0172] In some embodiments, the present disclosure provides a A conjugated therapeutic anti-CA19-9 antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a VH domain and a VL domain, wherein the VH domain and the VL domain respectively comprise an amino acid sequence selected from the group consisting of: residues 20-142 of SEQ ID NO:2 and residues 20-130 of SEQ ID NO:4; residues 20-142 of SEQ ID NO:6 and residues 20-129 of SEQ ID NO:8; residues 20-142 of SEQ ID NO:10 and residues 20-130 of SEQ ID NO:12; and residues 20-145 of SEQ ID NO:14 and residues 23-130 of SEQ ID NO:16.

[0173] In some embodiments, the present disclosure provides a method for combining sLe a Therapeutic anti-CA19-9 antibodies or functional fragments thereof, wherein the antibodies or functional fragments have one or more CDRs depicted as listed in Table 2. In some other embodiments, the present disclosure provides therapeutic anti-CA19-9 antibodies or functional fragments thereof, which antibodies or functional fragments thereof comprise one or more CDRs (particularly CDR3) and can be used with sLe as described in U.S. Pat. No. 9,475,874. aSpecific Binding. In some aspects, the therapeutic anti-CA19-9 antibodies or functional fragments thereof in the methods provided herein may include the CDC activity and / or ADCC activity of any of the clonal isolates 5B1, 9H3, 5H11, or 7E3 as described in U.S. Pat. No. 9,475,874.

[0174] In some embodiments, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof in the method, wherein the antibody comprises a VH chain domain having the CDR1, CDR2 and CDR3 amino acid sequences of clonal isolates 5B1, 9H3, 5H11 or 7E3 as described in U.S. Patent No. 9,475,874. Such a VH domain may comprise amino acid residues 55-62, 70-77 and 116-131 of SEQ ID NO: 2, or alternatively amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 6, or alternatively amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 10, or alternatively amino acid residues 45-52, 70-77 and 116-134 of SEQ ID NO: 14.

[0175] In some embodiments, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof in the method, wherein the antibody comprises a VL chain domain having the CDR1, CDR2 and CDR3 amino acid sequences of clonal isolates 5B1, 9H3, 5H11 or 7E3 as described in U.S. Patent No. 9,475,874. Such a VL domain may comprise amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 4, or alternatively amino acid residues 45-52, 70-72 and 109-119 of SEQ ID NO: 8, or alternatively amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 12, or alternatively amino acid residues 49-53, 72-74 and 111-120 of SEQ ID NO: 16.

[0176] In some embodiments, the therapeutic anti-CA19-9 antibody or its functional fragment in the method is a monoclonal antibody. In some embodiments, the therapeutic anti-CA19-9 antibody or its functional fragment in the method is an IgG or IgM isotype. In other embodiments, the therapeutic anti-CA19-9 antibody or its functional fragment in the method is an antibody of the IgG1, IgG2, IgG3 or IgG4 subclass.

[0177] In some embodiments, the therapeutic anti-CA19-9 antibody or its functional fragment in the method can be, but is not limited to, Fab, Fab', F(ab')2, Fabc, scFV, diabody, triabody, small molecule antibody or single domain antibody (sdAB). In some aspects, the present disclosure provides diabodies for the method, wherein the diabodies comprise the amino acid sequence SEQ ID NO: 18 or 20. In some aspects, such diabodies of the present disclosure can be encoded by a polynucleotide having a nucleic acid sequence SEQ ID NO: 17 or 19. With respect to antibodies and their functional fragments, various forms, changes and modifications are well known in the art. The sLe of the present disclosure a A CA19-9 specific antibody fragment may include any of these various antibody forms, changes and modifications. Examples of such various forms and terms known in the art are listed below.

[0178] In some embodiments, the antibodies useful in the methods provided herein can be antibodies that bind to CA19-9 and / or sialyl Lewis a The antibody or its functional fragment thereof that binds to CA19-9 and / or sialyl Lewis antigen is a polypeptide that binds to ... a The antibody or its functional fragment thereof that binds to CA19-9 and / or sialyl Lewis antigen. a A binding antibody or a functional fragment thereof, wherein the antibody comprises a light chain consisting of the light chain amino acid sequence of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059; and a heavy chain consisting of the heavy chain amino acid sequence of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059.

[0179] In another embodiment, the anti-CA19-9 antibody in the method provided herein can be an antibody or a functional fragment thereof, wherein the antibody heavy chain or light chain or a functional fragment thereof has one or more complementary determining regions (CDRs) of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059. On the other hand, the anti-CA19-9 antibody in the method provided herein can include complement dependent cytotoxicity (CDC) activity and / or antibody dependent cell-mediated cytotoxicity (ADCC) activity of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059. Methods for evaluating the specificity, affinity and / or avidity of an antibody or a functional fragment thereof are well known in the art, and exemplary methods are provided herein.

[0180] In some embodiments, the anti-CA19-9 antibody in the methods provided herein may comprise an antibody or a functional fragment thereof having less than six CDRs. In some embodiments, the therapeutic anti-CA19-9 antibody or a functional fragment thereof comprises one, two, three, four or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and / or VL CDR3 of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059. In a specific embodiment, the therapeutic anti-CA19-9 antibody or a functional fragment thereof comprises one, two, three, four or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of clonal isolate No. SW-1116-19-9 (also referred to as 1116-NS-19-9, NS-19-9 or NS19-9), which is described in U.S. Pat. No. 4,471,057, which is incorporated by reference in its entirety.

[0181] In some embodiments, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof, wherein the antibody or functional fragment comprises a variable heavy (VH) chain domain having CDR1, CDR2 and CDR3 amino acid sequences of the VH chain domain CDR1, CDR2 and CDR3 sequences of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059. In another embodiment, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof, wherein the antibody comprises a variable light (VL) chain domain having CDR1, CDR2 and CDR3 amino acid sequences of the VL chain domain CDR1, CDR2 and CDR3 sequences of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059. In other embodiments, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof, wherein the antibody comprises a VL chain domain having CDR1, CDR2 and CDR3 amino acid sequences of the VL chain domain CDR1, CDR2 and CDR3 sequences of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059; and a VH chain domain having CDR1, CDR2 and CDR3 amino acid sequences of the VH chain domain CDR1, CDR2 and CDR3 sequences of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059.

[0182] In some embodiments, the present disclosure provides a An isolated antibody or a functional fragment thereof that binds to sLe, wherein the antibody or the functional fragment thereof comprises a VH domain having the amino acid sequence of the variable heavy chain of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059. In some embodiments, the present disclosure provides a method for binding to sLe a The present invention relates to a therapeutic anti-CA19-9 antibody or a functional fragment thereof that binds to sLe, wherein the antibody or the functional fragment thereof comprises a VL domain having an amino acid sequence of a variable light chain of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059. a A conjugated therapeutic anti-CA19-9 antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a VL domain having the amino acid sequence of the variable light chain of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059; and a VH domain having the amino acid sequence of the variable heavy chain of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059.

[0183] In some embodiments, the antibodies useful in the methods provided herein can be antibodies that bind to CA19-9 and / or sialyl Lewis a The antibody or its functional fragment that binds to CA19-9, wherein the antibody comprises a heavy chain consisting of the heavy chain amino acid sequence of the cloned isolate NS19-9. In another embodiment, the antibody that can be used in the methods provided herein can be an antibody that binds to CA19-9 and / or sialyl Lewis a The antibody or its functional fragment that binds to CA19-9, wherein the antibody comprises a light chain consisting of the light chain amino acid sequence of the cloned isolate NS19-9. In other embodiments, the antibody that can be used in the methods provided herein can be a light chain that binds to CA19-9 and / or sialyl Lewis a A binding antibody or a functional fragment thereof, wherein the antibody comprises a light chain consisting of the light chain amino acid sequence of the clonal isolate NS19-9, and a heavy chain consisting of the heavy chain amino acid sequence of the clonal isolate NS19-9.

[0184] In some embodiments, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof in the method, wherein the antibody comprises a VH chain domain having the CDR1, CDR2 and CDR3 amino acid sequences of the clonal isolate NS19-9. In some embodiments, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof in the method, wherein the antibody comprises a VL chain domain having the CDR1, CDR2 and CDR3 amino acid sequences of the clonal isolate NS19-9. In another embodiment, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof in the method, wherein the antibody comprises a VH chain domain having the CDR1, CDR2 and CDR3 amino acid sequences of the clonal isolate NS19-9, and a VL chain domain having the CDR1, CDR2 and CDR3 amino acid sequences of the clonal isolate NS19-9.

[0185] In some other aspects, the present disclosure provides a method for a An isolated antibody or functional fragment thereof that binds to sLe, wherein the antibody or functional fragment thereof comprises a VH domain having a variable heavy chain amino acid sequence of the clone isolate NS19-9. In some embodiments, the present disclosure provides a method for binding to sLe a In another embodiment, the present disclosure provides a therapeutic anti-CA19-9 antibody or a functional fragment thereof that binds to sLe aA conjugated therapeutic anti-CA19-9 antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a VL domain having a variable light chain amino acid sequence of the clonal isolate NS19-9, and a VH domain having a variable heavy chain amino acid sequence of the clonal isolate NS19-9.

[0186] In some embodiments, the therapeutic antibody or functional fragment thereof in the method can be produced by suitable means (e.g., recombinantly, biologically, or synthetically) and purified by suitable means (e.g., chromatography, centrifugation, tag-based affinity separation, or differential dissolution rate) as described in U.S. Pat. No. 9,475,874, which is incorporated herein by reference in its entirety.

[0187] The therapeutic antibodies in the methods provided herein comprise Fab fragments. Fab fragments refer to monovalent fragments consisting of VL, VH, CL and CH1 domains; F(ab')2 fragments are bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; Fd fragments consist of VH and CH1 domains; Fv fragments consist of the VL and VH domains of a single arm of an antibody; and dAb fragments (Ward et al., Nature 341:544-546, (1989)) consist of the VH domain.

[0188] The therapeutic antibodies in the methods provided herein include and may have one or more binding sites. If there are more than one binding site, the binding sites may be identical or different from each other. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, and "bispecific" or "bifunctional" antibodies have two different binding sites.

[0189] The therapeutic antibodies in the methods provided herein can also be single-chain antibodies. Single-chain antibodies (scFv) refer to antibodies in which the VL and VH regions are connected via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous polypeptide chain, wherein the linker is long enough to allow the protein chain to fold itself and form a monovalent antigen binding site (see, e.g., Bird et al., Science 242: 423-26 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-83 (1988)). Diabodies refer to bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises a VH domain and a VL domain connected by a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing each domain to pair with a complementary domain on the other polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121-23 (1994)). If the two polypeptide chains of a diabody are identical, the diabody produced by pairing them will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare diabodies with two different antigen-binding sites. Similarly, triabodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which antigen-binding sites may be the same or different.

[0190] The present disclosure also provides therapeutic antibodies or functional fragments thereof, which are derivatives of 5B1, 9H3, 5H11 and / or 7E3 as described in U.S. Pat. No. 9,475,874 and NS19-9 as described in U.S. Pat. No. 4,471,057, wherein the antibodies or functional fragments bind to CA19-9 and sLe a Combination. Mutations can be introduced into the nucleotide sequence encoding the antibodies or functional fragments thereof of the present disclosure using standard techniques well known to those skilled in the art, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis to produce amino acid substitutions. In some aspects, the derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule.

[0191] In some embodiments, the disclosure provides therapeutic antibodies or their functional fragments, which have naturally occurring amino acids, conservative substitutions, non-naturally occurring amino acids, amino acid analogs and modified forms of mimetics, as long as such antibodies or functional fragments retain functional activity as defined herein. In one embodiment, the derivative has a conservative amino acid substitution carried out on one or more predicted non-essential amino acid residues. Conservative amino acid substitutions are a kind of conservative amino acid substitutions in which the amino acid residue is replaced by an amino acid residue with a side chain with a similar charge. Families of amino acid residues with side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody or functional fragment thereof can be expressed, and the activity of the antibody or functional fragment can be determined.

[0192] In some embodiments, the present disclosure provides therapeutic antibodies or functional fragments thereof having modified fucosylation, galactosylation and / or sialylation of the Fc fragment contained within the antibody or functional fragment of the present disclosure. Such modifications of the Fc fragment can affect Fc receptor-mediated activity, as discussed in Peipp et al., Blood, 112(6):2390-2399 (2008). For example, glycoengineered therapeutic antibodies lacking core fucose residues from Fc N-glycans exhibit robust ADCC and higher efficacy at lower concentrations compared to fucosylated counterparts. Shields et al., J. Biol. Chem., 277(30):26733-40 (2002); Okazaki et al., J Mol Biol.,336:1239-1249 (2004); Natsume et al., J. Immunol. Methods., 306:93-103 (2005). Methods for modifying the fucosylation, galactosylation and / or sialylation of antibodies or functional fragments thereof are well known in the art. For example, defucosylation methods can be divided into three methods: (1) conversion of the N-glycosylation pathway of non-mammalian cells to a "humanized" non-fucosylated pathway; (2) inactivation of the N-glycan fucosylation pathway of mammalian cells and (3) in vitro chemical synthesis of non-fucosylated N-glycoproteins or enzymatic modification of N-glycans to non-fucosylated forms, as described in Yamane-Ohnuki et al., MAbs., 1(3):230-236 (2009). It will be appreciated that any of these methods, or any other method well known in the art, may be used to generate antibodies or functional fragments thereof with modified fucosylation, galactosylation and / or sialylation.

[0193] 6.2.1.2. The peptides and peptidomimetics in the methods serve as therapeutic agents.

[0194] Peptides and peptidomimetics provide the structural diversity required for selective and high affinity interactions with target proteins. The present disclosure provides peptides and peptidomimetics as therapeutic agents in the methods. In some embodiments, the therapeutic agents that can be used in the methods provided herein are peptides or peptidomimetics that have the ability to inhibit, block, reverse, reduce or attenuate elevated CA19-9 expression in the pancreas, elevated EGFR signaling pathways in the pancreas, sialyl Lewis in the pancreas, a Exemplary peptides or peptidomimetics suitable for use in the method include peptides or peptidomimetics that specifically bind to a target selected from the group consisting of CA19-9 and sialyl Lewis a In one embodiment, an exemplary peptide suitable for use in the method is a blocking or neutralizing peptide. In another embodiment, an exemplary peptidomimetic suitable for use in the method is a blocking or neutralizing peptidomimetic.

[0195] The therapeutic peptide in the method can be based on the sequence of the binding region between two proteins. The linear sequence can be derived from the ring in the structured domain, or from the disordered region between the protein end or the restricted domain. In order to achieve the desired effect, the therapeutic peptide can be targeted to the appropriate site in the organism, tissue or cell via merging with the targeting sequence, and combined with the expected protein target in vivo. In one embodiment, the therapeutic peptide is a short peptide sequence, for example, no more than 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100 amino acid whose function is combined and blocked to reach the desired activity as provided herein. These linear sequences can be determined based on available structural information on the target protein, such as structure-based design of peptides or peptidomimetics. In addition, the peptide libraries and array technologies described in Section 6.2.1.2 can be used to screen and select therapeutic peptides and peptidomimetics for preventing, treating, alleviating or managing targets associated with pancreatitis as described in paragraph

[0189] .

[0196] The activity of therapeutic peptides or variants thereof can be identified, tested and validated from peptide or peptidomimetic libraries in any of the assays described in Section 6.3 and the Examples. Therapeutic peptides can be screened and identified from peptide or peptidomimetic libraries according to the assays described in Section 6.3 and Section 6.2.1.2. The peptides can be produced recombinantly or synthetically according to methods well known to those of ordinary skill in the art, for example, as described in WO 2007 / 019545; WO 2015 / 009820A2; and WO 2009 / 094172 A2, which are hereby incorporated by reference in their entirety.

[0197] Peptide libraries and peptidomimetics libraries from which therapeutic peptides can be identified and selected are well known to those of ordinary skill in the art. For example, a suitable peptide library can be a biological peptide library, wherein the peptide library has a DNA sequence encoding the peptide sequence of a genotype or the peptide sequence, and the DNA sequence is related to the expression of the peptide as a part of the normal structure of the library. Exemplary biological peptide libraries include biological peptide libraries displayed by bacteriophage, bacteria, ribosomes, mRNA, yeast, cDNA, retroviruses, baculoviruses and mammalian cells. Suitable peptide libraries can also be chemical peptide libraries, wherein the peptide library is synthetically produced. Exemplary chemical peptide libraries include combinatorial peptide libraries, such as single bead single compound (OBOC) libraries and position scanning synthetic peptide combinatorial libraries (PS-SPCL). For example, linear combinatorial chemical libraries such as polypeptide libraries are formed by combining chemical building blocks (amino acids) sets in various possible ways for a given compound length (i.e., the number of amino acids in the polypeptide compound). Millions of chemical compounds can be synthesized by such combination mixing of chemical building blocks. Detailed protocols for generating peptide libraries and for selecting target binding and / or target blocking peptides from various peptide libraries are described in Kehoe JW et al., Chem. Rev. 2005; 105:4056; Hill HR et al., Mol. Microbiol. 1996; 20:685; Smith GP et al., Chem. Rev. 1997; 97:391; Cabilly S. Mol. Biotechnol. 1999; 12:143; Fukunaga K et al., J. Nucleic Acids. 2012; 2012:9; Gray BP et al., Chem Rev. 2014; 114(2):1020-1081, all of which are incorporated herein by reference in their entirety.

[0198] In certain embodiments, the peptides used in the methods of the present invention may contain certain structural or functional motifs. The motif of a peptide refers to a group of continuous amino acids in the amino acid sequence of a peptide chain and / or a group of linear or spatially adjacent amino acids in the secondary and / or tertiary structure of the peptide. Because the motif can be formed in whole or in part due to protein folding, the adjacent amino acids in the described motif can be separated by 0, 1 or more, 5 or more, 10 or more, 15 or more or 20 or more amino acids in the linear amino acid sequence of the peptide. For example, a therapeutic peptide can be a peptide or a variant thereof of any CDR sequence of any anti-CA19-9 as provided herein.

[0199] In certain embodiments, the peptides used in the methods of the present invention include peptides with 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity to any therapeutic peptide provided herein. In order to determine the percent identity of two amino acid sequences, the sequences are aligned to achieve the best comparison purpose. The amino acid residues at the corresponding amino acid positions are then compared. When the position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules at the position are the same. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions × 100 / total number of positions). In one embodiment, the two sequences have the same length. In alternative embodiments, the sequences have different lengths, and therefore the percent identity refers to a comparison of a shorter sequence with a portion of a longer sequence, wherein the portion is the same length as the shorter sequence.

[0200] Peptides with additional modifications can also be used in the methods provided herein for preventing, treating, alleviating or managing pancreatitis. For example, peptides of the above structural motifs can be synthesized with one or more (D)-amino acids. The selection of (L)-amino acids or (D)-amino acids included in the peptides useful in the methods provided herein depends in part on the desired characteristics of the peptide. For example, the incorporation of one or more (D)-amino acids can confer increased stability to the peptide in vitro or in vivo. The incorporation of one or more (D)-amino acids can also increase or decrease the binding activity of the peptide, for example, as measured using a bioassay as described herein or other methods well known in the art.

[0201] The corresponding sequence of (D)-amino acids is used to replace all or part of the (L)-amino acid sequence, so that the corresponding part of the peptide chain presents an optical isomeric structure. Inversion of all or part of the (L)-amino acid sequence presents a reverse analog of the peptide. The combination of enantiomeric (L to D, or D to L) replacement and sequence inversion presents a reverse-reverse analog of the peptide. It is known to those skilled in the art that enantiomeric peptides, their reverse analogs and their reverse-reverse analogs maintain a significant topological relationship with the parent peptide, and in particular the parent and its reverse-reverse analog often obtain a high degree of similarity. This relationship and similarity can be reflected in the biochemical properties of the peptide, in particular the high binding of the corresponding peptide and analogs to receptor proteins. For example, the synthesis of characteristic retro-inverse analogs of peptides has been discussed in Methods of Organic Chemistry (Houben-Weyl), Synthesis of Peptides and Peptidomimetics - Workbench Edition Vol. E22c (Goodman M. Ed.) 2004 (George Thieme Verlag Stuttgart, New York), and references cited therein, all of which are hereby incorporated by reference in their entirety.

[0202] Therapeutic peptides provided herein also include peptides with amino acid modifications. Amino acid "modification" refers to the change of naturally occurring amino acids to produce non-naturally occurring amino acids. Derivatives of peptides with non-naturally occurring amino acids that can be used with the methods provided herein can be produced by chemical synthesis or by incorporating non-natural amino acids site-specifically into peptides during biosynthesis, as described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, 1989 Science, 244: 182-188, the reference is hereby incorporated by reference in its entirety.

[0203] Peptidomimetics that are structurally similar to therapeutically useful peptides (peptide mimetics) can be used to produce an equivalent therapeutic or prophylactic effect. In general, peptidomimetics are structurally similar to paradigm polypeptides (i.e., polypeptides having biochemical properties or pharmacological activity), but have one or more peptide bonds that are optionally replaced by a bond selected from the group consisting of: --CH2-NH--, --CH2S--, --CH2-CH2--, --CH=CH- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CH2SO--, the replacements being performed by methods known in the art and further described in the following references: Spatola, AF in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," edited by B. Weinstein, Marcel Dekker, New York, p. 267 (1983); Spatola, AF, Vega Data (March 1983), Vol. 1, No. 3, "Peptide Backbone Modifications" (review); Morely, JS, Trends Pharmacol. Sci (1980) pp. 463-468 (review); Hudson, D. et al., (1979) Int J Pept Prot Re 14:177-185 (--CH2—NH--, --CH2—CH2--); Spatola, AF et al., (1986) Life Sci 38:1243-1249 (--CH2—S--); Hann, MM, (1982) J Chem Soc Perkin Trans I 307-314 (--CH=CH--, cis and trans); Almquist, RG et al., (1980) J Med Chem 23:1392 (--COCH2--); Jennings-White, C et al., (1982) Tetrahedron Lett 23:2533 (--COCH2--); Szelke, M et al., European Application EP 45665 (1982) CA:97:39405 (1982) (--CH(OH)CH2--); Holladay, MW et al., (1983) Tetrahedron Lett 24:4401-4404 (--C(OH)CH2--); and Hruby, VJ, (1982) Life Sci 31:189-199 (--CH2—S--); each of which is incorporated herein by reference.

[0204] In another embodiment of the peptidomimetic, the non-peptide bond is --CH2NH--. Such peptide mimetics have significant advantages over peptide embodiments, including, for example, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, etc.

[0205] A variety of designs of peptide mimics are possible. For example, it is particularly envisioned that the necessary conformation is stabilized by a non-peptide cyclic peptide, U.S. Patent No. 5,192,746 of Lobl et al., U.S. Patent No. 5,576,423 of Aversa et al., U.S. Patent No. 5,051,448 of Shashoua and U.S. Patent No. 5,559,103 of Gaeta et al. (all incorporated herein by reference) describe a variety of methods for producing such compounds. The synthesis of non-peptide compounds simulating peptide sequences is also known in the art. Eldred et al., J.Med.Chem.37:3882 (1994) (incorporated herein in its entirety by reference) describes non-peptide antagonists simulating peptide sequences. Similarly, Ku et al., J.Med.Chem 38:9 (1995) (incorporated herein in its entirety by reference) further illustrates the synthesis of a series of such compounds.

[0206] In addition, the therapeutic peptide can be a peptide with a mutation in the peptide sequence. The mutation provided herein can be a substitution, including conservative or non-conservative substitutions of internal or terminal amino acid residues; deletion, including internal deletion and terminal residue deletion; addition, including internal addition and addition to both ends of the peptide, all of which can be carried out according to methods well known to those of ordinary skill in the art, such as Molecular Cloning: A Laboratory Manual (4th edition); described in ISBN978-1-936113-42-2.

[0207] Conservative or non-conservative amino acid substitutions may be made at one or more amino acid residues in the therapeutic peptide. Conservative and non-conservative substitutions may also be made. Conservative substitutions are substitutions that occur within a family of amino acids that are related in their side chains. Non-conservative substitutions are substitutions where an amino acid from one family is replaced with an amino acid from a different family. Genetically encoded amino acids can be divided into four families: (1) acidic = Asp (D), Glu (G); (2) basic = Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic) = Cys (C), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y); (4) uncharged polar = Asn (N), Gln (Q), Ser (S), Thr (T). Non-polar can be subdivided into: strongly hydrophobic = Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F); and moderately hydrophobic = Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W). Alternatively, the amino acid repertoire can be grouped as (1) acidic = Asp (D), Glu (G); (2) basic = Lys (K), Arg (R), His (H); (3) aliphatic = Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Ser (S), Thr (T), wherein Ser (S) and Thr (T) are optionally grouped individually as aliphatic-hydroxy; (4) aromatic = Phe (F), Tyr (Y), Trp (W); (5) amide = Asn (N), Glu (Q); and (6) sulfur-containing = Cys (C) and Met (M). (See, e.g., Biochemistry, 4th edition, L. Stryer, ed., WH Freeman and Co., 1995, which reference is incorporated herein by reference).

[0208] In certain embodiments, the peptide used in the present method can be an isolated or purified peptide. An isolated or purified peptide refers to a peptide that is substantially free of at least one component of the peptide when it is present in the cell or microorganism that produces the peptide, or substantially free of at least one component of the peptide when it is first synthesized in the form of a mixture. Thus, the peptide may be substantially free of cellular material (such as lipids, DNA and / or carbohydrates) or other contaminating proteins from the cell or tissue source from which the protein or peptide is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free" includes the preparation of peptides, wherein the peptide is separated from one or more components of the mixture from which the peptide is isolated. Thus, peptides that are substantially free of cellular material include preparations of peptides having less than about 30%, 20%, 10% or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When the peptide is recombinantly produced, the peptide may be substantially free of culture medium, such as culture medium that accounts for less than about 20%, 10% or 5% of the volume of the protein preparation. When producing peptides by chemical synthesis, the peptides can be substantially free of chemical precursors or other chemicals, for example, the peptides are separated from chemical precursors or other chemicals involved in protein synthesis. Therefore, the preparations of such peptides have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the peptide of interest. For example, peptides provided herein and / or peptides for methods such as those provided herein can be peptides that are separated or purified, or peptides that have not yet been separated or purified.

[0209] 6.2.2 Patient populations, biomarkers, and treatment outcomes

[0210] As discussed above, pancreatitis can be divided into chronic pancreatitis and acute pancreatitis. In some embodiments, the difference between acute pancreatitis and chronic pancreatitis is the duration and persistence of pancreatic inflammation and pancreatic damage. Acute pancreatitis refers to a single (sometimes sudden or short-lived) pancreatitis attack, often accompanied by severe abdominal pain. Chronic pancreatitis refers to irreversible inflammation and fibrosis symptoms that persist in the pancreas, resulting in long-term damage to the exocrine and / or endocrine functions of the pancreas. Therefore, chronic pancreatitis can encompass pancreatitis in which inflammation persists in the pancreas, or even if inflammation has subsided, the resulting damage to the pancreas (such as damage characterized by fibrosis, calcification and ductal injury) still affects pancreatic function. It can be clearly seen from the above and below discussion that the causes and mechanisms of developing into acute pancreatitis and chronic pancreatitis are overlapping. Therefore, it will be understood by those skilled in the art that acute pancreatitis can lead to chronic pancreatitis, and the onset of acute pancreatitis can occur in chronic pancreatitis patients. Additionally, patients who have had resolution of one or more episodes of acute pancreatitis may develop chronic pancreatitis.

[0211] According to various markers described herein, acute pancreatitis can be further divided into different stages. For example, according to the Atlanta classification, acute pancreatitis can be further divided into mild acute pancreatitis and severe acute pancreatitis (Edward L. Bradley, Arch Surg. 1993; 128 (5): 586-590), which is incorporated herein by reference in its entirety. Mild acute pancreatitis refers to acute pancreatitis associated with minimal organ dysfunction, no pancreatic tissue necrosis, no organ failure and / or a self-limiting course. Severe acute pancreatitis refers to pancreatitis with organ failure, local pancreatic complications (such as pseudocysts) and / or tissue necrosis (necrotizing pancreatitis). Acute pancreatitis has been further classified using various scoring systems, including the Ranson criteria, the Glasgow-Imrie prognostic criteria, the CT severity index, the APACHE II classification system, and the Balthazar CT enhancement scoring system, as disclosed in Chapter 37 of GI / Liver Secrets Plus, 4th edition, McNally PR, ed., 2010, on acute pancreatitis, and in Carroll JK et al., Am Fam Physician 2007; 75: 1513-20, which are incorporated herein by reference in their entirety. In some embodiments, scores from various acute pancreatitis scoring systems can be used to classify acute pancreatitis. For example, meeting less than 3 of the 11 criteria of the Ranson system can indicate mild acute pancreatitis, while meeting 3 or more of the 11 criteria in the Ranson system can indicate severe acute pancreatitis. In one embodiment, meeting 3 or more of 8 criteria in the Glasgow-Imrie scoring system can indicate severe acute pancreatitis, while meeting 3 or less criteria in the same system can indicate mild acute pancreatitis. In another embodiment, a score of 8 or more points in the 14 criteria of the APACHE II scoring system can indicate severe acute pancreatitis, while a score of less than 8 points in the same system can indicate mild acute pancreatitis. In other embodiments, the standards and scoring systems are well known to those of ordinary skill in the art and have been described in GI / LiverSecrets Plus, 4th edition, McNally PR, Chapter 37 of 2010 on acute pancreatitis; Carroll JK et al., AmFam Physician 2007; 75: 1513-20 and Matull WR et al., J Clin Pathol 2006; 59: 340-344 in detail, the references are all incorporated herein by reference in their entirety. The method provided herein can be used for patients at any stage of acute pancreatitis classified by any classification system disclosed herein.

[0212] Alternatively, according to the revised Atlanta classification (Banks PA et al., Gut 2013; 62: 102-111), acute pancreatitis can be further divided into mild acute pancreatitis, moderate acute pancreatitis and severe acute pancreatitis, which is incorporated herein by reference in its entirety. In the revised Atlanta classification, mild acute pancreatitis refers to acute pancreatitis in which the patient has no organ failure and no local or systemic complications; moderate acute pancreatitis refers to acute pancreatitis in which the patient suffers from transient organ failure (organ failure that resolves within 48 hours) and / or local or systemic complications without persistent organ failure; and severe acute pancreatitis refers to acute pancreatitis in which the patient suffers from persistent organ failure (single organ failure or multiple organ failure for more than 48 hours). The methods provided herein can be used for patients with mild acute pancreatitis, moderate acute pancreatitis and severe acute pancreatitis according to the revised Atlanta classification disclosed and cited herein.

[0213] The classification of mild acute pancreatitis, moderate acute pancreatitis, and severe acute pancreatitis can also be determined in the scoring system. In one embodiment of such a scoring system for the classification of acute pancreatitis, the patient is evaluated with a computed tomography severity index (CTSI). In the CTSI, the patient is first evaluated with a Balthazar score, wherein a normal pancreas is scored as 0, an enlarged pancreas is scored as 1, inflammatory changes in the pancreas and peripancreatic fat are scored as 2, a single, poorly defined peripancreatic fluid collection is scored as 3, and two or more poorly defined peripancreatic fluid collections are scored as 4. The patient is further evaluated with a pancreatic necrosis score, wherein no pancreatic necrosis is scored as 0, no more than 30% pancreatic necrosis is scored as 2, more than 30% but no more than 50% pancreatic necrosis is scored as 4, and more than 50% pancreatic necrosis is scored as 6. In some embodiments, the sum of the Balthazar score and the pancreatic necrosis score can then be used to classify acute pancreatitis, wherein a total score of 0 to 3 is classified as mild acute pancreatitis, a total score of 4 to 6 is classified as moderate acute pancreatitis, and a total score of 7 to 10 is classified as severe acute pancreatitis. The methods provided herein can be used for patients with mild acute pancreatitis, moderate acute pancreatitis, and severe acute pancreatitis according to the CTSI classification disclosed herein.

[0214] In addition, according to Banks PA et al., Gut 2013; 62: 102-111, acute pancreatitis can be subdivided into two types: interstitial edematous pancreatitis and necrotizing pancreatitis. Interstitial edematous pancreatitis refers to acute pancreatitis with diffuse (or occasional localized) enlargement of the pancreas due to inflammatory edema. Necrotizing pancreatitis refers to acute pancreatitis with necrosis of pancreatic parenchyma, peripancreatic tissue, or both. The methods provided herein can be used for patients with interstitial edematous pancreatitis or necrotizing pancreatitis disclosed herein.

[0215] Similarly, according to the Cambridge classification system, chronic pancreatitis can be further divided into mild chronic pancreatitis, moderate chronic pancreatitis, and severe chronic pancreatitis (also referred to as Cambridge classification or Cambridge stages I, II, and III, respectively), as described in Sai JK et al., World J Gastroenterol. 2008 Feb 28;14(8):1218-21; Samer M et al., Gut. 1984 Jul;25(7):756-9; Axon AT et al., Gut. 1984 Oct;25(10):1107-12, all of which are incorporated herein by reference in their entirety. Mild chronic pancreatitis refers to pancreatitis with 3 or more abnormal branches on endoscopic retrograde cholangiopancreatography (ERCP) findings and 2 or more of one of the following groups on ultrasound or computed tomography (CT) findings: lumen <10 mm, ductal irregularity, focal acute necrosis, parenchymal heterogeneity, increased echogenicity of the ductal wall, and irregular head or body contour. Moderate chronic pancreatitis refers to pancreatitis with more than 3 abnormal side branches and abnormal main duct on ERCP findings and 2 or more of one of the following groups on ultrasound or CT findings: lumen <10 mm, ductal irregularity, focal acute necrosis, parenchymal heterogeneity, increased echogenicity of the ductal wall, and irregular head or body contour. Severe chronic pancreatitis refers to at least mild and moderate chronic pancreatitis abnormalities, and in addition selected from ERCP examination results of large lumen> 10mm, filling defects in the duct, duct obstruction (stenosis), and duct dilatation or irregularity, and selected from large lumen> 10mm, filling defects in the duct, duct obstruction (stenosis), duct dilatation or irregularity, stones and / or pancreatic calcification, and invasion of adjacent organs. The method provided herein can be used for patients with mild chronic pancreatitis, moderate chronic pancreatitis, and severe chronic pancreatitis disclosed herein.

[0216] As described above and below, the methods provided herein can improve any one or more of the pancreatitis markers described herein in pancreatitis patients. When referring to pancreatitis markers, the term "improvement (improve / improving / improvement)" includes any changes in pancreatitis markers, as long as the changes are in the direction of markers from subjects with pancreatitis to markers or normal reference ranges for subjects without pancreatitis. Therefore, for pancreatitis markers associated with the severity of pancreatitis, such "improvement" of the pancreatitis markers will mean a reduction in pancreatitis markers. Examples of such related markers include immune cell invasion, collagen deposition, fibrotic tissue, fat necrosis, interstitial edema, acinar and vascular destruction, interstitial hemorrhage, ductal dilatation, acinar cell homogenization, sLe a The expression level of CA19-9, the expression level of serum lipase, serum amylase, serum C-reactive protein, weight loss, food malabsorption, pancreatic atrophy, pancreatic enlargement, pancreatic inflammation, bile reflux and pancreatitis permeability. For pancreatitis markers that are inversely correlated with the severity of pancreatitis, such "improvement" of the pancreatitis marker will mean an increase in the pancreatitis marker. Examples of inversely correlated pancreatitis markers include the percentage of pancreatic function, the number of pancreatic acini and the number of acinar cells. Further disclosure of markers and treatment outcomes is provided herein.

[0217] In some specific embodiments, the disclosure further provides that the methods provided herein can improve the patient's treatment outcome by resolving the patient's pancreatitis or reversing the stage of pancreatitis as classified by any classification system described herein, for example, from severe acute / chronic pancreatitis to moderate acute / chronic pancreatitis, from severe acute / chronic pancreatitis to mild acute / chronic pancreatitis, or from moderate acute / chronic pancreatitis to mild acute / chronic pancreatitis. Such improvements due to the methods provided herein can also be a slowing of progression of the patient's pancreatitis stage, such as when compared to similar patients who are not treated or treated with a placebo, the progression from one stage to another is not rapid, or can be a stabilization of the patient's pancreatitis stage, such as the patient's pancreatitis staying at mild, moderate or severe pancreatitis and the disease does not deteriorate to the next stage.

[0218] Alternatively, chronic pancreatitis can be classified according to the Marseilles-Rome classification modified by Sarlesas. Based on epidemiology, molecular biology and morphology, this classification divides chronic pancreatitis into four groups. In this classification system, chronic pancreatitis is classified as calcific chronic pancreatitis, characterized by irregular fibrosis, intraductal protein plug, intraductal calculus and / or ductal injury; obstructive chronic pancreatitis, characterized by glandular changes, uniform fibrosis, ductal dilatation, acinar atrophy and / or improvement with elimination of pancreatic duct obstruction; inflammatory chronic pancreatitis, characterized by mononuclear cell infiltration, exocrine parenchymal destruction, diffuse fibrosis and / or atrophy; and asymptomatic pancreatic fibrosis, characterized by silent diffuse lobular perifibrosis. The method provided herein can be used for chronic pancreatitis patients at any stage classified according to the Marseilles-Rome classification scheme, as discussed in GI / Liver Secrets Plus, the 4th edition, McNally PR, 2010, about the 38th chapter of chronic pancreatitis.

[0219] Various physiological, biological, histological, serological and / or signaling parameters can be used as biomarkers to monitor the course of treatment and adjust the dosage of therapeutic agents described herein (such as the therapeutic agents described in Section 6.2.1). Additionally or alternatively, biomarkers can be used to select patients for treatment using the methods disclosed herein. In this section, various such biomarkers that can be used with the methods provided herein are described.

[0220] As disclosed above, the potential symptoms of pancreatitis are pancreatic inflammation and pancreatic tissue damage. Exemplary pancreatic tissue damage in pancreatitis patients includes damage to islets of Langerhans, acinar cells, pancreatic blood vessels, pancreatic ducts, pancreatic duct cells, exocrine components in the pancreatic organ system, endocrine components in the pancreatic organ system, and / or other pancreatic tissues. Pancreatic damage can have microscopic or even macroscopic structural manifestations.

[0221] As mentioned above, pancreatitis includes, for example, acute pancreatitis and chronic pancreatitis, which can be caused or associated with various factors and / or pathophysiology respectively. For example, acute pancreatitis can be caused by the obstruction of tissue or organ, including but not limited to gallstones, microlithiasis (bile mud formation), ampulla or pancreatic tumor, papillary stenosis, worms or foreign bodies blocking ampulla, Oddi sphincter dysfunction, common bile duct cyst, duodenal diverticulum, and pancreas divisum may also be present. Acute pancreatitis can also be induced by toxins such as alcohol (ethanol, methanol), scorpion venom, organophosphorus pesticides and other drugs. In addition, acute pancreatitis can be caused by trauma, such as abdominal accidental blunt trauma and iatrogenic trauma (for example postoperative trauma). Other causes of acute pancreatitis may include metabolic abnormalities, such as hypertriglyceridemia and hypercalcemia; hereditary conditions, such as cystic fibrosis and hereditary pancreatitis; infectious diseases, such as parasites, viruses, bacterial infections; vascular diseases, such as ischemia (e.g., after cardiac surgery), atherosclerotic embolism, vasculitis (e.g., systemic lupus erythematosus and polyarteritis nodosa); other miscellaneous or idiopathic causes, such as peptic ulcer disease, Crohn's disease, Reye's syndrome, and hypothermia. Among the various causes, gallstones and alcohol are the most common causes of acute pancreatitis. The methods provided herein can be used to prevent, treat, alleviate or manage pancreatitis, including acute pancreatitis caused by any cause or any combination of causes disclosed herein.

[0222] Similarly, chronic pancreatitis can be caused by or associated with a variety of factors and / or pathophysiologies, such as alcohol consumption or alcohol abuse, autoimmune diseases, metabolic abnormalities (such as hyperparathyroidism (hypercalcemia), diabetes or hypertriglyceridemia), nutritional or tropical factors, tissue or organ obstruction caused by strictures (such as trauma or previous episodes of pancreatitis) or malignancies, hereditary diseases (including hereditary pancreatitis and cystic fibrosis), and other idiopathic causes. The methods provided herein can be used to prevent, treat, alleviate or manage pancreatitis, including chronic pancreatitis caused by any cause or any combination of causes disclosed herein.

[0223] Because pancreatitis can have various reasons, the methods provided herein can be used to prevent, treat, alleviate or manage pancreatitis (including acute and chronic pancreatitis), which is the result of a selective cause or a selective cause group. Similarly, the methods provided herein can be used to prevent, treat, alleviate or manage pancreatitis (including acute and chronic pancreatitis), which is the result of excluding any specific factor or cause or excluding the cause of a group of selective factors or causes. For example, the methods provided herein can be used to prevent, treat, alleviate or manage pancreatitis (including acute and chronic pancreatitis), which is not caused by or has nothing to do with any cancer or neoplastic disorder. In some embodiments, the methods provided herein can be used to prevent, treat, alleviate or manage pancreatitis in a patient with pancreatitis, wherein the patient does not suffer from cancer or has not yet developed into cancer, such as pancreatic cancer or neoplastic disorder, such as pancreatic neoplastic disorder.

[0224] Pancreatitis can be measured and observed by determining, measuring or observing various markers of pancreatic inflammation and pancreatic damage. Such markers may include histological markers indicating histological changes in the pancreas; serological markers indicating blood abnormalities; signaling markers indicating changes in signaling pathways that underlie the pathological mechanism of pancreatitis or changes in signaling pathways that are a pathological phenotype of pancreatitis; or physiological markers indicating physiological signs or changes in pancreatitis patients (e.g., physiological signs that can be easily observed without invasive procedures or blood draws).

[0225] 6.2.2.1. Serological, urine, and stool markers of pancreatitis

[0226] One or more specific substances in the blood or urine of patients with pancreatitis, the ratio of the level of one substance in the blood or urine relative to another substance, the total level of one substance in the blood or urine (e.g., the total level of a specific enzyme), or a ratio involving such total levels can be used as a biomarker for pancreatitis. The substance used as a marker can be a cell, tissue, protein (such as an enzyme), DNA, RNA, microRNA, lipid, metabolite, or any other substance that can reflect pancreatitis inflammation or damage. Examples of such serological pancreatitis markers include serum lipase levels, serum CA19-9 levels, serum amylase levels (including levels of amylase, isoamylase and total amylase), serum C-reactive protein levels, carbohydrate-depleted transferrin, trypsinogen activation products (including, for example, trypsinogen activation peptide, trypsin 1, trypsin 2, the complex of trypsin 1 or trypsin 2 with alpha-antitrypsin (also known as trypsin-1-AAT and trypsin-2-AAT), elastase 1, phospholipase A2, procalcitonin (a precursor of calcitonin), lipids, inflammatory cytokines and chemokines, microRNAs, peptides (RA1609 and RT2864, as disclosed in Walgren JL et al., Toxicol Sci. 2007 Mar;96(1):184-93), which reference is incorporated herein by reference in its entirety), alcohols and alcohol metabolites (fatty acid ethyl esters), and toxins or toxin metabolites. Elevated levels of any serological marker disclosed herein, or any combination of serological markers, can be used in the methods provided herein.

[0227] Exemplary urine abnormalities as markers for pancreatitis can include amylase, isoamylase, trypsinogen activation peptide, carboxypeptidase activation peptide, and phospholipase A2. Any urine marker with elevated levels can be used alone in the methods provided herein, or in any arrangement or combination with any marker disclosed herein.

[0228] Additionally, markers of pancreatitis also encompass stool markers, including stool elastase 1, stool chymotrypsin, and stool fat content. Any elevated level of a stool marker can be used alone in the methods provided herein, or in any arrangement or combination with any marker disclosed herein.

[0229] Other markers from other body fluid sources can also be used as markers for pancreatitis. In some embodiments, markers from pancreatic juice (including CA19-9, cathepsin B, cathepsin L, cathepsin S, lysosomal hydrolases and carboxypeptidase B activation peptide (CACAP)) can be used as markers for pancreatitis. Any pancreatic juice marker of elevated levels can be used alone in the methods provided herein, or with any marker disclosed herein in any arrangement or combination.

[0230] As discussed above, one of the serological markers is the level of serum amylase. The normal serum amylase of healthy people can vary according to the method for measuring amylase and the laboratory for determination. Various commercial kits can be used to measure serum amylase levels, such as the amylase assay kit (colorimetric) (ab 102523) from Abeam and the amylase activity assay kit (MAK009-1KT) from Sigma-Aldrich, Inc. In healthy individuals, an exemplary normal range of serum amylase is 23-85U / L, although in some laboratories, an exemplary normal serum amylase can be 0-140U / L. Various levels of serum amylase can be used as an indicator of pancreatitis. In some embodiments, ≥140 U / L, ≥150 U / L, ≥160 U / L, ≥170 U / L, ≥180 U / L, ≥190 U / L, ≥200 U / L, ≥210 U / L, ≥220 U / L, ≥230 U / L, ≥240 U / L, ≥250 U / L, ≥260 U / L, ≥270 U / L, ≥280 U / L, ≥290 U / L In some embodiments, the serum amylase level of ≥200U / L is used as a marker for pancreatitis. In another embodiment, the serum amylase level of ≥140U / L is used as a marker for pancreatitis.

[0231] Because serum amylase levels can vary according to the assay method and the laboratory being assayed, the ratio between the serum amylase of a pancreatitis patient and the serum amylase of a healthy control subject can be used as a surrogate marker for pancreatitis. For example, the ratio between the serum amylase of a pancreatitis patient and the serum amylase upper limit of a healthy control subject can be used as a marker in the method provided herein. In some embodiments, pancreatitis markers include >=1, >=1.5, >=2, >=2.5, >=3, >=3.5, >=4, >=4.5, >=5, >=6, >=7, >=8, >=9, >=10 or higher exemplary amylase ratios (i.e., the ratio relative to the upper limit of a healthy individual in a pancreatitis patient). In a specific embodiment, ratio >=3 is used as a pancreatitis marker. In another embodiment, ratio >=5 is used as a pancreatitis marker.

[0232] As discussed above, another serological marker is the level of serum lipase. The normal serum lipase level of healthy people can vary according to the method for measuring lipase and the laboratory for measuring. Various commercial kits can be used to measure serum lipase levels, such as the lipase activity assay kit (colorimetric) (ab102524) from Abeam, the lipase assay kit (DZ132A-K) from Diazyme Laboratories, Inc. and the lipase activity assay kit (MAK046-1KT) from Sigma-Aldrich, Inc. The exemplary range of normal serum lipase in healthy individuals is 12-70U / L, although in some laboratories exemplary normal serum lipase can be as high as 0-160U / L. Various levels of serum lipase can be used as an indicator of pancreatitis. In some embodiments, ≥140 U / L, ≥150 U / L, ≥160 U / L, ≥170 U / L, ≥180 U / L, ≥190 U / L, ≥200 U / L, ≥210 U / L, ≥220 U / L, ≥230 U / L, ≥240 U / L, ≥250 U / L, ≥260 U / L, ≥270 U / L, ≥280 U / L, ≥290 U / L In some embodiments, the serum lipase level of ≥100U / L is used as a marker for pancreatitis. In some embodiments, the serum lipase level of ≥100U / L is used as a marker for pancreatitis. In some embodiments, the serum lipase level of ≥100U / L is used as a marker for pancreatitis. In some embodiments, the serum lipase level of ≥100U / L is used as a marker for pancreatitis. In some embodiments, the serum lipase level of ≥100U / L is used as a marker for pancreatitis.

[0233] Because serum lipase levels can change according to the assay method and the laboratory being assayed, the ratio between the serum lipase of a pancreatitis patient and the serum lipase of a healthy control subject can be used as a surrogate marker for pancreatitis. For example, the ratio between the serum lipase upper limit of a pancreatitis patient and the serum lipase of a healthy control subject can be used as a marker in the method provided herein. In some embodiments, pancreatitis markers include >=1, >=1.5, >=2, >=2.5, >=3, >=3.5, >=4, >=4.5, >=5, >=6, >=7, >=8, >=9, >=10 or higher exemplary lipase ratios (i.e., the ratio relative to the upper limit of a healthy individual in a pancreatitis patient). In a specific embodiment, ratio >=3 is used as a pancreatitis marker. In another embodiment, ratio >=5 is used as a pancreatitis marker.

[0234] Additional serological markers are the levels of serum CA19-9. Various commercial kits can be used to measure serum CA19-9 levels, such as the CA19-9 human ELISA kit (Catalog No. EHCA199) from Thermo Fisher Scientific, the human CA-19-9 ELISA test kit (Project No. 6909-16) from Diagnostic Automation / Cortez Diagnostics Inc., and the human cancer antigen CA19-9 ELISA kit (ab 108642) from Abcam. As known to those of ordinary skill in the art, serum CA19-9 is also routinely determined in CA 19-9 radioimmunoassays.

[0235] An exemplary normal range of serum CA19-9 in a healthy individual is 0-37 U / ml. Various levels of serum CA19-9 can be used as an indicator of pancreatitis. In some embodiments, ≥37 U / ml, >37 U / ml, ≥40 U / ml, ≥50 U / ml, ≥60 U / ml, ≥70 U / ml, ≥80 U / ml, ≥90 U / ml, ≥100 U / ml, ≥110 U / ml, ≥120 U / ml, ≥130 U / ml, ≥140 U / ml, ≥150 U / ml, ≥160 U / ml, ≥170 U / ml, ≥180 U / ml, ≥190 U / ml, ≥200 U / ml, ≥210 U / ml, ≥220 U / ml In some embodiments, the serum CA19-9 level of more than 37U / ml but not more than 40U / ml is used as a pancreatitis marker. In another embodiment, the serum CA19-9 level of more than 40U / ml but not more than 100U / ml is used as a pancreatitis marker. In another embodiment, serum CA19-9 levels exceeding 100 U / ml are used as a pancreatitis marker.

[0236] As known to those of ordinary skill in the art, C-reactive protein (CRP; also known as high-sensitivity C-reactive protein (hs-CRP) or ultrasensitive C-reactive protein (us-CRP)) is a protein found in plasma and is produced by the liver in response to inflammation. Therefore, CRP can be used as a marker for pancreatitis, which, as discussed above, is a disease of inflammation and damage to the pancreas. The level of CRP can be correlated with the level of inflammation in the body. Therefore, the higher the CRP level, the more inflammation in the body. The normal range of serum CRP in healthy people is below 1 mg / L. Serum CRP levels between 1 and 2.9 mg / L can indicate moderate levels of inflammation in a human subject, between 3 and 10 mg / L can indicate high levels of inflammation in a human subject, and more than 10 mg / L can indicate severe levels of inflammation in a human subject. In some embodiments, the pancreatitis markers in the methods provided herein include exemplary serum CRP levels exceeding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg / L. Various commercial kits can be used to measure serum CRP levels, such as human C-reactive protein / CRPQuantikine ELISA kits (Cat. No. DCRP00) from R&D Systems Inc., CRP (people) ELISA kits (ENZ-KIT102-0001) from Enzo Life Sciences, Inc., and human C-reactive protein ELISA kits (RAB0096-1KT) from Sigma-Aldrich, Inc.

[0237] It will be further understood by those of ordinary skill in the art that various other serological markers or other markers from body fluids can be used to identify therapeutic agents, characterize pancreatitis, identify pancreatitis patients and determine the therapeutic outcome of the treatment methods for pancreatitis. Therefore, it will be understood by those of ordinary skill in the art that various other known pancreatitis markers can be used in the methods provided herein. Such pancreatitis markers are disclosed in publications such as Chapters 37 and 38 on acute pancreatitis and chronic pancreatitis, respectively, of GI / Liver Secrets Plus, 4th edition, McNally PR ed., 2010; Matull WR et al., J Clin Pathol 2006; 59:340-344; Basnayake C. et al., Aust Prescr 2015; 38:128-30; Carroll JK et al., Am Fam Physician 2007; 75:1513-20; Kaphalia BS, Chapter 16 of Biomarkers in Toxicology, ISBN:978-0-12-404630-6, (2014); Jasdanwala S et al., Integr Mol Med, 2015; 2(3):189-195; Büchler MW et al., BMC Gastroenterology 2009,9:93;Duggan SN et al., World J Gastroenterol 2016;22(7):2304-2313;Etemad B et al., Gastroenterology 2001;120:682-707, all of which are incorporated herein by reference in their entirety. It will be further understood by one of ordinary skill in the art that any marker and any arrangement or combination of markers disclosed above and below can be used in the methods provided herein.

[0238] Therefore, the present disclosure provides patients suitable for the methods, including patients with elevated one or more serological markers, urine markers, and fecal markers (including listed markers) as described in paragraphs

[0219] to

[0230] . More specifically, the methods provided herein can be used for patients with any one or more of the markers with listed values ​​as described in paragraphs

[0219] to

[0230] .

[0239] The present disclosure further provides that the methods provided herein can improve patient treatment outcomes by reversing or improving any one or more or any combination of these serological markers, urine markers, and fecal markers of pancreatitis as described in paragraphs

[0219] to

[0230] , such as lowering serum amylase, lowering serum lipase, or lowering serum CRP.

[0240] 6.2.2.2. Histological markers

[0241] As discussed above, markers based on pancreatic histological abnormalities (e.g., histological staining or IHC staining from pancreatic tissue samples) can also be used in methods provided herein. Such histological markers include abnormal expression of one or more proteins in the pancreas, the presence or abnormal number of a certain type of non-pancreatic cells (e.g., immune cells, including leukocytes, macrophages, and neutrophils) in the pancreas, the abundance of certain cells in the pancreas or the abnormal changes in quantity, and structural abnormalities of pancreatic cells or pancreatic tissue (e.g., abnormalities in pancreatic parenchyma, pancreatic ducts, islets, and acinar cells / tissues). Pancreatic structural abnormalities may be located in specific pancreatic regions or structures, such as pancreatic parenchyma, pancreatic ducts, islets, acinar cells / tissues, pancreatic exocrine components, pancreatic endocrine components, or cysts.

[0242] Examples of histological markers in the pancreas include parenchymal fibrosis, acinar atrophy, ductal deformation, intraductal calcification, invasion or infiltration of immune cells (e.g., invasion / infiltration of macrophages, neutrophils, and leukocytes), collagen deposition, fibrotic tissue, fat necrosis, interstitial edema, acinar cell destruction, glandular enlargement (edema), irregular sclerosis, loss of pancreatic parenchyma, pancreatic vascular destruction, interstitial hemorrhage, ductal dilatation, large fusion foci of fat necrosis, focal hemorrhage, acinar cell necrosis, homogenization of acinar cells, sLe, as shown by histological staining or IHC staining of the pancreas or pancreatic tissue. a The expression level of CA19-9 and the expression level of CA19-9. These histological markers can be used in various stages of acute or chronic pancreatitis. Any elevated level of histological marker can be used alone in the methods provided herein, or in any arrangement or combination with any marker disclosed herein.

[0243] In some embodiments, histological markers of chronic pancreatitis may include irregular sclerosis in the pancreas, loss of exocrine parenchyma in the pancreas. In other embodiments, fat necrosis, glandular enlargement (edema), large fusion foci of fat necrosis around the pancreas, focal hemorrhage, and acinar cell necrosis may be used as markers of acute pancreatitis.

[0244] As is well known to those of ordinary skill in the art, various histological or IHC assays for histological markers have been developed and can be used in the methods provided herein, such as, for example, Matull WR et al., J Clin Pathol 2006; 59:340-344; Basnayake C. et al., Aust Prescr 2015; 38:128-30; Carroll JK et al., Am Fam Physician 2007; 75:1513-20; Kaphalia BS, Chapter 16 of Biomarkers in Toxicology, ISBN:978-0-12-404630-6, (2014); Jasdanwala S et al., Integr Mol Med, 2015; 2(3):189-195; Büchler MW et al., BMC Gastroenterology 2009, 9:93; Duggan SN et al., World J Gastroenterology 2009, 9:94; Assays disclosed in J Gastroenterol 2016; 22(7): 2304-2313; Etemad B et al., Gastroenterology 2001; 120: 682-707, each of which is incorporated herein by reference in its entirety.

[0245] Because imaging techniques often measure histological structures, it will be appreciated by those skilled in the art that histological markers are often associated with structural abnormalities detected by imaging, and the structural abnormalities can be used as markers for pancreatic inflammation and pancreatic damage. Therefore, markers, for example, can be used as histological markers for pancreatitis in the methods provided herein for abnormalities detected in various imaging techniques. Pancreatic structure can be imaged as described in Sections 6.2.2.2 and 6.3.2.2 below. Exemplary histological markers for pancreatic inflammation and pancreatic damage detected by imaging include pancreatic atrophy, bile duct or pancreatic duct obstruction (such as gallstones), pancreatic duct dilatation, pancreatic calcification, biliary stenosis, pancreatic parenchyma and duct scarring, pancreatic duct stenosis or any combination thereof.

[0246] In some embodiments, the histological markers in the methods provided herein include abnormal pancreatic morphology shown in computed tomography (CT), which reflects inflammation or damage caused by pancreatitis or reflects complications associated with pancreatitis. For example, histological markers reflecting pancreatitis that can be observed in CT imaging include pancreatic calcification, such as calcification in the pancreatic duct or parenchyma; dilation of the pancreatic duct (such as the main pancreatic duct); and pancreatic atrophy, such as atrophy of the pancreatic parenchyma. Histological markers reflecting complications of pancreatitis that can be observed in CT imaging include pseudocysts, dilatation of the common bile duct, thrombosis of the splenic vein, gastric varices, pseudoaneurysms, duodenal stenosis, malignant tumors, and any combination thereof.

[0247] Similarly, the histological markers in the methods provided herein include abnormal pancreatic morphology shown in endoscopic retrograde cholangiopancreatography (ERCP), which reflects inflammation or damage caused by pancreatitis or reflects complications associated with pancreatitis. For example, the histological markers reflecting pancreatitis that can be observed in ERCP imaging include: dilation and irregularity of pancreatic ductules and branches in mild pancreatitis; dilation and irregularity of the main pancreatic duct in moderate pancreatitis; and tortuosity, stenosis, calcification and cysts in the pancreas of severe pancreatitis. In some embodiments, pancreatic histological markers found in ERCP (including the number of ductal abnormalities, thriving side branches, filling defects in the ducts and ductal abnormalities (such as stenosis, dilation, irregular branch ducts and ductal stones in multiple regions)) can be used as pancreatitis markers in the methods provided herein.

[0248] In other embodiments, the histological markers in the method provided herein include the pancreas abnormality shown in endoscopic ultrasonography (EUS), which reflects the inflammation or damage caused by pancreatitis or reflects the complication associated with pancreatitis. In some specific embodiments, the histological markers of mild pancreatitis observed in EUS include the high echo strands in the slight irregularity and expansion, high echo duct edge and pancreatic parenchyma of the main pancreatic duct. The histological markers of severe pancreatitis observed in EUS include pancreatic lobular outline and main pancreatic duct dilatation. Other pancreatitis markers observed in EUS include the pancreatitis markers described in Rosemont standard, such as the high echo focus with strands, with honeycombed lobules, high echo focus, lobules, cysts, high echo strands, stones, irregular main pancreatic duct outline, high echo main pancreatic duct edge, side branch dilatation or its any combination.

[0249] In other embodiments, the histological markers in the methods provided herein include pancreatic abnormalities shown in magnetic resonance imaging (such as magnetic resonance cholangiopancreatography (MRCP)), which reflect inflammation or damage caused by pancreatitis or reflect complications associated with pancreatitis. Such histological markers observable in MRCP of the pancreas include dilated ducts, communicating pseudocysts, short strictures, pancreatic or ductal stones, or any combination thereof.

[0250] Additional histological markers shown in imaging tests also include Etemad B et al., Gastroenterology 2001; 120: 682-707; Duggan, SN et al., World J Gastroenterol 2016; 22 (7): 2304-2313; and GI / Liver Secrets Plus, 4th edition, McNally PR, ed., 2010, Chapter 37 and Chapter 38, respectively, on acute pancreatitis and chronic pancreatitis, the references are hereby incorporated by reference in their entirety. For the methods as provided herein, one or more histological markers can be used alone or in any combination of any number of other markers disclosed herein.

[0251] Thus, the present disclosure provides patients suitable for use in the methods, including patients having any one or more of the histological markers described in paragraphs

[0233] to

[0242] , including the listed histological markers.

[0252] The present disclosure further provides that the methods provided herein can improve patient treatment outcomes by reversing or improving any one or more of these pancreatitis histological markers as described in paragraphs

[0233] to

[0242] , or any combination thereof.

[0253] 6.2.2.3. Functional markers

[0254] Because the severity of pancreatitis can be reflected in the function of the pancreas, the marker of pancreatitis can also include functional markers. Functional markers refer to any markers directly or inversely related to pancreatic function. As will be understood by those skilled in the art, the two main functions of the pancreas are pancreatic exocrine function and pancreatic endocrine function. Therefore, the present disclosure provides a defect in pancreatic exocrine function, a defect in pancreatic endocrine function, or both can be used as a marker of pancreatitis in the method provided herein. As also known to those of ordinary skill in the art, the bicarbonate content of pancreatic exocrine secretion is high. Therefore, in the case of the administration of cholecystokinin (CCK), the secretin stimulation test can be used to directly measure pancreatic function by monitoring the volume of these pancreatic secretions and the bicarbonate concentration after the injection of secretin. In some embodiments, the bicarbonate level less than 50mEq / L in the secretin stimulation test (with or without CCK administration) can be a marker for patients with pancreatitis, and can be used in the method provided herein. In other embodiments, bicarbonate levels of less than 5 mEq / L, 10 mEq / L, 15 mEq / L, 20 mEq / L, 25 mEq / L, 30 mEq / L, 35 mEq / L, 40 mEq / L, 45 mEq / L, 50 mEq / L, 55 mEq / L, 60 mEq / L, 65 mEq / L, 70 mEq / L, 75 mEq / L, or 80 mEq / L in a secretin stimulation test (with or without CCK administration) can be used as a marker for patients with pancreatitis and used in the methods provided herein.

[0255] Alternatively, pancreatic function can be determined by measuring pancreatic enzymes in serum and feces or any metabolites of enzymes in serum or urine after oral administration of the compound. Because these markers reflect the level of pancreatic dyspepsia, the more severe the disease, the more sensitive the marker can be for detecting or diagnosing pancreatitis. Such functional markers that can be used for the methods provided herein include serum trypsinogen, wherein the extremely low value (e.g., 20ng / mL or lower) of the patient marks late chronic pancreatitis and steatorrhea; fecal chymotrypsin, wherein fecal chymotrypsin is less than 3U / g of feces indicating late chronic pancreatitis; fecal elastase, wherein fecal elastase is less than 100mcg / g of feces indicating severe pancreatic insufficiency; and fecal fat, wherein fecal fat is greater than 7g / d indicating pancreatitis and fecal fat is greater than 20g / d indicating severe pancreatitis. Other exemplary values ​​as functional markers of pancreatitis include serum trypsinogen less than 5, 10, 15, 20, 25, 30, 35, or 40 ng / mL; fecal chymotrypsin less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 U / g of stool; fecal elastase less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mcg / g of stool; fecal fat greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / d; or any combination thereof.

[0256] One of ordinary skill in the art will appreciate that any functional marker that may be associated with pancreatitis, including those described herein, may be used in the methods provided herein. Other functional markers that can be used in the methods provided herein include those described in John G Lieb II and Peter V Draganov, World J Gastroenterol. 2008 May 28; 14(20):3149-3158; Clain JE et al., Surg Clin North Am 1999; 79:829-845; Couper R. Can J Gastroenterol 1997; 11:153-156; Layer P et al., J Clin Gastroenterol 1999; 28:3-10; Toskes PP. Curr Gastroenterol Rep 1999; 1:145-153; and functional markers described in Chapter 38 on chronic pancreatitis in GI / Liver Secrets Plus, 4th edition, McNally PR ed., 2010; all of which are incorporated herein by reference in their entirety. For the methods as provided herein, one or more functional markers can be used alone or in any combination with any number of other markers disclosed herein.

[0257] Therefore, the present disclosure provides patients suitable for the method, including patients having any one or more of the functional markers described in paragraphs

[0245] to

[0247] (including the listed functional markers).

[0258] The present disclosure further provides that the methods provided herein can improve the patient's treatment outcome by reversing or improving any one or more of these pancreatitis functional markers as described in paragraphs

[0245] to

[0247] . In some specific embodiments, the present disclosure provides that the methods provided herein can improve the patient's treatment outcome by increasing the bicarbonate level in the secretin stimulation test, increasing serum trypsinogen, increasing fecal chymotrypsin, increasing fecal elastase, reducing fecal fat, or any combination thereof.

[0259] 6.2.2.4. Signal transduction markers

[0260] The present disclosure provides a method for detecting pancreatitis and CA19-9 and sialyl Lewis in the pancreas a The levels of CA19-9 and sialyl Lewis a It is a factor in the potential mechanism of pancreatitis.

[0261] In addition, the disclosure provides that the CA19-9 that rises in the pancreas activates the epidermal growth factor receptor (EGFR) signal transduction pathway in the pancreas. The activation of EGFR signal transduction is triggered by ligand-induced receptor dimerization, and then the intrinsic kinase domain of a receptor cross-phosphorylates the specific residues in the C-terminal tail of the partner receptor, thereby providing a scaffold for the recruitment of other proteins (effectors and adapter proteins) in the EGFR signal transduction pathway. This is via the Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains on the effector protein and the phosphotyrosine motifs presented on the intracellular tyrosine kinase domain of EGFR. The activated adapter and effector protein will further stimulate its corresponding signal transduction cascade (including RAS, RAF, MEK and ERK axes of signal transduction pathways, phosphatidylinositol 3 kinases (PI3K), phospholipase Cγ protein axes of signal transduction pathways and PKB / Akt kinase pathways and STAT signal transduction pathways), which leads to a variety of biological responses, including cell proliferation, angiogenesis, migration, survival and adhesion.

[0262] Therefore, the present disclosure provides that markers of pancreatitis can include all indicators of activated EGFR signaling pathways, including changes in expression levels, position changes, binding partner changes, or any biochemical or biophysical changes (such as phosphorylation, ubiquitination, and conformational changes) of molecules in the EGFR signaling pathway. In some embodiments, pancreatitis markers suitable for the methods provided herein can be Akt phosphorylation in the pancreas, EGFR phosphorylation in the pancreas, Erk1 / 2 phosphorylation in the pancreas, RAS activation in the pancreas (e.g., GTP load of RAS), RAF phosphorylation in the pancreas, and MEK phosphorylation in the pancreas. Other markers of activated EGFR signaling pathways suitable for use in the methods provided herein are further described in Seshacharyulu, P et al., Expert Opin Ther Targets. 2012 Jan; 16(1): 15-31; Normanno N et al., Gene. 2006 Jan 17; 366(1): 2-16; Oda K et al., Mol Syst Biol. 2005; 1: 2005.0010; Luije G et al., Oncology. 2009; 77(6): 400-10; Yarden, Y et al., 2007; 131(5): 1018-1018, all of which are incorporated herein by reference in their entirety.

[0263] In addition, because the present disclosure provides that pancreatitis is associated with the level of CA19-9 in the pancreas, and elevated CA19-9 is a factor in the potential mechanism causing pancreatitis, it will be understood by those of ordinary skill in the art that CA19-9 and any other signaling markers in pancreatitis that can be induced in response to elevated CA19-9 levels can be used as markers in the methods provided herein. In addition, in the methods provided herein, CA19-9 can be used alone or in combination with any marker disclosed above and below.

[0264] Therefore, the present disclosure provides patients suitable for the methods, including patients having any one or more of the signaling markers described in paragraphs

[0250] to

[0253] (including the listed signaling markers). The present disclosure further provides that the methods provided herein can improve the treatment outcomes of patients by reversing or improving any one or more of these pancreatitis signaling markers as described in paragraphs

[0250] to

[0253] .

[0265] 6.2.2.5. Physiological markers

[0266] Because pancreatitis can affect the physiology of the subject and cause other observable symptoms, any marker reflecting the physiological symptoms of a pancreatitis patient can be used in the methods provided herein. Such physiological markers include weight loss, pain such as abdominal pain, food malabsorption, pancreatic atrophy, pancreatic enlargement, pancreatic inflammation, and bile reflux. Such physiological markers can be determined by direct observation or based on a description of the patient's symptom sensation. Therefore, the present disclosure provides that patients suitable for the method include patients with any one or more of these physiological markers, and the methods provided herein can improve the patient's treatment outcome by reversing or improving any one or more of these pancreatitis signal transduction markers.

[0267] In certain embodiments, any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more markers described herein can be used in any arrangement or combination in the methods provided herein. In other embodiments, any number of markers provided herein can be used in any arrangement or combination for determination, selection of patient populations (e.g., using any one or more markers as patient selection criteria), dosing regimens (e.g., increasing or decreasing dosage based on improvement or deterioration of one or more markers), treatment outcomes (e.g., assessing improvement of one or more markers) and / or therapeutic agents (e.g., using one or more markers to confirm the functional properties of therapeutic agents) in the methods provided herein.

[0268] 6.2.3 Dosage regimen, pharmaceutical composition and route of administration

[0269] In one embodiment, the methods provided herein use a pharmaceutical composition comprising a therapeutic agent that can be systemically administered to prevent, treat, alleviate or manage pancreatitis. Such administration can be parenteral or transmucosal, such as oral, buccal, nasal, sublingual, submucosal or transdermal. In some embodiments, administration is parenteral, such as via intravenous or intraperitoneal injection, intraarterial, intramuscular administration, intradermal administration, and subcutaneous administration.

[0270] For other routes of administration (such as by use of a perfusate, injection into an organ, or other local administration), pharmaceutical compositions will be provided that produce similar levels of the therapeutic agent, as further described below.

[0271] The pharmaceutical composition of the therapeutic agent provided herein can include a therapeutic agent and a pharmaceutically acceptable carrier for treatment of an effective amount. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopoeia or other commonly recognized foreign pharmacopoeias, for use in animals and more specifically for people. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with a therapeutic agent. Such drug carriers can be sterile liquids, such as saline solutions in water and oil, including those oils of petroleum, animals, plants and synthetic sources (such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). When the pharmaceutical composition is administered intravenously, saline solutions can be carriers. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition may also include a small amount of a wetting agent or emulsifier or a pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, slow-release formulations, etc. The composition may be formulated as a suppository with conventional adhesives and carriers (such as triglycerides). The therapeutic agents provided herein may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with free amino groups, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and salts formed with free carboxyl groups, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EWMartin, which is incorporated herein by reference as a whole. Such preparations will include a therapeutically effective amount of a therapeutic agent (e.g., in a purified or unpurified form) and a suitable amount of a carrier to provide a form for appropriate administration to the patient. One of ordinary skill in the art will appreciate that the formulation can be selected to suit the mode of administration.

[0272] Also provided herein are formulations for increasing transmucosal adsorption of therapeutic agents such as peptides. Pharmaceutical compositions suitable for oral administration can be provided in capsules or tablets; provided as powders or granules; provided as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); provided as edible foams or whips; or provided as emulsions. Tablets or hard gelatin capsules may contain lactose, starch or its derivatives, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or its salts. Soft gelatin capsules may contain vegetable oils, waxes, fats, semisolid or liquid polyols. Solutions and syrups may contain water, polyols and sugars.

[0273] The therapeutic agent intended for oral administration may be coated or mixed with a material that delays disintegration and / or absorption of the active agent in the gastrointestinal tract (e.g., glyceryl monostearate or glyceryl distearate may be used). Thus, sustained release of the active agent may be achieved over many hours and, if desired, the active agent may be protected from degradation in the stomach. Pharmaceutical compositions for oral administration may be formulated to promote release of the active agent at a specific gastrointestinal location due to specific pH or enzymatic conditions.

[0274] Pharmaceutical compositions suitable for transdermal administration can be provided with discrete patches, and the patches are intended to maintain close contact with the epidermis of the recipient over a long period of time. Pharmaceutical compositions suitable for topical administration can be provided with ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In order to be topically applied to skin, mouth, eyes or other external tissues, topical ointments or creams can be used. When formulated into ointments, active ingredients can be used together with paraffin or water miscible ointment bases. Alternatively, active ingredients can be formulated into emulsifiable pastes together with oil-in-water bases or water-in-oil bases. Pharmaceutical compositions suitable for topical administration to eyes include eye drops. In these compositions, active ingredients can be dissolved or suspended in a suitable carrier, for example, in an aqueous solvent.

[0275] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions or suspensions, which may include antioxidants, buffers, antibacterial agents and / or make compositions substantially isotonic with the blood of the intended recipient. Other components that may be present in such compositions include, for example, water, alcohol, polyols, glycerol and vegetable oils. Compositions suitable for parenteral administration may be present in unit doses or multidose containers (e.g., sealed ampoules and vials), and may be stored under freeze drying (lyophilization) conditions, only requiring the addition of sterile liquid carriers (e.g., sterile saline solutions) immediately before use for injection. Provisional injection solutions and suspensions may be prepared by sterile powders, granules and tablets. In one embodiment, an automatic syringe including an injectable solution of a therapeutic agent may be provided for emergency use in ambulances, emergency rooms, and even for self-administration in a home environment, such as pancreatic patients staying at home.

[0276] In one embodiment, the composition is formulated as a pharmaceutical composition suitable for intravenous administration to a human being according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic (such as lidocaine) to alleviate pain at the injection site.

[0277] Typically, the ingredients are supplied separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. When the composition is to be administered by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is to be administered by injection, an ampoule of sterile saline can be provided so that the ingredients can be mixed prior to administration.

[0278] Suppositories usually contain the active ingredient in the range of 0.5% to 10% by weight; oral formulations may contain 10% to 95% active ingredient.

[0279] The present disclosure also provides a pharmaceutical package or kit, which comprises one or more containers, wherein the containers are filled with one or more ingredients of a pharmaceutical composition of one or more therapeutic agents provided herein. Optionally, associated with such containers may be a notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of a drug or biological product, the notice reflecting approval by the agency to manufacture, use or sell for human administration.

[0280] In another embodiment, for example, the therapeutic agent can be delivered in a controlled release system. For example, the therapeutic agent can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, a liposome, or other modes of administration. In one embodiment, a pump can be used (see Langer, Science 249: 1527-1533 (1990); Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574, each of which is incorporated herein by reference in its entirety). In another embodiment, the therapeutic agent can be delivered in a vesicle, such as a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); WO 91 / 04014; U.S. Pat. No. 4,704,355; Lopez-Berestein, ibid., pp. 317-327). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1953; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105, each of which is incorporated herein by reference in its entirety).

[0281] In yet another embodiment, the controlled release system can be placed in the vicinity of the therapeutic target (i.e., target cell, tissue, or organ), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, pp. 115-138, 1984, which is incorporated herein by reference in its entirety). Other controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533, which is incorporated herein by reference in its entirety).

[0282] In another embodiment, the therapeutic agent, if appropriately formulated, may be administered by nasal, buccal, oral, rectal, ocular, transdermal, parenteral, inhalation, or sublingual administration.

[0283] In a specific embodiment, it can be useful to locally administer the therapeutic agent provided herein to an area in need of treatment; this can be achieved by, for example, but not limited to, local infusion during surgery, local administration (e.g., by injection, by catheter, by suppository, or by implant), wherein the implant is a porous, nonporous, or gel-like material, including a membrane such as a silicone rubber membrane or fiber. Non-limiting examples of this embodiment include stents or other stents coated with the therapeutic agent provided herein, which are implanted into a portion of a vascular system, a catheter, etc.

[0284] The selection of an effective dose will be readily determined by the skilled artisan based on consideration of several factors, which are known to those of ordinary skill in the art. Such factors include the specific form of the therapeutic agent and its pharmacokinetic parameters, such as bioavailability, metabolism, half-life, etc., which are determined in conventional development procedures typically used to obtain regulatory approval for therapeutic agents. Other factors that consider dosage include the condition or stage of the disease to be treated or the benefit to be achieved in a normal individual, the patient's weight, the route of administration, whether the administration is acute or chronic, and concomitant medications and other well-known factors that affect the efficacy of the administered agent.

[0285] Although the recipient of the therapeutic agent contemplated for the entire purpose of this article is a human, the methods herein are equally applicable to other mammals, particularly domesticated animals, livestock, companion animals, and zoo animals. However, the methods provided herein are not restrictive, and the benefits can be applied to any mammal. In other aspects of the methods provided herein, any therapeutic agent as described in Section 6.2.1 can be used in any combination or arrangement with the formulations provided herein.

[0286] In certain embodiments, the present disclosure provides a pharmaceutical composition in the form of a dosage unit suitable for use in the methods provided herein, comprising an amount of a therapeutic agent in the range of about 0.1 pg to 30 mg, 0.5 pg to 25 mg, 1 pg to 20 mg, 500 pg to 10 mg, 1 ng to 10 mg, 500 ng to 10 mg, 1 μg to 10 mg, 500 μg to 10 mg, 1 mg to 5 mg, or 1 mg to 10 mg of the therapeutic agent, and a pharmaceutically acceptable carrier. In one embodiment, the amount of the therapeutic agent is in the range of about 0.5 pg to 1 mg.

[0287] According to the type and severity of the disease, as envisioned in the method provided herein, a series of dosages can be applied to the patient. In one embodiment, the dosage of about 1mg to 12mg therapeutic agent is applied to the patient. In certain embodiments, the dosage is in the range of about 2mg to about 12mg, about 2mg to about 11mg, about 3mg to about 10mg, about 4mg to about 9mg, about 5mg to about 8mg or about 6mg to about 7mg. In certain embodiments, the dosage is about 2mg to about 8mg. In certain embodiments, the dosage is about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 11mg or about 12mg. In certain embodiments, the dosage is 4mg.

[0288] The dosage of therapeutic agent applied to the patient can be based on the patient's weight. In one such embodiment, the therapeutic agent dosage between about 0.1 μg / kg and about 120 μg / kg body weight is applied to the patient. In certain embodiments, dosage is about 0.4 μg / kg to about 110 μg / kg, about 0.5 μg / kg to about 100 μg / kg, about 1 μg / kg to about 95 μg / kg, about 5 μg / kg to about 90 μg / kg, about 10 μg / kg to about 85 μg / kg, about 15 μg / kg to about 80 μg / kg, about 20 μg / kg to about 75 μg / kg, about 25 μg / kg to about 70 μg / kg, about 30 μg / kg to about 65 μg / kg, about 35 μg / kg to about 60 μg / kg or about 40 μg / kg to about 50 μg / kg body weight. In certain embodiments, the dosage of the therapeutic agent is in the range of about 0.2 μg / kg to about 120 μg / kg body weight. In certain embodiments, the dosage is in the range of about 1 μg / kg to about 60 μg / kg, about 10 μg / kg to about 30 μg / kg, about 20 μg / kg to about 50 μg / kg, or about 30 μg / kg to about 60 μg / kg. In certain embodiments, the dosage is about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 13 μg / kg, about 14 μg / kg, about 15 μg / kg, about 16 μg / kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 21 μg / kg, about 22 μg / kg, about 23 μg / kg, about 24 μg / kg, about 25 μg / kg, about 26 μg / kg, about 27 μg / kg, about 28 μg / kg, about 29 μg / kg, or about 30 μg / kg. In certain embodiments, the dosage is less than about 80 μg / kg, less than about 50 μg / kg, less than about 30 μg / kg, less than about 20 μg / kg, less than about 15 μg / kg, or less than about 10 μg / kg. In certain embodiments, the dosage is 10 μg / kg. In certain embodiments, the dosage is 30 μg / kg. In other embodiments, the therapeutic agents provided herein can be systemically administered at a dosage of between about 1 ng and about 300 μg / kg body weight, about 5-150 μg / kg body weight, or about 10-100 μg / kg body weight per administration.

[0289] In other embodiments of the methods provided herein, the therapeutic agent is administered in a dosage range of about 0.1 mg / kg / dose to about 10 mg / kg / dose. In some embodiments, the therapeutic agent is administered in a dosage range of about 0.1 mg / kg / dose, about 0.2 mg / kg / dose, about 0.3 mg / kg / dose, about 0.4 mg / kg / dose, about 0.5 mg / kg / dose, about 0.6 mg / kg / dose, about 0.7 mg / kg / dose, or about 0.8 mg / kg / dose, about 0.9 mg / kg / dose, about 1 mg / kg / dose, about 2 mg / kg / dose, about 3 mg / kg / dose, about 4 mg / kg / dose, about 5 mg / kg / dose, about 6 mg / kg / dose, about 7 mg / kg / dose, or about 8 mg / kg / dose, about 9 mg / kg / dose, about 10 mg / kg / dose, or more.

[0290] In certain embodiments, the therapeutic agent used in the methods of the present invention may be administered in a prophylactic effective amount. A prophylactic effective amount refers to an amount of a therapeutic agent sufficient to prevent pancreatitis and / or damage, effects or symptoms caused by pancreatitis. When used in conjunction with an amount of a therapeutic agent, a prophylactic effective amount may encompass an amount that improves overall prevention or enhances the efficacy of a prophylactic against pancreatitis or provides a synergistic effect with another prophylactic agent that can prevent pancreatitis.

[0291] In addition, the therapeutic agent in the method provided herein can be applied at regular time intervals over time. In some such embodiments, therapeutic agents are applied every day, every other day, once a week, once every 10 days, once every two weeks, once every three weeks, once every four weeks, once every six weeks, or once every eight weeks. In other embodiments, as long as clinical needs, the application of therapeutic agents can be repeated every hour, every day, or at appropriate time intervals (e.g., every 1-12 hours, every 6-12 hours; every 2-6 days, every 2-4 days; every 1 to 12 weeks, every 1 to 3 weeks). In additional embodiments, therapeutic agents are applied once a month (monthly), once every two months (bimonthly), once every three months, once every four months, once every five months, or once every six months.

[0292] In some embodiments, the therapeutic agent provided in the methods described herein is administered for about 1 week to about 48 weeks. In certain embodiments, the therapeutic agent provided in the methods described herein is administered for about 1 week to about 44 weeks; about 1 week to about 40 weeks; about 1 week to about 36 weeks; about 1 week to about 32 weeks; about 1 week to about 28 weeks; about 1 week to about 24 weeks; about 1 week to about 20 weeks; about 1 week to about 16 weeks; or about 1 week to about 12 weeks. In other embodiments, the therapeutic agent provided in the methods described herein is administered for about 1 week to about 48 weeks; about 4 weeks to about 44 weeks; about 8 weeks to about 40 weeks; about 12 weeks to about 36 weeks; about 16 weeks to about 32 weeks; or about 20 weeks to about 28 weeks. In certain embodiments, the therapeutic agents provided in the methods described herein are administered for about 1 week to about 24 weeks; about 2 weeks to about 24 weeks; about 4 weeks to about 24 weeks; about 6 weeks to about 24 weeks; about 8 weeks to about 24 weeks; about 10 weeks to about 24 weeks; about 12 weeks to about 24 weeks; about 14 weeks to about 24 weeks; about 16 weeks to about 24 weeks; about 18 weeks to about 24 weeks; about 20 weeks to about 24 weeks; or about 22 weeks to about 24 weeks.

[0293] In some specific embodiments, the therapeutic agents provided in the methods described herein can be used for long-term administration. In certain embodiments, administration can continue as long as the patient has pancreatitis.

[0294] In other embodiments, an effective amount of a therapeutic agent and a pharmaceutically acceptable carrier may be packaged in a single-dose vial or other container. In additional embodiments, a therapeutic agent capable of exerting the activities described herein is used long-term to combat chronic pancreatitis.

[0295] As is well known to those of ordinary skill in the art, the formulations, routes of administration and dosages of therapeutic proteins including therapeutic antibodies and therapeutic polypeptides may be different. Various formulations, routes of administration and dosages specific for therapeutic proteins (including therapeutic antibodies and therapeutic polypeptides) are well known in the art. Exemplary formulations, routes of administration and dosages of therapeutic antibodies are described in WO2015112886 and WO2017019957; Exemplary formulations, routes of administration and dosages of therapeutic polypeptides are described in WO2007019545 and WO2015009820, all of which are incorporated herein by reference in their entirety.

[0296] 6.3 Assays for testing therapeutic agents

[0297] Various assays can be used to determine the activity and efficacy of the therapeutic agents described in Section 6.2.1 for use in the methods of treatment provided herein. Various marker assays are also provided herein. The disclosure further provides that the activity, effectiveness and efficacy of the various therapeutic agents described in Section 6.2.1 can be determined based on biomarkers or patient treatment outcomes as described in Section 6.2.2. In addition, one of ordinary skill in the art will recognize that the suitability of the various therapeutic agents in the methods provided herein can be confirmed by various in vitro and in vivo pancreatitis assays known in the art or provided herein.

[0298] 6.3.1 Binding assay

[0299] Binding of the therapeutic agents of the methods provided herein to CA19-9 or sialyl Lewis can be readily determined using a variety of routine assays. a Binding between molecules or between any two molecules (such as between two proteins). Exemplary conventional binding assays include immunohistochemistry assays, enzyme immunoassays (EIA) or ELISA, FACS assays, immunoblotting assays, surface plasmon resonance (SPR) assays, co-immunoprecipitation assays, chromatography or gel shift assays, isothermal titration calorimetry, various fluorescence assays (such as fluorescence correlation spectroscopy and fluorescence polarization / anisotropy), competitive binding assays, equilibrium dialysis, fluorescence resonance energy transfer (FRET), and any other assay known to those of ordinary skill in the art for testing binding between two molecules. Details of these general assays are described below and in Wu, Ge. Chapter 5, General Protein Binding Assay Formats, in Assay Development: Fundamentals and Practices, Online ISBN: 9780470583128; Print ISBN: 9780470191156 (2010); Wan H et al., Journal of Liquid Chromatography & Related Technologies, (2007) 30: 681-700, both of which are hereby incorporated by reference in their entirety.

[0300] As is well known to those of ordinary skill in the art, immunohistochemical assays are routinely used to determine the interaction between therapeutic agents (e.g., proteins such as antibodies) and targets in cells or tissues. In the example of IHC assays, IHC assays are usually determined by using color development or fluorescent signals well known in the art as final measurements to determine proteins (such as antibodies) that are in situ bound to target molecules in cells or tissues. In some embodiments, a tissue sample is contacted with a protein (such as a primary antibody) that is expected to bind to a specific target for a period of time sufficient to allow protein-target binding to occur. The bound protein (such as a bound primary antibody) can be detected by direct labeling on the protein itself, such as radioactive labeling, fluorescent labeling, hapten labeling (such as biotin) or enzymes (such as horseradish peroxidase or alkaline phosphatase). Alternatively, unlabeled proteins (such as unlabeled primary antibodies) are used in combination with labeled secondary antibodies, and the secondary antibodies include antiserum, polyclonal antiserum, or monoclonal antibodies specific to the protein (such as primary antibodies). Therefore, enzyme-labeled secondary antibodies can be used, which specifically recognize proteins (e.g., recognize the antibody isotype of a primary antibody). The secondary antibody reacts with a protein bound to the sample (e.g., a primary antibody bound to the sample) and then generates and / or detects a signal. The second layer of detection antibodies can be labeled with an enzyme (such as peroxidase) that reacts with the chromogen 3,3'-diaminobenzidine (DAB) to produce a brown precipitate at the reaction site. The final signal (such as a chromogenic signal, fluorescence, and radioactivity) from the label on the secondary antibody can be used to determine the level, intensity, and specificity of binding between a protein (such as a primary antibody) and a target in a cell or tissue. Automated systems for IHC processing are commercially available and can routinely perform the assay in a medium to high throughput format. Leica BOND Autostainer and Leica Bond Refine Detection systems are examples of such automated systems.

[0301] Alternatively, enzyme immunoassay (EIA) or ELISA can be routinely used to determine the binding between two proteins (such as therapeutic agents and CA19-9). Both EIA and ELISA assays are known in the art. Various ELISA assay formats are available, see, for example, U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653, which are incorporated herein by reference in their entirety. These include non-competitive types and single-site assays and dual-site assays or "sandwich" assays in traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target proteins. Sandwich assays are also commonly used assays. ELISA assay technology has a variety of variations. For example, in order to test the binding between protein X and protein Y, protein X can be fixed on a solid substrate, and protein Y or a sample comprising protein Y is contacted with fixed protein X. After an appropriate incubation period, lasting a period of time sufficient to allow the formation of a binding complex of protein X and protein Y, a second antibody specific for protein Y labeled with a reporter molecule capable of producing a detectable signal is then added and incubated for a time sufficient to allow another complex of protein X, protein Y and the labeled second antibody to form. Any unreacted material is washed away, and the binding between protein X and protein Y is determined by observing the signal produced by the reporter molecule. The results can be qualitative (by simply observing a visible signal) or can be quantitative (by determining the level of binding for a series of increasing concentrations of protein X or protein Y and fitting a binding curve as known to those of ordinary skill in the art).

[0302] In some embodiments of EIA or ELISA assays, the enzyme is conjugated to the second antibody. In other embodiments, a fluorescently labeled second antibody can be used instead of an enzyme-labeled second antibody to produce a detectable signal in the form of an ELISA assay. When activated by light irradiation of a specific wavelength, the fluorescent dye-labeled antibody absorbs light energy, induces an excited state in the molecule, and then emits light with a characteristic color detectable by a detection device. As in EIA and ELISA, the fluorescently labeled antibody is allowed to bind to the complex formed by a pair of binding proteins tested. After washing away the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength, and under appropriate controls known to those of ordinary skill in the art, the observed fluorescence indicates the level, intensity and specificity of the binding. Immunofluorescence and EIA techniques are both very mature in the art and are disclosed herein. As well known to those skilled in the art, it is conventional to carry out EIA or ELISA assays to test the combination between two molecules, and it is conventional to adopt such assays for high-throughput formats.

[0303] Similarly, immunoblotting assays can be used to determine the binding between two proteins, such as a therapeutic agent and CA19-9. The details of immunoblotting assays are further described in Section 6.3.2.5. In one embodiment, CA19-9 can be on a membrane, and the binding between a therapeutic antibody and CA19-9 can be determined in any immunoblotting assay described in Section 6.3.2.5.

[0304] In addition, FACS assays can be used to determine the binding between two proteins (such as therapeutic agents and CA19-9). The details of FACS assays are further described in Section 6.3.2.5. In one embodiment, cells that do not normally express CA19-9 can be engineered to express one or more CA19-9, and the binding between therapeutic agents (such as therapeutic antibodies, therapeutic proteins or therapeutic peptides) and CA19-9 can be determined in any FACS assay described in Section 6.3.2.5 by measuring the increase in the binding of therapeutic agents to cells expressing CA19-9 compared to the binding of control cells that do not express CA19-9.

[0305] The combination between therapeutic agent and CA19-9 can also be determined in immunoprecipitation assay, coprecipitation assay or immunocoprecipitation assay. In immunoprecipitation assay, an antibody (such as a therapeutic antibody) for a specific target protein (such as CA19-9) forms an immune complex with the target in a sample (such as a cell lysate). The immune complex is then captured or precipitated on a beaded support fixed with an antibody-binding protein (such as protein A or G), and any protein not precipitated on the beads is washed away. Finally, the target protein (and antibody, if the antibody is not covalently attached to beads and / or denaturing buffer is used) is eluted from the support and analyzed by various means, such as by mass spectrometry (with or without enzyme digestion) or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (often combined with protein blotting).

[0306] Co-immunoprecipitation is an extension of immunoprecipitation, which is based on the potential of immunoprecipitation reaction to capture and purify the main target (such as CA19-9) and other macromolecules in the sample solution that are bound to the main target by interaction. For example, if protein X can be combined with protein Y, the immunoprecipitation of protein X as described above will cause some protein Y to be co-immunoprecipitated because protein Y is combined with protein X. Protein Y can be eluted from the immunoprecipitated complex and analyzed by various means, such as by mass spectrometry (with or without enzyme digestion) or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (often combined with Western blot). Therefore, the combination between protein X and protein Y can be determined. Except that antibodies are not used in co-precipitation determination, co-precipitation determination is carried out according to the principle and form substantially the same as immunoprecipitation or immuno-co-precipitation determination. For example, glutathione-agarose beads can be used to precipitate GST fusion protein X. Protein Y that binds to protein X can be identified in the precipitant along with protein X by various means such as in immunoprecipitation, and such co-precipitation of protein X and protein Y would indicate that the two proteins can interact.

[0307] In immunoprecipitation assays, co-immunoprecipitation assays or co-precipitation assays, the binding partners of the precipitated target protein can also be detected or measured by mass spectrometry (combination LC-MS including liquid chromatography and mass spectrometry) to identify thousands of binding partners in one assay. For example, if CA19-9 is a target protein precipitated in any precipitation assay provided herein, any and all interaction partners bound to CA19-9 can be eluted and mass spectrometry (including LC-MS) can be performed for identification. Therefore, if CA19-9 has been contacted with a molecular library, all molecules from the library that can be bound to CA19-9 can be immediately identified in one assay. As well known to those skilled in the art, it is conventional to perform immunoprecipitation assays, co-immunoprecipitation assays or co-precipitation assays to test the combination between two molecules, and it is conventional to adopt such assays for high throughput formats.

[0308] In addition, the binding between therapeutic agents and CA19-9 can also be determined in a surface plasmon resonance (SPR) assay. In an SPR assay, a target molecule (most often a protein) is immobilized on a prepared gold sensor surface, and a sample containing potential interaction partners in solution is injected onto the surface through a series of flow cells. During the interaction, polarized light is directed toward the sensor surface, and the angle of minimum intensity reflected light is detected. This angle changes as the molecules bind and dissociate, and thus the interaction curve is recorded in real time in a sensorgram. In the case of sialyl Lewis aIn one example of such an SPR assay for binding to a therapeutic antibody, a biotinylated monovalent sLe a (Cat. No. 02-044) or polyvalent sLe a -PAA-biotin (Cat. No. 01-044) was coupled to the flow cell of a separate SPA biosensor chip. The flow cell blocked with HSA and medium containing free biotin was used as a reference cell. Binding kinetic parameters can be calculated using sLe a -PAA-biotin coated flow cells are determined by diluting several known concentrations of antibodies in HBS-EP buffer (10mmol / L HEPES, pH 7.4, 150mmol / L NaCl, 3.4mmol / L EDTA, 0.005% surfactant P20). Then, the estimated values ​​of association rate and dissociation rate are generated using the curve fitting software provided by the Biacore instrument, from which the affinity is calculated. Commercial SPR measurement systems are available, such as Biacore3000 (GE Healthcare). Examples of implemented SPR measurements are described in U.S. Patent 9,475,874. As is well known to those skilled in the art, it is routine to perform SPR measurements to test the binding between two molecules.

[0309] Alternatively, the binding of therapeutic agents to CA19-9 (or sialyl Lewis a ) are conventional, as described in Hellman LM et al., Nat Protoc. 2007; 2(8): 1849-1861; Mallik R et al., J Chromatogr A. 2010 Apr 23; 1217(17): 2796-2803; Koeplinger KA et al., Analytical Biochemistry (1996) 243(1): 66-73; Chan J et al., Mol Cell Proteomics. 2012 Jul; 11(7): M111.016642. Bloustine J et al., Biophys J. 2003 Oct; 85(4): 2619-2623, all of which are incorporated herein by reference in their entirety. Isothermal titration calorimetry was used to measure the interaction of therapeutic agents with CA19-9 (or sialyl Lewis a ) are also conventional, as described in Freyer MW et al., Methods Cell Biol. 2008; 84: 79-113, both of which are hereby incorporated by reference in their entirety.

[0310] In addition, fluorescence assays such as fluorescence correlation spectroscopy and fluorescence polarization / anisotropy are used to measure the binding of therapeutic agents to CA19-9 (or sialyl Lewis a ) are conventional, such as Langowski J, Methods Cell Biol. 2008; 85: 471-84; Lagerkvist AC et al., Protein Science (2001), 10: 1522-1528; Melo AM et al., Quantifying Lipid-Protein Interaction by Fluorescence Correlation Spectroscopy (FCS). In: Engelborghs Y., Visser A. (eds.) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology (Methods and Protocols), Vol. 1076. Humana Press, Totowa, NJ; Hall MD et al., Methods and Applications in Fluorescence, 2016; Vol. 4, No. 2, 022001; Gijsbers A et al., J Vis Exp. 2016 Oct 21; (116); James NG et al., Methods Mol Biol. 2014; 1076: 29-42; Jameson DM et al., Methods Mol Biol. 2005; 305: 301-22, all of which are hereby incorporated by reference in their entirety.

[0311] One of ordinary skill in the art can also use conventional competitive binding assays to measure the binding of therapeutic agents to CA19-9 (or sialyl Lewis a ) as described in Hu L et al., Anal Chem. 2013 May 21; 85(10):5071-7; Crolla LJ et al., Clinical Chemistry, Chapter 3, pages 57-68, ACS Symposium Series, Vol. 36, ISBN 13:9780841203457; Schmidt KJ et al., Laboratory Medicine, October 1972, pages 42-45, all of which are hereby incorporated by reference in their entirety.

[0312] Similarly, fluorescence resonance energy transfer (FRET) is used to measure the binding of therapeutic agents to CA19-9 (or sialyl Lewisa ) is also conventional, as described in Margineanu A et al., Sci Rep. 2016 Jun 24; 6:28186; Vercruysse T et al., RNA Biology, 8:2,316-324; Du Y et al., Assay Drug Dev Technol. 2013 Jul; 11(6):367-381, all of which are incorporated herein by reference in their entirety. Similarly, in the methods provided herein, bioluminescence resonance energy transfer (BRET) is used to measure the interaction between a therapeutic agent and CA19-9 (or sialyl Lewis a ) are conventional, as described in Cui B et al., MBio, 5(3), e01050-14; Dimri S et al., Methods Mol Biol. 2016; 1443:57-78; Mild JG et al., Mol Biotechnol (2018). pp. 1-11, all of which are incorporated herein by reference in their entirety.

[0313] Other well-known conventional binding assays that can be used are described in Ligand-Binding Assays: Development, Validation, and Implementation in the Drug Development Arena, Khan MN and Findlay JW, eds., ISBN: 9780470041383; Golemis E Protein-protein interactions: Amolecular cloning manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press, p ix, 682. (2002); Phizicky EM et al., Protein-protein interactions: Methods for detection and analysis. Microbiol Rev (1995) 59: 94-123, all of which are hereby incorporated by reference in their entirety.

[0314] 6.3.2 Determination of therapeutic activity against pancreatitis

[0315] As disclosed above, the root causes of pancreatitis are pancreatic inflammation and pancreatic damage. Therefore, the determination of the effectiveness of the method provided herein for evaluating the prevention, treatment, alleviation or management of pancreatitis can include one or more steps of the method provided herein, such as applying the therapeutic agent provided herein to the determination system through various dosing regimens; Determine one or more readings from the determination system; and compare the readings between the therapeutic agent treatment group and the control (placebo) treatment group (or reference range). The change in the reading is used to determine the activity of the therapeutic agent for preventing, treating, alleviating or managing pancreatitis.

[0316] As disclosed in Section 6.2.2, pancreatitis can be determined and observed by determining, measuring or observing various indicators and / or markers. Thus, the assay systems described in this section can use one or more readouts based on one or more markers as discussed above in Section 6.2.2 Biomarkers.

[0317] 6.3.2.1. Immunohistochemistry (IHC) or histological staining assays for pancreatitis

[0318] As disclosed in Section 6.2.2, the markers of pancreatitis include abnormalities in pancreatic histology (e.g., histological staining or IHC staining from pancreatic tissue samples). Such histological markers include abnormal expression of one or more proteins in the pancreas, the presence or abnormal number of a certain type of non-pancreatic cells (e.g., immune cells, including leukocytes, macrophages, and neutrophils) in the pancreas, the abundance of certain cells in the pancreas or the abnormal changes in the quantity, and structural abnormalities of pancreatic cells or pancreatic tissue (e.g., abnormalities in pancreatic parenchyma, pancreatic ducts, islets, and acinar cells / tissues). Therefore, the determination of the effectiveness of the method provided herein for assessing the prevention, treatment, alleviation, or management of pancreatitis includes determinations for histopathological staining and / or for detecting such abnormal IHC determinations in pancreatic histology.

[0319] In some embodiments of the assays in the methods provided herein, a therapeutic agent is administered according to the methods provided herein to a model assay system as further discussed in Section 6.3.3 below, and one or more histological markers are determined by an assay as described in this section. Therapeutic agents that reverse abnormalities in histological markers in pancreatitis or that change histological markers toward normal reference levels or reference levels of healthy control subjects can be used in the methods provided herein.

[0320] As is well known to those of ordinary skill in the art, it is routine to perform IHC assays to determine any histological pancreatitis markers. For IHC assays, it is well known in the art that antibodies are generally used to detect and visualize cell antigens in situ by color development or fluorescence methods. A primary antibody or antiserum (such as a polyclonal antiserum and a monoclonal antibody that specifically binds to a target protein) can be used to detect the level of a target in an IHC assay. In some embodiments, a tissue sample is contacted with an antibody for a specific target for a period of time sufficient to allow antibody-target binding to occur. The antibody can be detected by direct labeling on the antibody itself, such as radioactive labeling, fluorescent labeling, hapten labeling (such as biotin) or an enzyme (such as horseradish peroxidase or alkaline phosphatase). Alternatively, an unlabeled primary antibody is used in combination with a labeled secondary antibody (such as an antiserum, a polyclonal antiserum or a monoclonal antibody that is specific to the primary antibody). Then an enzyme-labeled secondary antibody can be applied, and the secondary antibody specifically recognizes the antibody isotype of the primary antibody (second layer). The secondary antibody reacts with the primary antibody, and a detection signal such as a substrate chromogen can then be generated. The second layer of antibodies can be labeled with an enzyme (such as peroxidase) that reacts with the chromogen 3,3'-diaminobenzidine (DAB) to produce a brown precipitate at the reaction site. Due to the potential signal amplification by applying a signal amplification system, this method is sensitive and versatile. IHC protocols and kits are well known in the art and are commercially available. Automated systems for slide preparation and IHC processing are commercially available. Leica BOND Autostainer and Leica Bond Refine Detection systems are examples of such automated systems.

[0321] The assay provided herein can also use conventional histopathological staining, utilize molecules other than antibodies to visualize pancreatic histological structure and detect abnormalities in pancreatic histology. In histopathological staining, one or more staining dyes are used to mark cells, tissues, nucleic acids, proteins, lipids and / or other cells or tissue components to help perform microscopic examination of pancreatic histology. The typical procedures of histopathological staining include fixation, processing, embedding, slicing, staining, washing / development, such as Alturkistani et al., Histological Stains: A Literature Review and Case Study, Glob J Health Sci., (March 2016), 8 (3): disclosed in 72-79, or commercially available from Leica Biosystems (e.g., from Leica's Infinity staining kit system). Exemplary histopathological staining is hematoxylin-eosin staining (H&E), which is routinely used for all tissue specimens to reveal potential tissue structure and conditions. It is clear to those skilled in the art that histopathological staining and IHC can be combined in various arrangements and combinations for the assays envisioned in the present disclosure.

[0322] Thus, in some embodiments of assays for therapeutic agents or methods provided herein, therapeutic agents are administered according to the methods provided herein to a model assay system as further discussed in Section 6.3.3 below, and abnormalities in the pancreas are determined by histopathological staining and / or IHC staining as described in this section. Therapeutic agents that reduce, alter, or reverse abnormalities in pancreatic histology toward normal reference pancreatic histology or reference pancreatic histology of a healthy control subject can be used in the methods provided herein.

[0323] 6.3.2.2. Imaging measures of pancreatitis

[0324] As discussed above, pancreatitis markers can also be detected by imaging assays. As well known to those skilled in the art, the abnormality in pancreatic structure can be imaged, for example, by ultrasound (such as EUS), computed tomography, endoscopic retrograde cholangiopancreatography (ERCP) and magnetic resonance imaging (such as MRCP) (with or without contrast agent enhancement). Exemplary pancreatitis markers detected by imaging are described in Section 6.2.2.2 above. Therefore, in some embodiments of the assay for the activity of therapeutic agents provided herein, the therapeutic agent is administered to a model assay system as further discussed in Section 6.3.3 below according to the method provided herein, and the abnormality in the pancreas is determined by imaging assays as described in this section. Reduce, improve, change or reverse the abnormality of pancreatitis markers (described in Sections 6.2.2 and 6.2.1) detected by imaging assays, so that the therapeutic agent that tends to the normal reference range of markers or the reference range of healthy control subjects can be used in the method provided herein.

[0325] As is well known to those of ordinary skill in the art, it is routine to perform imaging assays to determine any of the pancreatitis markers provided herein. Detailed protocols for imaging assays are well known to those of ordinary skill in the art and have been described in Etemad B et al., Gastroenterology 2001; 120: 682-707; Duggan, SN et al., World J Gastroenterol 2016; 22(7): 2304-2313; and Chapters 37 and 38 on acute pancreatitis and chronic pancreatitis, respectively, of GI / Liver Secrets Plus, 4th edition, McNally PR ed., 2010; Sai JK et al., World J Gastroenterol 2008 February 28; 14(8): 1218-1221; Banks PA et al., Gut 2013; 62: 102-111; Iglesias-Garia J et al., Gut 2006; 55: 1661-1684, all of which are hereby incorporated by reference in their entirety.

[0326] 6.3.2.3. Serological marker-based pancreatitis assay

[0327] As is well known to those skilled in the art, the serological, urine and fecal markers provided in Section 6.2.2.1 can be detected by biochemical assays such as immunoassays. Such assay formats can include ELISA assays, test strip tests, bead-based FACS assays, Western blot assays, chromatographic assays (e.g., FPLC and HPLC), spectrometric assays, colorimetric assays, and mass spectrometry.

[0328] In some embodiments of the assays in the methods provided herein, a therapeutic agent is administered according to the methods provided herein to a model assay system as further discussed in Section 6.3.3 below, and one or more serological markers are determined by an assay as described in this section. Therapeutic agents that reverse abnormalities in serological markers in pancreatitis or that shift serological marker levels toward a normal reference range or a reference range for healthy control subjects can be used in the methods provided herein.

[0329] Serum amylase, lipase, CRP and other markers provided in Section 6.2.2.1 can be detected using immunoassays such as enzyme immunoassay (EIA) or ELISA. Both EIA and ELISA assays are known in the art, for example, for measuring various tissues and samples, including blood, urine, serum or pancreatic tissue. Various ELISA assay formats are available, see, for example, U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653, which are incorporated herein by reference in their entirety. These include non-competitive types and single-site assays and dual-site assays or "sandwich" assays in traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target proteins. Sandwich assays are commonly used assays. There are a variety of variations in sandwich assay techniques. For example, in a typical forward assay, an unlabeled antibody is fixed to a solid substrate, and the sample to be tested is contacted with the fixed antibody. After a suitable incubation period, which lasts for a period sufficient to allow the formation of an antibody-antigen complex, a second antibody specific for the antigen, labeled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient to form another complex of the antibody-antigen labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observing the signal produced by the reporter molecule. The result can be qualitative (by simple observation of visible signals) or can be quantitative (by comparison with a control sample containing a known amount of target protein).

[0330] In some embodiments of EIA or ELISA assays, an enzyme is conjugated to a second antibody. In other embodiments, a fluorescently labeled second antibody can be used instead of an enzyme-labeled second antibody to produce a detectable signal in the form of an ELISA assay. When activated by irradiation with light of a specific wavelength, the fluorescent dye-labeled antibody absorbs light energy, induces an excited state in the molecule, and then emits light with a characteristic color detectable by a detection device. As in EIA and ELISA, a fluorescently labeled antibody is allowed to bind to the first antibody-target protein complex. After washing away the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength, and the observed fluorescence indicates the presence or quantity of the target protein of interest. Immunofluorescence and EIA techniques are both very mature in the art and disclosed herein.

[0331] In one embodiment, the assay provided herein uses a quantitative sandwich enzyme immunoassay technique. The monoclonal antibodies provided herein that are specific for pancreatitis markers are pre-coated on a microplate. Standards and samples (e.g., blood or serum samples) are pipetted into the wells, and any markers present are bound by fixed antibodies. After washing away all unbound substances, an enzyme-linked polyclonal antibody specific for the marker is added to the wells. After washing to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells, and color development is proportional to the amount of markers bound in the initial step, which is related to the marker level in the sample (e.g., blood or serum sample). Stop color development and measure the intensity of the color. The measurement is used as a substitute for the marker level in the sample (e.g., blood or serum sample).

[0332] As well known to those of ordinary skill in the art, it is conventional to carry out EIA or ELISA determination to determine that any pancreatitis marker provided herein. Exemplary commercial determinations for measuring serum amylase, serum lipase, CRP and other pancreatitis markers have been provided in Section 6.2.2.1. Therefore, in some embodiments of the determination in the method provided herein, according to the method provided herein, therapeutic agents are applied to the model determination system as further discussed in Section 6.3.3 below, and the level of serological markers (such as amylase, lipase or CRP) is determined by the determination as described in this section. The therapeutic agent that reduces marker levels or changes marker levels to normal reference range or healthy control subject reference range can be used in the method provided herein.

[0333] In some embodiments, the methods provided herein can be used with certain specific amylase, lipase, and CRP assays, as are well known to those of ordinary skill in the art. Examples of such specific assays for amylase, lipase, CRP and other serological markers are described in Kaphalia BS, Chapter 16, Biomarkers of acute and chronic pancreatitis in Biomarkers in Toxicology, Gupta R. ed., ISBN: 978-0-12-404630-6 (2014); Sutton PA et al., Ann R Coll Surg Engl 2009;91:381-384; Matull WR et al., J Clin Pathol 2006;59:340-344; Basnayake C et al., Aust Prescr 2015;38:128-30; and Jasdanwala S et al., Integr Mol Med, 2015;2(3):189-195, all of which are hereby incorporated by reference in their entirety.

[0334] 6.3.2.4. Measurement of pancreatic function

[0335] In some embodiments of the assays in the methods provided herein, a therapeutic agent is administered to a model assay system as further discussed in Section 6.3.3 below according to the methods provided herein, and pancreatic function is determined by an assay as described in this section. Therapeutic agents that restore pancreatic function or change the level of pancreatic function toward a normal reference range or a reference range for healthy control subjects can be used in the methods provided herein. Various detailed protocols for measuring pancreatic function are well known to those of ordinary skill in the art and are routinely performed, for example, as described in John G Lieb II and Peter V Draganov, World J Gastroenterol. 2008 May 28;14(20):3149-3158; Clain JE et al., Surg Clin North Am 1999;79:829-845; Couper R. Can J Gastroenterol 1997;11:153-156; Layer P et al., J Clin Gastroenterol 1999;28:3-10; Toskes PP. Curr Gastroenterol Rep 1999;1:145-153; Chapter 38 on chronic pancreatitis in GI / Liver Secrets Plus, 4th edition, McNally PR ed., 2010; all of which are hereby incorporated by reference in their entirety.

[0336] 6.3.2.5. Determination of signal transduction markers

[0337] In some embodiments of the assays in the methods provided herein, a therapeutic agent is administered to a model assay system as further discussed in Section 6.3.3 below according to the methods provided herein, and one or more signaling markers are determined by an assay as described in this section. Therapeutic agents that reduce signaling marker levels or change signaling marker levels to normal reference ranges or healthy control subject reference ranges can be used in the methods provided herein. Signaling markers that can be used for assays are described in Section 6.2.2.4 above.

[0338] In some embodiments, immunoblotting can be used to detect signaling markers with antibodies described herein. In some embodiments of immunoblotting, proteins can (but not necessarily) be separated by electrophoresis and transferred to a membrane (usually a nitrocellulose membrane or a PVDF membrane), such as in a Western blot assay. Similar to IHC assays, a primary antibody or antiserum (such as polyclonal antiserum and monoclonal antibodies) that specifically targets a signaling marker can be used to detect the level of a signaling marker. In some embodiments, the membrane is contacted with a primary antibody for a specific target for a period of time sufficient for antibody-antigen binding to occur, and the bound antibody can be detected by direct labeling on the primary antibody itself, such as with a radioactive label, a fluorescent label, a hapten label (such as biotin) or an enzyme (such as horseradish peroxidase or alkaline phosphatase). In other embodiments, an unlabeled primary antibody is combined with a labeled secondary antibody specific for the primary antibody for indirect determination as described above for IHC assays. As described herein, a secondary antibody can be labeled, for example, with an enzyme or other detectable label such as a fluorescent label, a luminescent label, a colorimetric label or a radioisotope. Immunoblotting protocols and kits are well known in the art and are commercially available. Automated systems for immunoblotting, such as the iBind Western system (ThermoFisher, Waltham, MA USA 02451) for Western blotting are commercially available. Immunoblotting includes but is not limited to Western blotting, intracellular protein blotting and dot blotting. Dot blotting is a simplified procedure in which protein samples are not separated by electrophoresis, but are directly placed on the film. Intracellular protein blotting involves cells being seeded in microtiter plates, fixed / permeabilized cells, and subsequently using a labeled primary antibody or an unlabeled primary antibody, followed by detection with a labeled secondary antibody as described herein.

[0339] In other embodiments, the level of signaling markers can also be detected with antibodies described herein in flow cytometry assays (including fluorescence activated cell sorting (FACS) assays). Similar to IHC or immunoblotting assays, primary antibodies or antisera (such as polyclonal antisera and monoclonal antibodies) that specifically target signaling markers can be used to detect the level of signaling markers in FACS assays. In some embodiments, cells are stained with a primary antibody for a period of time sufficient for antibody-antigen binding to occur, and the bound antibody can be detected by a direct label on the primary antibody, such as a fluorescent label or a hapten label (such as biotin) on the primary antibody. In other embodiments, an unlabeled primary antibody is combined with a fluorescently labeled secondary antibody specific for the primary antibody for indirect determination as described above for IHC assays. FACS provides a method for sorting or analyzing a mixture of fluorescently labeled biological cells one cell at a time according to the specific light scattering and fluorescence characteristics of each cell. Therefore, the flow cytometer detects and reports the intensity of the fluorescent dye-labeled antibody, which indicates the level of the signaling marker in each cell. Therefore, antibodies to signaling markers can be used to detect the level of signaling markers. Cytoplasmic signaling markers can also be observed by staining permeabilized cells. Methods for performing FACS staining and analysis are well known to those skilled in the art and are described by Teresa S. Hawley and Robert G. Hawley in Flow Cytometry Protocols, Humana Press, 2011 (ISBN 1617379506, 9781617379505). It is routine for those of ordinary skill in the art to perform FACS assays and immunoblot assays to determine any signaling marker provided herein.

[0340] As known to those of ordinary skill in the art, the levels of signaling markers can also be routinely measured using immunoassays such as enzyme immunoassays (EIA) or ELISA as described above in Section 6.3.2.3.

[0341] 6.3.3 Model systems used for determination

[0342] 6.3.3.1. Animal models

[0343] The present disclosure provides an animal model (e.g., a transgenic mouse model), wherein the animal (e.g., mouse) is genetically engineered to express CA19-9 in the pancreas and develop pancreatitis. The following disclosure is provided using mice as an exemplary animal model, but those skilled in the art will appreciate that the present disclosure includes and is applicable to other animals, such as monkeys, pigs, dogs, rats, and / or rabbits. The present disclosure further provides that CA19-9 is produced by gradually adding different sugar moieties to a type 1 precursor chain, ultimately linking the α1,4 of fucose to GlcNAc. As used herein, Fut3 refers to a fucosyltransferase having the ability to add a fucose moiety through an α1,4 bond. In one embodiment of a transgenic mouse pancreatitis model, mouse embryonic stem cells (mESCs) can be produced, wherein the mESCs express Fut3 and β3GalT5 via an inducible promoter that depends on the tissue-specific expression of Cre, the recombination of a LoxP-flanked termination box (LSL), and the administration of doxycycline (Dox). Mice can be generated from mESCs with and without Cre transfection to produce strains that are dependent on or independent of Cre expression. In these transgenic mice, the expression of Fut3 and β3GalT5 can be induced by administering Dox, and thus the expression of CA19-9 can be induced. Therefore, the present disclosure provides mice in which CA19-9 expression can be induced in the entire pancreas of mice, mice in which CA19-9 expression can be induced in focal areas of the pancreas of mice, and mice in which CA19-9 expression can be induced in the whole body of mice. These mice are referred to as CA19-9 mouse models, and each can be used in the methods provided herein.

[0344] In all CA19-9 mouse models in which CA19-9 expression is induced in the pancreas, non-pancreatic tissues exhibit normal histology after a period of Dox treatment relative to genetically negative littermate controls. Induction of CA19-9 expression in the pancreas of the mouse model leads to pancreatitis, including multiple physiological, histological and serological signs of pancreatitis (e.g., acinar atrophy, immune cell invasion, collagen deposition, elevated lipase and amylase), while pancreatitis cannot be observed in Dox-treated genetically negative controls or untreated mice. In addition, CA19-9 mice in which CA19-9 expression has been induced throughout the mouse exhibit weight loss and pancreatic atrophy after CA19-9 expression, resulting in weight loss of more than 20% and subsequent euthanasia in some cases. In CA19-9 mice in which CA19-9 expression has been induced throughout the mouse, all tissues except the pancreas remain healthy, indicating that pancreatitis in CA19-9 mice after CA19-9 expression is not autoimmune-mediated.

[0345] CA19-9 mice in which CA19-9 expression has been induced throughout the pancreas develop hyperosmotic pancreatitis and elevated levels of amylase and lipase without weight loss or pancreatic weight loss. The immune cell types present in the spleen, lymph nodes, and pancreas of CA19-9 mice in which CA19-9 expression has been induced throughout the pancreas are comparable to the bombesin model of acute pancreatitis (discussed below). Both the CA19-9 mice in which CA19-9 expression has been induced throughout the pancreas and the bombesin model exhibit treatment-dependent influx of inflammatory monocytes and macrophages in the pancreas, without any changes in immune cell types represented in the spleen or lymph nodes. CA19-9 mice in which CA19-9 expression has been induced in focal areas of the pancreas develop smaller pancreatitis lesions and elevated levels of amylase and lipase without weight loss or pancreatic weight loss, and exhibit the expected CA19-9 expression pattern relative to genetic controls and untreated controls. All CA19-9 mouse models exhibit chronic pancreatitis with multiple histological markers of chronic pancreatitis. CA19-9 can also be detected in the plasma of CA19-9 mouse models with CA19-9 expression throughout the mouse body and in the plasma of CA19-9 mouse models with CA19-9 expression throughout the pancreas of the mouse. In one embodiment, CA19-9 mice with CA19-9 expression throughout the pancreas provide a high penetration of the pancreatitis phenotype without weight loss. As used herein, the penetration of pancreatitis refers to the percentage of area in the pancreas affected by pancreatitis: the higher the percentage of area affected, the higher the penetration.

[0346] In addition, pancreatitis models suitable for the methods provided herein include clinical human pancreatitis patients. Such patients for clinical testing can be identified based on the pancreatitis markers and classification schemes described in Section 6.2.2 above.

[0347] 6.3.3.2. Cell Model

[0348] Alternatively, cell models that reproduce some or all of the signaling pathways or mechanisms in pancreatitis can be used in the methods provided herein to identify therapeutic agents, confirm the activity of therapeutic agents in reducing or increasing signaling markers as described above, determine the therapeutic results of therapeutic agents in pancreatitis, and test the efficacy of therapeutic agents in pancreatitis. In addition, as discussed above, any cell model provided herein can be used in any arrangement or combination with the assay disclosed herein. These cell models suitable for the methods provided herein include mouse pancreatic ductal adenocarcinoma (PDAC) cell lines (e.g., by introducing Fut3 and / or β3GalT5 expression in mouse PDAC cells) and human PDAC cell lines (such as Suit2) that have been engineered to express CA19-9. The present disclosure provides natural or recombinant PDAC cells expressing CA19-9 that reproduce some or all of the signaling pathways and mechanisms that are the basis of human pancreatitis, and can be used in the methods and assays provided herein.

[0349] In addition, primary acinar cells isolated from animals (such as pigs, monkeys or humans) or acinar cell lines (such as rat pancreatic acinar cell line AR42J) can be used as cell models in the methods provided herein to identify therapeutic agents, confirm the activity of therapeutic agents in reducing or increasing signaling markers as described above, determine the therapeutic outcome of therapeutic agents in pancreatitis, and test the efficacy of therapeutic agents in pancreatitis. Single, double and large clusters of acinar cells were obtained using a collagenase digestion protocol described in Burnham DB et al., J Physiol 349:475-482, 1984; Criddle DN et al., J Gastroenterol Hepatol 21 Suppl 3:S14-17, 2006; Peikin SR et al., Am J Physiol 235(6):E743-749, 1978; Schultz GS et al., Exp Cell Res 130(1):49-62, 1980; Williams JA et al., Am J Physiol 235(5):517-524, 1978, all of which are hereby incorporated by reference in their entirety. The present disclosure provides that acinar cells recapitulate some or all of the signaling pathways and mechanisms that underlie human pancreatitis and can be used in the methods and assays provided herein.

[0350] Three-dimensional (3D) cell culture, whether in vitro 3D culture or 3D structure isolated from animal pancreas, can also be used as cell model in the method provided herein as discussed above. Such 3D culture reproduces some or all signal transduction pathways and mechanisms as the basis of human pancreatitis, including intracellular signal transduction and intercellular signal transduction, and can be used in the method and determination provided herein. 3D structure isolated from animal pancreas includes pancreatic lobule, pancreatic organoid and pancreatic slice.

[0351] As known to those of ordinary skill in the art, pancreatic lobules can be prepared by spreading them out through injection of Krebs-Ringer bicarbonate buffer into the loose connective tissue of the pancreas, and then individually excising the lobules by microdissection under a stereomicroscope. Because most surgical trauma is limited to ducts and blood vessels, this process can minimize damage to acinar cells. The excised lobules retain the overall acinar structure of the tissue and its small size, and allow oxygen and solutes to easily penetrate from the incubation solution. According to this method, the lobules can be kept in culture for several hours and used for subsequence determination (including high-throughput determination). Procedures and methods for isolating pancreatic lobules suitable for assay are described in detail in Barreto SG et al., Am J Physiol Gastrointest Liver Physiol 299(1):G10-22, 2010; Barreto SG et al., Am J Physiol Gastrointest Liver Physiol 297(2):G333-339, 2009; Flowe KM et al., Pancreas 9(4):513-517, 1994; Linari G et al., Pharmacol Res Society 43(3):219-223, 2001; Scheele GA et al., J Biol Chem 250(7):2660-2670, 1975; Schloithe AC et al., Neurogastroenterol Motil 20(9):1060-1069, 2008, all of which are hereby incorporated by reference in their entirety.

[0352] As is well known, organoids refer to three-dimensional cell, tissue or organ cultures. Pancreatic organoids can be rapidly generated from resected pancreatic tumors, pancreatic tissues and pancreatic biopsies after digestion with collagenase XI and seeding into growth factor-reduced Matrigel. Adjusting the culture medium with the growth factor R-spondin promotes the main ductal cell population. These pancreatic organoids can survive cryopreservation and exhibit ductal and disease stage-specific characteristics. In addition, pancreatic organoids from mice accurately reproduce physiologically relevant aspects of disease progression in vitro. After orthotopic transplantation, pancreatic organoids are able to regenerate normal ductal structures. In a specific embodiment for preparing pancreatic organoids, the ducts from the pancreas can be harvested after enzymatic digestion with 0.012% collagenase XI (Sigma) and 0.12% dispase (GIBCO) in DMEM containing 1% FBS (GIBCO) and embedded in 100% growth factor-reduced (GFR) Matrigel (BD) to maintain isolated pancreatic organoids. Detailed procedures for isolating and culturing pancreatic organoids are described in Boj SF et al., Cell 160(1-2):324-338, 2015; Huch M et al., EMBO J 32(20):2708-2721, 2013, both of which are incorporated herein by reference in their entirety.

[0353] To obtain pancreatic slices, low melting point agarose gel can be injected into the pancreatic duct of anesthetized mice via duodenal puncture and common bile duct cannulation. The pancreas can then be removed and trimmed. Agarose makes the pancreas strong enough and then sliced ​​using a vibrating slicer with a thickness of 80-140 μm. In addition, agarose is porous and can therefore provide free exchange of tissue with buffer, thereby ensuring optimal culture health for up to two days. The detailed process of isolating and culturing pancreatic slices is described in Huang YC et al., J Physiol 589 (Pt 2): 395-408, 2011; Huch M et al., EMBO J 32 (20): 2708-2721, 2013, both of which are incorporated herein by reference in their entirety.

[0354] These cells and 3D cell structures can be used in any assay provided herein to measure any pancreatitis marker provided herein, such as signaling markers, histological markers, physiological markers, functional markers, and serological markers, or any combination thereof. In some specific embodiments, the cells and 3D cell structures are used to test therapeutic agents using EGFR signaling (e.g., phosphorylation of EGFR, phosphorylation of Erk1 / 2, phosphorylation of Akt, or any combination thereof) as a measurement of the assay. In other specific embodiments, the cells and 3D cell structures are used to test therapeutic agents using CA19-9, sialyl Lewis aor any combination thereof as a measure in the assay to test the therapeutic agent.

[0355] 6.3.4 High-throughput screening assay

[0356] The determination using cells and 3D cell structures can be a high-throughput determination. As known to those of ordinary skill in the art, when using cells or 3D cell structures as model systems, any determination described in Section 6.3.1, Section 6.3.2 and the embodiments can be used for high-throughput determination. By measuring any of the signal transduction markers, histological markers and serological markers described herein, the determination can be designed to use 36 holes, 64 holes, 96 holes, 384 holes and 1536 well plates for screening. In order to establish such high-throughput determinations, liquid handling systems for transferring therapeutic agents and other determination reagents are known in the art, such as the PlateMate Plus system from Matrix (Hudson, NH) and the MiniTrak and Multiprobe II HTEX from the PerkinElmer system. Data measured based on any of the signal transduction markers, histological markers and serological markers described herein can be collected using commercially available imaging systems such as the ViewLux imaging system (PerkinElmer). In specific embodiments, wild-type cells, wild-type 3D cell structures, cells from healthy subjects, or 3D cell structures from healthy subjects can be used as controls in high-throughput assays to indicate normal or reference ranges for measurements of any of signaling markers, histological markers, and serological markers.

[0357] In some specific embodiments, any binding assay provided herein can be routinely adapted into a high-throughput assay format to easily test thousands of pairs of binding partners (e.g., a therapeutic agent with CA19-9 (or sialyl Lewis a ) between); to target CA19-9 (or sialyl Lewis a ) to screen a molecular library for therapeutic agents that bind to CA19-9; or to identify or screen a library for CA19-9.

[0358] Alternatively, any assay provided herein can be combined with mass spectrometry (such as LC-MS) to identify CA19-9 or therapeutic agents from a library of potential molecules. For example, immunoprecipitation, co-immunoprecipitation, or co-precipitation assays can be combined with mass spectrometry (MS) or liquid chromatography-mass spectrometry (LC-MS) to precipitate anti-sialylated Lewis a CA19-9 can be identified by using antibodies or by precipitating CA19-9 to identify molecules that bind to CA19-9. In one embodiment of such an adaptive high-throughput assay, a sialyl Lewis antibody can be used to identify CA19-9. aCo-immunoprecipitation with antibodies was combined with LC-MS to identify the list of CA19-9.

[0359] As is well known to those of ordinary skill in the art, one or more of the assays provided herein can be used in a high throughput format to test the binding of therapeutic agents to CA19-9 (or sialyl Lewis a ) and testing the effect of therapeutic agents on one or more markers in the model system provided herein is routine. Detailed protocols and various formats for adaptive assays of high-throughput assays are described in Szymański P et al., Int J Mol Sci. 2012; 13(1): 427-452; Klumpp, M. et al., Journal of biomolecular screening, 11(6), 617-633; Mayr, LM et al., Journal of Biomolecular Screening, 2008; 13(6), 443-448; Mayr, LM et al., 2009; 9(5), 580-588; D.Cronk, D, Chapter 8, in Drug Discovery and Development (Second Edition), 2013, Hill RG ed., ISBN: 978-0-7020-4299-7, all of which are incorporated herein by reference in their entirety.

[0360] Example

[0361] Example 1

[0362] Materials used in the embodiments described in the Examples were prepared, manufactured, acquired, purchased, identified, evaluated, verified, and / or obtained as follows.

[0363] Human specimens:

[0364] Human specimens were collected at the Technical University of Dresden (Ethikkommission an der Technischen The samples were obtained with the approval of the Human Ethics Committee of the University of Illinois at Chicago (Institute of Pathology, University Hospital Dresden, Dresden, Germany) and the Institutional Review Board (IRB) of the Cold Spring Harbor Laboratory. Normal pancreatic tissue was obtained from the islet transplantation program of the University of Illinois at Chicago and the University of Miami Miller School of Medicine. Tissue microarrays were prepared from samples of patients with chronic pancreatitis obtained in Dresden (Institute of Pathology, University Hospital Dresden) after appropriate informed consent. Informed consent was obtained from each patient after review by the Human Ethics Committee: Technical University of Dresden (EK59032007). Chronic pancreatitis samples were collected from patients undergoing surgery from 1993 to 2009. A pathologist (DA) confirmed the diagnosis of chronic pancreatitis. The pathologist determined the final diagnosis based on histological examination, and PDAC in the disease group was excluded with a median follow-up time of 24 months. For patients with pancreatitis, the inclusion criteria for all cohorts included written informed consent given by the patient and pre-existing chronic pancreatitis. If one or more of the following criteria are met and there is no other more likely diagnosis, then chronic pancreatitis is diagnosed (Mayerle, J. et al., Dtsch Arztebl Int 110, 387-393, (2013), which is hereby incorporated by reference as a whole): recurrent pancreatic pain with recorded elevated amylase or lipase activity for more than one year, and radiological evidence supporting the diagnosis, pancreatic calcification, histological evidence of chronic pancreatitis, clear changes in pancreatic duct morphology, and severe abnormalities in pancreatic function tests with dyspepsia. Calcification was identified on CT scans, and a diagnosis of diabetes was recommended according to the WHO definition, and exocrine insufficiency was determined by fecal elastase measurement or concurrent pancreatic enzyme supplementation. Exclusion criteria were other accompanying malignant diseases, curative treatment of malignant diseases in the last two years before recruitment, cystic diseases associated with the pancreas, pregnancy, or patients who were unable to give informed consent. CA19-9 determinations were performed centrally in certified clinical laboratories.

[0365] Antibody:

[0366] CD4 (100428, clone GK1.5), CD8 (100714, clone 53.6.7), TcRβ clone (109222, clone H57-597), TcRγδ (118106, clone GL3), CD19 (152406, clone 1D3), CD45 (103138, clone 30-F11), NKp46 (137716, clone 29A1.4), CD11b (101224, clone M1 / 70 ), CD11c (117318, clone N418), Ly6C (128016, clone HK1.4), Ly6G (27606, clone 1A8), PDGFRa (APA5), anti-rat BV421 (4054) and streptavidin (405214) were purchased from Biolegend, CA19-9 (r5B-1, Mabvax (Sawada, R. et al. Clin Cancer Res 17, 1024-1032, (2011); Viola-Villegas, NT, et al. Journal of nuclear medicine: official publication, Society of Nuclear Medicine 54, 1876-1882, (2013), both of which are incorporated herein by reference in their entirety)), CA19-9 (ATCC hybridoma 1116-NS-19-9HB8059), phosphorylated EGFR (8G6.2, EMD Millipore, IHC), phosphorylated ERK1 / 2 (4370, Cell Signaling), ERK1 / 2 (4695, Cell Signaling), phosphorylated Akt (4060, 2965, Cell Signaling), Akt (4685, Cell Signaling), phosphorylated ribosomal S6 (4858, Cell Signaling), Signaling) and S6 ribosomal protein (2317, Cell Signaling), phosphorylated EGFR (3777, Cell Signaling), EGFR (71655, Cell Signaling), EGFR (4267, Cell Signaling), recombinant mouse EGFR-Fc (RnD), Ki67 (Spring Bioscience M3062), CK19 (TROMA III, DSHB), eGFP (A-11122, Thermofisher), FLAG (Sigma, F7425), human isotype control (Synagis), mouse isotype control (MOPC21).

[0367] Example 2

[0368] clone:

[0369] Human FUT3 cDNA was PCR amplified, subcloned into pBabe-Neo, and confirmed by Sanger sequencing. Human FUT3 and β3GalT5 (Dharmacon, CCSB-broad-spectrum lentiviral expression OHS6085-213580074) were PCR amplified and subcloned into pBabe-Neo and ColA1 targeting cassette as T2A fusion protein (Fut3-T2A-β3GalT5) (Beard C et al., Genesis. 2006; 44(1):23-8).

[0370] Embryonic stem cell targeting:

[0371] D34 mES cells (129 / C57B1 / 6J F1 hybrid) containing a ColA1 pre-targeted FRT site (Beard, C., Genesis 44, 23-28, (2006), which is incorporated herein by reference in its entirety) and a CAGS-LSL-rtTA3-IRES-mKate2 (Dow LE et al., PLoS One. 2014; 9(4):e95236, which is incorporated herein by reference in its entirety) allele for recombinase-mediated cassette exchange were used to introduce the TRE-driven Fut3-T2A-β3GalT5-IRES-eGFP construct. Briefly, mES cell line D34 was thawed at passage 3 in mES cell culture medium containing LIF onto irradiated DR4 mouse embryonic fibroblast feeders in gelatin-coated tissue culture plates and switched to 2i conditions 48 hours later (Ying, QL et al., Nature 453, 519-523 (2008), which is incorporated herein by reference in its entirety). mES cells were trypsinized and reattached to irradiated DR4 feeders at 37°C for 45 minutes. Electroporation was performed using 2.5 μg FlpE and 5 μg of a targeting cassette containing a TRE-Fut3-T2A-β3GalT5-IRES-eGFP transgene. 600,000 mES cells were resuspended in electroporation buffer (P3, Amaxa from Lonza), mixed with DNA, transferred to electroporation strip wells, and electroporated using an Amaxa electroporation program optimized for mES cells. After electroporation, mES cells were transferred to 6 cm plates containing irradiated DR4 feeders. Selection for hygromycin B (140 μg / ml, Roche) was started after 48 hours, and surviving colonies were selected for expansion and evaluation after 11 days. The mES cell clones were treated with Geneticin in a separate plate to confirm the correct recombination 364 and the restoration of neomycin sensitivity. In addition, in an additional plate, mES cells were transfected with CRE and treated with doxycycline to confirm Cre-dependent mKate fluorescence as well as Cre-dependent and Dox-dependent eGFP fluorescence.

[0372] Generate mice from mES cells with and without Cre transfection to generate strains that are dependent or independent of Cre expression. R osa26:CAGGS-rtTA-mKate and ColAl:TRE- F ut3-β3GalT5-GFP (R; F-whole body), Pdxl-OR p48- C re, R osa26:CAGGS- LSL-rtTA371 mKate and ColAl:TRE- LSL - F ut3-β3GalT5-GFP (C PDX / P48 ; R LSL ; F LSL - focal pancreas) and Pdxl-Cre, Rosa26:CAGGS- LSL -rtTA-mKate and ColAl:TRE- F ut3-β3GalT5-GFP (C PDX ; R LSL ; F- whole pancreas) of mice.

[0373] For Southern blotting g DNA Isolation:

[0374] Harvest mES cell gDNA from 15 clones (Gentra, Puregene). In short, the cells were washed twice with PBS, and 300 μl of cell lysis solution was added to each well, and the cells were scraped using a cell lifter. The lysate was transferred to a new test tube and incubated at 37°C for 45 minutes. 100 μl of protein precipitation solution was added, and the test tube was inverted ten times. The lysate was centrifuged at maximum speed for 2 minutes, and the supernatant was transferred to a new test tube. 300 μl of isopropanol was added, and the test tube was inverted 50 times, and then centrifuged at maximum speed for 2 minutes. The supernatant was carefully poured out, blotted on absorbent paper, and air-dried. The DNA precipitate was resuspended in 50 μl of water, incubated at 65°C for one hour, incubated at room temperature while gently shaking for 4 hours, and then stored at -20°C.

[0375] Southern blotting and PCR:

[0376] ES cell gDNA was digested with EcoRI (NEB) at 37°C for 18 hours with gentle shaking. The digested gDNA was run on a 0.8% TAE agarose gel at 20-35V for 22 hours. The gel was stained with ethidium bromide while rotating for 10 minutes and imaged before washing with water. The gel was incubated in a denaturing solution (0.5N NaOH, 1.5M NaCl) while rotating for 30 minutes and then washed in water. The gel was incubated in a neutralizing solution (1M Tris, 3M NaCl, pH7.0) for 30 minutes while rotating for 30 minutes and then washed in water. The gel was transferred to a nylon membrane (Schleicher & Schuell Nytran) by capillary flow overnight in 20×SSC at room temperature. The membrane was rinsed in 2×SSC and placed on Whatman paper to air dry, followed by UV cross-linking for 60 seconds. The membrane was placed in 10 ml of Pre-Hyb solution in a Robbins tube and incubated at 68°C for 30 minutes while rotating. Pre-hyb washes were repeated at 42°C while rotating. The membrane was then incubated overnight at 42°C in a hybridization solution containing the appropriate probes while rotating. Probes were generated for eGFP (482958 CPM, Rediprime) and puromycin (186409 CPM, Rediprime). The membrane was then washed twice in 3×SSC / 0.5% SDS at 68°C for 30 minutes while rotating and then exposed to the membrane overnight at -80°C. gDNA was also sent to Transnetyx for genotyping of eGFP to exclude clones with signals higher than the verified single copy insertion. Clones FB8 and FB12 were selected for tetraploid complementation (NYU), both of which produced germline-transmitted founders. Both clones showed identical CA19-9 expression patterns, and clone FB12 founder was selected for further evaluation.

[0377] Mouse:

[0378] KPC mice (Kras + / LSL-G12D ;Trp53 + / R172H; Pdxl-Cre) (Hingorani SR et al., Cancer Cell. 2003; 4(6):437-50; Hingorani SR et al., Cancer Cell. 2005; 7(5):469-83) and backcrossed to a C57B1 / 6J background (The Jackson Laboratory, strain 000664). All animal experiments were performed according to procedures approved by the IACUC at Cold Spring Harbor Laboratory. Mice were included in the experimental study at 6 to 8 weeks of age. Doxycycline was administered to mice via drinking water (0.5-2 mg / ml) supplemented with 10 mg / ml sucrose for a study duration of up to one week. Doxycycline was administered via food for experimental studies over a week, designed to deliver 2-3 mg doxycycline per day (Harlan Teklad, diet TD.01306, doxycycline 625 mg), based on a consumption of 4-5 g / day per mouse. EdU (300 μg, Santa Cruz) was administered intraperitoneally twice a day for three consecutive days. Bombesin was administered intraperitoneally at a dose of 200 μl (10 μg / ml) every 8 hours for 48 hours, and C57B1 / 6J mice were allowed to recover for 24 or 72 hours. Blood glucose levels were measured on the 414-sided great saphenous vein using ACCU-CHECKNano (Roche).

[0379] Retroviral Production and Infection:

[0380] Phoenix cells (ATCC) were cultured at 4 × 10 cells / 10 cm tissue culture plate. 6Cells were inoculated. After 24 hours, the cells were 70-80% fused. One hour before transfection, the culture medium was replaced with 10% FBS, 25 μm chloroquine in DMEM. 10 μg of plasmid DNA in a total volume of 438 μl water was added to 62 μl CaCl2. When the DNA / CaCl2 mixture was added dropwise to 500 μl 2×HBS, air was bubbled through the solution at the same time. The mixture was incubated at room temperature for 30 minutes. The solution was vortexed and then added dropwise to the tissue culture plate. After 16 hours, the culture medium was replaced with 10% FBS supplemented DMEM. The virus supernatant was harvested and filtered (0.45 μm) at 24 hours and 48 hours. One volume of Retro-X concentrate (Clontech) was added to three volumes of virus supernatant and incubated overnight at 4°C. After centrifugation at 1500xg for 45 minutes at 4°C, the virus pellet was resuspended with appropriate culture medium for infection. To infect monolayer cells, the virus was resuspended in 10% FBS supplemented DMEM containing 10 μg / ml polybrene. Selection began 48 hours after infection. To infect organoids, the virus pellet was resuspended in an organoid culture medium containing 10 μg / ml polybrene and 10.5 μM Rho kinase inhibitor Y-27632 (Sigma). After single cell dissociation, spin inoculation was used at room temperature at 600RCF for 1 hour (100,000-250,000 cells), then resuspended in matrigel and propagated as an organoid in the presence of 10.5 μM Rho kinase inhibitor Y-27632 (Sigma) for 72 hours before organoid infection. Two days after infection, organoids were treated with 1 mg / ml geneticin (Life technologies) or blasticidin (10 μg / mL, final concentration) for selection.

[0381] Immunohistochemistry:

[0382] Formalin-fixed paraffin-embedded blocks were sectioned and dewaxed. Unless otherwise specified, antigen retrieval was performed in a pressure cooker in citrate buffer for 6 minutes. After 30 minutes of antigen retrieval in citrate buffer, phosphorylated EGFR IHC was performed as previously described (Ardito CM et al., Cancer Cell. 2012; 22 (3): 304-17). Endogenous peroxidase was quenched with 3% hydrogen peroxide in TBS for 20 minutes at room temperature. The slides were then rinsed in tap water and transferred to the Cadenza system. The slides were blocked in 3% BSA in TBST for 1 hour at room temperature and incubated overnight in the primary antibody. The slides were washed 3 times in TBST and then incubated in horseradish peroxidase-conjugated secondary antibodies at room temperature for one hour. The slides were washed 3 times in TBST and disassembled from the Cadenza system. ImmPACT DAB (carrier) was incubated for 1-10 minutes, then washed with water and incubated in hematoxylin counterstain. Slides were then dehydrated and coverslipped in cytoseal 60.

[0383] Automated immunohistochemistry was performed on formalin-fixed paraffin-embedded 5 μm sections collected on Plus slides (Fischer Scientific) and stored at room temperature. Before staining, the slides were incubated in a 60 ° C convection oven for 1 hour. Unless otherwise specified and as previously described (Perez-Aso M et al., FASEB J. 2016; 30 (1): 457-65), chromogenic immunohistochemistry was performed on the Ventana Medical Systems Discovery XT platform, using Ventana's reagents and detection kits for online dewaxing. In brief, the sections were dewaxed in xylene (3 changes), rehydrated by graded alcohol (3 changes of 100% ethanol, 3 changes of 95% ethanol) and rinsed in distilled water. Heat-induced epitope retrieval was performed in a 1200-watt microwave oven at 100% power in 10 mM sodium citrate buffer (pH 6.0) for 20 minutes. The sections were cooled for 30 minutes and then rinsed in distilled water. Endogenous peroxidase activity was blocked. Primary antibodies were diluted 1:1000-1:10000 in Tris-buffered saline (25 mM Tris, 0.15 M NaCl) and incubated for 10 hours at room temperature. Secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) were diluted 1:200 in Tris buffer and incubated for 1 hour at 37°C. The complexes were visualized with 3,3 diaminobenzidine and enhanced with copper sulfate. Slides were washed in distilled water, counterstained with hematoxylin, dehydrated and mounted with permanent medium. Negative controls used antibody diluent alone instead of primary antibody.

[0384] Histological quantification:

[0385] Slides were scanned using an Aperio CS2 scanner (Leica), and total occupied pixel area was calculated in Imagescope (Leica). Affected pancreatitis areas on H&E-stained slides were annotated manually, and uninvolved and affected areas were measured. Pancreatitis areas were defined by the presence of infiltrating lymphocytes, intralobular swelling, acinar to ductal metaplasia, fibrosis, and adipocyte replacement. The number of Ki67-positive nuclei was manually counted in five representative high-power fields of at least four animals per group.

[0386] Serological measurements:

[0387] Amylase and lipase levels in plasma (lithium heparin, BD biosciences) were measured by IDEXX pathology services. CA19-9 levels were measured on the MagPIX multiple ELISA platform (Luminex) using a human cancer biomarker assay (EMD Millipore). Blood glucose levels were measured on the lateral great saphenous vein using an Accu-Chek Nano monitor (Roche). The level of CA19-9 directed antibodies was measured by coating high binding polystyrene flat-bottom plates with 275 units of CA19-9 (MyBioSource, MBS318287) at room temperature overnight. The CA19-9 coated plates were washed in 0.05% Tween20 in PBS and blocked in 1% BSA in the wash buffer, and then 10 μl of mouse plasma was incubated in a final volume of 100 μl of blocking buffer.

[0388] Fecal measurements:

[0389] Fresh fecal samples were collected and processed using manual disruption (Biomasher II) in each respective assay buffer. Chymotrypsin (BioVision) and elastase (EnzChek, ThermoFisher) were measured according to the manufacturers' instructions.

[0390] Organoids, Acinar Explants, Monolayer Cell Lines, and Culture Conditions:

[0391] Detailed procedures for the isolation and propagation of pancreatic ductal organoid cultures have been described previously (Boj Sylvia F et al., Cell. 2015; 160(l-2): 324-38). Briefly, ducts from the pancreas were harvested after enzymatic digestion with 0.012% collagenase XI (Sigma) and 0.12% dispase (GIBCO) in DMEM containing 1% FBS (GIBCO) and embedded in 100% growth factor-reduced (GFR) matrix gel (BD). Organoids were treated with doxycycline at a final concentration of 100ng-1μg / ml. When necessary (e.g., growth curves), organoids were dissociated into single cell suspensions with 2mg / ml dispase, followed by 10 minutes of TrypLE incubation at 37°C. Cells were filtered with a 40μm cell strainer. Cell concentrations were determined using acridine orange and propidium iodide on a Nexcelom Cellometer. Organoid single cells were seeded at 500 cells per well in 10% Matrigel in 384-well plates for growth curve analysis using CellTiter-Glo. Antibody blocking experiments were performed using 2-4 μg of antibody.

[0392] After brief digestion for 5-10 minutes in advanced DMEM / F12 (GIBCO) containing 5 mg / ml collagenase II (GIBCO), 1 mg / ml dispase (GIBCO), 2.50% FBS (GIBCO) and 1 mg / ml soybean trypsin inhibitor (GIBCO), acinar transplantation was performed on minced pancreatic tissue. Acinar clusters were monitored every 3-5 minutes while digesting at 37°C until the acinar mass reached a cluster size of approximately 20-200 cells. Acinar clusters were washed once and then seeded in advanced DMEM / F12 (GIBCO) containing 1×Glutamax (GIBCO), 1×Penicillin / Streptomycin (P / S, GIBCO), 1×HEPES (GIBCO), 3×NEAA (GIBCO) and 0.1%BSA (Sigma-Aldrich).

[0393] Monolayer cell lines from mouse pancreatic tumors were prepared by dissecting primary tumors from adjacent normal pancreas and manually mincing into approximately 1 mm 3The minced tissue was enzymatically digested in 100 mg / ml collagenase V at 37°C for 45 minutes while rotating. The digestion was quenched with 10% FBS, P / S in DMEM and the digested material was centrifuged at 300 RCF for 5 minutes. The supernatant was discarded and the digested material was resuspended in 10% FBS, P / S, DMEM and seeded on 10 cm tissue culture plates for cryopreservation and propagation. Human pancreatic cancer cell lines Capan2, Suit2, Miapaca2 (ATCC) and hM1-2D were propagated in 10% FBS, P / S in RPMI (GIBCO).

[0394] Single Cell Suspensions of Primary Mouse Tissues for Flow Cytometry:

[0395] Spleens and lymph nodes were mechanically dissociated, homogenized, and passed through a nylon cell strainer (BD Falcon) in FACS buffer (eBioscience). Pancreases were mechanically dissociated and digested with 0.4 mg / mL liberase, 0.05 mg / mL collagenase D, and 0.1 mg / mL DNase I (Roche) at 37°C in DMEM (Gibco) supplemented with 10% FCS for 30 minutes, and the suspension was passed through a nylon cell strainer.

[0396] Flow cytometric analysis:

[0397] Staining was performed by incubating cells with antibodies in FACS buffer (eBioscience) at 4°C on a plate shaker for 15 minutes. Multicolor flow cytometric analysis was performed using an LSR Fortessa cytometer (BD Biosciences). The gating strategy for all flow cytometric experiments is further described below.

[0398] Western blotting:

[0399] Immunoblotting was performed using standard techniques. Briefly, protein lysates were separated on 4-12% Bis-Tris NuPAGE gels (Life technologies), transferred to PVDF membranes (Millipore), and blocked for 1 hour in 3% BSA (Sigma) in 0.1% Tween20 in TBS. The membranes were then incubated overnight with shaking at 4°C in the primary antibody, followed by incubation for one hour in the appropriate secondary antibody conjugated to horseradish peroxidase and detection by ECL (GE Healthcare). The normalized changes in the indicated protein targets were quantified relative to the loading control cofilin and untreated time points.

[0400] Isolation of proteins from cell lines:

[0401] The tissue culture plates were washed three times with ice-cold PBS and lysed on the plate in 1% Triton X-100, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA with 1× protease and phosphatase inhibitor cocktail on ice for 30 minutes. The lysate was transferred to a microcentrifuge tube and clarified at 16100 RCF for 10 minutes at 4°C and aliquoted into new tubes for storage at -20°C.

[0402] Isolation of proteins from organoids:

[0403] Liquid overlay medium was drawn from organoids and ice-cold cell recovery solution (Corning) containing 0.5× complete mini protease inhibitor cocktail (Roche, EDTA-free) and 0.5× PhosSTOP (Roche). Organoids were incubated with 0.5× protease and phosphatase inhibitors on ice in CRS for 10 minutes, then centrifuged at 1500RCF for 5 minutes at 4°C. The supernatant was discarded and the organoids were washed once in CRS with 0.5× protease and phosphatase inhibitors. The organoid pellet was snap-frozen in liquid nitrogen and stored at -80°C. In 1% Triton X-100, 50mM Tris-HCl pH 7.5, 150mMNaCl, 5mM EDTA, the organoids were lysed with 1× protease and phosphatase inhibitor cocktail on ice for 30 minutes, centrifuged at 16100RCF at 4°C for 10 minutes, and aliquoted into new tubes for storage at -20°C.

[0404] Immunoprecipitation:

[0405] CA19-9 immunoprecipitation was performed in IP buffer containing 20 mM HEPES, 150 mM NaCl in the presence of 0.5× complete mini protease inhibitor cocktail (Roche, EDTA-free). The monoclonal antibody was conjugated to M-270 epoxy Dynabeads (Thermo Fisher) according to the manufacturer's instructions. 100 μl of mAb-conjugated Dynabeads were washed 3 times in IP buffer and incubated with 500 μg of whole cell lysate from monolayer cell lines in 1 ml IP buffer in a 1.5 ml LoBind microcentrifuge tube (Eppendorf) or with 4 ml of organoid conditioned medium in a 5.0 ml LoBind tube (Eppendorf) while rotating at 4°C for one hour. The samples were bound to a magnet for one minute before removing the supernatant and washing the beads 3 times in IP buffer.

[0406] Flag immunoprecipitation was performed in TBS (50 mM Tris, pH 7.6; 150 mM NaCl) using 5 ml of conditioned medium, 10 μg of rabbit anti-Flag antibody (Sigma) and protein G-conjugated Dynabeads (ThermoFisher) according to the manufacturer's instructions. Immunoprecipitated proteins were gently shaken for 1 hour using DYK peptide (100 μg / ml) or eluted on ice with ice-cold, amine-based, low pH IgG elution buffer (ThermoFisher) and quenched with 10× TBS.

[0407] EGFR immunoprecipitation was performed in 1% Triton X-100, 150 mM NaCl and 50 mM Tris, pH 7.5.

[0408] RNA Isolation, cDNA Synthesis, and Quantitative RT-PCR of Organoids:

[0409] RNA was extracted from cells and organoid cultures using Trizol and PureLink RNA isolation kits according to the manufacturer's instructions (Thermo Fisher). cDNA was synthesized using 1 μg of total RNA and TaqMan reverse transcription reagents (Applied Biosystems). All targets were amplified (40 cycles) using gene-specific Taqman primers and probe sets (Applied Biosystems) on a QuantStudio 6-flex real-time PCR instrument (Applied Biosystems). Relative gene expression was quantified using the DDCt method with QuantStudio real-time PCR software v1.1 (Applied Biosystems). Expression levels were normalized to Hprt. Exemplary genes analyzed include: Mm01187858_ml (EGFR); Mm00658541_ml (Her2); Mm01159999_ml (Erbb3); Mm01256793_ml (Erbb4); Mm00438696_ml (EGF); Mm00446232_ml (TGFa); Mm01354339_ml (Areg); Mm00514794_ml (Ereg); Mm00446968_ml (Hprt).

[0410] RNA sequencing and analysis

[0411] RNA sequencing was performed at the Judith Sulzberger Columbia Genome Center with 60× coverage and 100 bp paired-end reads on an Illumina HiSeq 2500 instrument. Kallisto 0.44 (Bray, NL et al., Nat Biotechnol 34, 525-527 (2016), which is hereby incorporated by reference in its entirety) was used with Ensembl The GRCh38 mouse transcriptome was combined with 03GALT5 (ENST00000380620.8, ENST00000475838.1, ENST00000380618.5, ENST00000398714.3, ENST00000615480.4, ENST00000615480.4), FUT3 (ENST00000458379.6, ENST00000458379.6, ENST00000303225.10, ENST00000398714.3, ENST00000615480.4, ENST00000615480.4), and FUT4 (ENST00000458379.6, ENST00000458379.6, ENST00000303225.11, ENST00000398714.3, ENST00000615480.4, ENST00000615480.4). Transcript abundance was quantified using cDNAs of ENST0000589620.5, ENST00000589620.5, ENST00000587048.1, ENST00000587048.1, ENST00000585715.1, ENST00000587183.1, ENST00000588539.1), eGFP (pBS31'-RBGpA\TRE), rtTA3 (pRosa-pA-CAGGs-L), and mKate2 (Akama-Garren, EH et al. Sci Rep 6, 16836, (2016), which is hereby incorporated by reference in its entirety).

[0412] Transcript abundance was summarized at the gene level using tximport 1.8 (R / Bioconductor (Soneson, C. et al., FlOO Research 4, 1036 (2015), which is hereby incorporated by reference in its entirety)). Gene level counts were differentially expressed using DESeq2 1.2 (R / Bioconductor (Love, MI et al., Genome Biol 15, 550, (2014), which is hereby incorporated by reference in its entirety)) with the following time point and condition comparisons: 4 hour genetic negative organoids vs. 0 hour genetic negative organoids, 24 hour genetic negative organoids vs. 0 hour genetic negative organoids, 24 hour genetic negative organoids vs. 4 hour genetic negative organoids, 4 hour C PDX ; R LSL ; F Organoids vs 0 hour C PDX ; R LSL ; F Organoid, 24 Hour C PDX ; RLSL ; F Organoids vs 0 hour C PDX ; R LSL ; F organoids and 24-hour C PDX ; R LSL ; F organoids for 4 hours C PDX ; R LSL ; F organoids. The reporter gene eGFP and the human transgenes β3GALT5 and FUT3 were excluded to avoid biased differential expression analysis (which pools information across genes). Due to their extreme differential expression, the genes Oprdl and Gstm5 were identified as outliers and were also excluded.

[0413] Genes were ranked using -log10 (q value) * symbol (log2 fold change), where q value is the p value corrected for multiple tests (Benjamini & Hochberg method). GSEA 3.0 (Subramanian, A. et al. Proc Natl Acad Sci USA 102, 15545-15550, (2005), which is hereby incorporated by reference in its entirety) was used with the following MSigDB6.1 databases: C2 KEGG, C5, C6 and H for gene set enrichment of the ranked gene lists for 24-hour treatment and 0-hour treatment. GSEA uses the following non-default parameters: -nperm 2000, -set_min 10, -set_max 1000. Before running GSEA, the differentially expressed genes in the comparison of 24-hour genetic negative organoids to 0-hour genetic negative organoids were removed from the ranked gene list.

[0414] For visualization, raw counts were normalized using the variance stabilizing transformation function of DESeq2 to control for library size and dispersion-mean relationship.

[0415] Trypsin digestion and iTRAQ labeling:

[0416] The beads were reconstructed with 20 μL of 50 mM triethylammonium bicarbonate buffer (TEAB). RapiGest was added to a final concentration of 0.1%, and tris (2-carboxyethyl) phosphine (TCEP) was added to a final concentration of 5 mM. The sample was then heated to 55°C for 20 minutes, cooled to room temperature, and methyl methylthiosulfonate (MMTS) was added to a final concentration of 10 mM. The sample was incubated at room temperature for 20 minutes to completely block free sulfhydryl groups. 2 μg of sequencing grade trypsin (Promega) was then added to the sample, and they were digested overnight at 37°C. After digestion, the supernatant was removed from the beads and vacuum dried. The peptide was reconstructed in 50 μL of 0.5 M TEAB / 70% ethanol and labeled with 8-plex iTRAQ reagent at room temperature for 1 hour (Ross PL et al., Mol Cell Proteomics. 2004; 3 (12): 1154-69). The labeled samples were then acidified to pH 4 using formic acid, combined, and concentrated in vacuo until approximately 10 μL remained.

[0417] Two-dimensional fractionation using spin columns:

[0418] According to the manufacturer's instructions with slight modifications, the peptides were fractionated using the Pierce high pH reverse phase peptide fractionation kit (ThermoScientific). In brief, the peptides were reconstructed in 150 μL 0.1% TFA, loaded onto a spin column, and centrifuged at 3000 g for 2 minutes. The column was washed with water, and then the peptides were eluted with the following percentages of acetonitrile (ACN) in 0.1% triethylamine (TEA): 5%, 7.5%, 10%, 12.5%, 15%, 20%, 30% and 50%. Each of the 8 fractions was then injected into a mass spectrometer at low pH using a capillary reverse phase LC.

[0419] Two-dimensional fractionation by LC:

[0420] Peptides were fractionated using a high-low pH reversed phase separation strategy (adapted from Gilar, M. et al., Journal of separation science 28, 1694-1703 (2005), which is hereby incorporated by reference in its entirety). For the first dimension (high pH), peptides were fractionated on a 10 cm x 1.0 mm column packed with Gemini 3u C18 resin (Phenomenex, Ventura, CA) at a flow rate of 100 μl / min. Mobile phase A consisted of 20 mM ammonium formate at pH 10, and mobile phase B consisted of 90% acetonitrile / 20 mM ammonium formate at pH 10. The sample was reconstituted with 50 μL of mobile phase A, and the entire sample was injected onto the column. Peptides were separated using a 35 minute linear gradient (from 5% B to 70% B), and then the mobile phase was increased to 95% B over 10 minutes. Fractions were collected every minute for 40 minutes and then combined into 8 fractions using the connection strategy described in Wang, Y. et al. Proteomics 11, 2019-2026 (2011), which is hereby incorporated by reference in its entirety. Each of the 8 fractions was then injected into a mass spectrometer separately at low pH using capillary reversed phase LC.

[0421] Mass Spectrometry (Velos):

[0422] An Orbitrap Velos Pro mass spectrometer (Thermo Scientific) equipped with a nano-ion spray source was coupled to an EASY-nLC system (Thermo Scientific). The nanoflow LC system was configured with a 180-μm inner diameter fused silica capillary trapping column containing 3 cm of Aqua 5-μm C18 material (Phenomenex) and a self-packed PicoFrit® column packed to 15 cm with Aqua 3-μm C18 material (Phenomenex) with an 8-μm emitter (New Objective, Woburn, MA). TM 100-μm analytical column. Mobile phase A consisted of 2% acetonitrile; 0.1% formic acid, and mobile phase B consisted of 90% acetonitrile; 0.1% formic acid. 3 μL of each sample dissolved in mobile phase A was injected onto the trapping column by an autosampler. The peptides were then separated using the following linear gradient steps at a flow rate of 400 nL / min: 5% B for 1 min, 5% B to 35% B over 70 min, 35% B to 75% B over 15 min, hold at 75% B for 8 min, 75% B to 8% B over 1 min, and finally hold at 8% B for 5 min.

[0423] The eluted peptides were electrosprayed directly into the Orbitrap Velos Pro mass spectrometer using an applied distal 2.3 kV spray voltage and a capillary temperature of 275 ° C. Each full scan mass spectrum (Res = 60,000; 380-1700 m / z) was followed by the MS / MS spectra of the first 12 substances. High energy collision dissociation (HCD) was used, the normalized collision energy was set to 35 for fragmentation, the isolation width was set to 1.2, and the activation time was 0.1. The duration of dynamic exclusion was set to 70 seconds, the exclusion list size was 500, the repeat count was 1, and the exclusion mass width was 10 ppm. We used monoisotopic precursors to select charge states of 2+ or greater, and all data were acquired in distribution mode.

[0424] Mass Spectrometry (Fusion):

[0425] An Orbitrap Fusion Lumos mass spectrometer (ThermoScientific) equipped with a nano-ion spray source was coupled to an EASY-nLC 1200 system (Thermo Scientific). The LC system was configured with a self-packed PicoFrit® with an 8-μm emitter (New Objective, Woburn, MA) filled to 25 cm with ReproSil-Pur C18-AQ, 1.9 uM material (Dr. Maish GmbH). TM 75-μm analytical column. Mobile phase A consisted of 2% acetonitrile; 0.1% formic acid, and mobile phase B consisted of 90% acetonitrile; 0.1% formic acid. Peptides were then separated at a flow rate of 200 nL / min using the following steps: 2% B to 6% B over 1 minute, 6% B to 30% B over 84 minutes, 30% B to 60% B over 9 minutes, 60% B to 90% B over 1 minute, hold at 90% B for 5 minutes, 90% B to 50% B over 1 minute, and then the flow rate was increased to 500 nL / min while holding at 50% B for 9 minutes.

[0426] The eluted peptides were electrosprayed directly into a Fusion Lumos mass spectrometer using an applied distal 2.3 kV spray voltage and a capillary temperature of 300°C. Full scan mass spectra (Res = 60000; 400-1600 m / z) were then selected for MS / MS using the "top speed" method. High energy collision dissociation (HCD) was used, with a normalized collision energy set to 35 for fragmentation, an isolation width set to 1.2, and a duration of dynamic exclusion set to 10 seconds with an exclusion mass width of 10 ppm. We used monoisotopic precursors to select charge states of 2+ or greater, and all data were acquired in distribution mode.

[0427] Database Search:

[0428] Peaklist (.MGF) files were generated by Mascot Distiller 2.6 (Matrix Science). Protein identification and quantification were performed using Mascot 2.6 (Perkins, DN, et al., Electrophoresis 20, 3551-3567 (1999), which is hereby incorporated by reference in its entirety) searches against the UniProt mouse or human reference proteome (60,725 and 93,799 sequences, respectively). Methylthiolation of cysteine ​​and iTRAQ modifications of N-terminal and lysine were set as fixed modifications, and methionine oxidation and deamidation (NQ) were set as variables. Trypsin was used as a cleavage enzyme, allowing for one missed cleavage. The mass tolerance of the intact peptide mass was set to 30ppm, and the mass tolerance of the fragment ion was set to 0.3Da. The search results were rescored using a randomized version of the same Uniprot human or mouse database to give a final FDR of 1% or 5%. Only peptides with expected values ​​<1% or 5% FDR were used to calculate iTRAQ ratios at the protein level weighted by intensity. Because these were protein IP experiments, global ratio normalization was not applied. Protein enrichment was then calculated by dividing the protein ratio of the real sample by the corresponding control sample ratio (a value of 1.8 was used as the cutoff for enrichment (>2SD)).

[0429] CA19-9 expression is elevated in patients with pancreatitis:

[0430] Pancreatitis is a serious and common medical condition that can lead to death or a chronic condition with significant morbidity, systemic inflammatory response, and multiple organ dysfunction syndrome. Pancreatitis is also associated with a 16.5-fold increased risk of pancreatic cancer (Lowenfels, AB et al. N Engl J Med 328, 1433-1437 (1993), which is hereby incorporated by reference in its entirety), which clearly indicates that further research into the etiology and treatment of pancreatitis is needed. Glycosylation changes, including elevations in CA19-9 (10-30%), have been noted in the serum of some pancreatitis patients, but this glycan epitope has no role in disease progression (Haglund, C. et al., Br J Cancer 53, 189-195 (1986); Makovitzky, J. Virchows Archiv. B, Cell pathology including molecular pathology 51, 535-544 (1986); both are hereby incorporated by reference in their entirety). We evaluated tissue microarrays (TMA) of resected pancreatitis specimens and found that CA19-9 was expressed primarily in the ducts (rather than in the acinar epithelial cells) in more than 90% of patients ( Figure 1A , 1B ), indicating its prominent presence in this disease. CA19-9 is produced by the stepwise addition of different sugar moieties to the type 1 precursor chain, culminating in the α1,4 linkage of fucose to N-acetylglucosamine (GlcNAc) ( Figure 1C ) and can be multivalently added to secretory proteins and cell surface proteins. The lack of the pseudogene Fut3 in rodents hinders the evaluation of the functional significance of CA19-9 in mice (Xia, B. et al., Anal Biochem 387, 162-170 (2009); Narimatsu, H. in Handbook of Glycosyltransferases and Related Genes (Naoyuki Taniguchi et al., ed.) 218-225 (Springer Japan, 2002); both are hereby incorporated by reference in their entirety). Fut3 is the only fucosyltransferase with the ability to add a fucose moiety through an α1,4 bond. We found that expression of FUT3 and β3GALT5 (Ishijima, N. et al. J Biol Chem 286, 25256-25264 (2011), which is hereby incorporated by reference in its entirety), which are required for the production of type I chain precursors, is essential for the production of CA19-9 (sialyl Lewis a , SLe a )Required by Figure 1C-1E). Expression of both FUT3 and β3GALT5 resulted in cell surface expression of CA19-9 to levels equivalent to those observed in a human cancer cell line (Colo205) (Figure 1E). To confirm that the CA19-9 epitope was expressed in an appropriate manner in the engineered mouse PDAC cells, they were compared to human PDAC cell lines and were found to also exhibit the expected membrane localization in culture (Figure 1F). Furthermore, after orthotopic transplantation, the CA19-9 conjugated protein was secreted in vivo, demonstrating that CA19-9 production levels were comparable to those in human PDAC cell line transplant recipients (Figure 1G).

[0431] To determine whether CA19-9 was spuriously attached to proteins in rodent expression systems, CA19-9-modified proteins present in engineered mouse PDAC cell lines were compared to endogenous CA19-9-modified proteins present in human PDAC cells. CA19-9 protein loading was immunoprecipitated (IP) and identified by mass spectrometry (MS) comparing CA19-9 expressing cells (FB) to empty vector transduced controls (Neo). Figure 2A). The relative protein abundance difference between CA19-9 positive and negative cells was determined using isobaric tags for relative and absolute quantification (iTRAQ) (Ross, PL et al. Mol Cell Proteomics 3, 1154-1169 (2004), which is hereby incorporated by reference in its entirety). The selection was made based on the use of two CA19-9 antibody clones (5B1, NS19-9) in the literature (Koprowski, H. et al. Somatic Cell Genet 5, 957-971 (1979); Sawada, R. et al. Clin Cancer Res 17, 1024-1032 (2011); both are hereby incorporated by reference in their entirety). Using any of the CA19-9 antibodies, CA19-9 protein carriers were defined as CA19-9 positive upregulation of at least 1.5-fold relative to negative samples. These analyses identified known CA19-9 protein carriers in FB mouse cells, including Mucl, Lgals3bp, CD44, and Muc5ac (Sperandio, M. et al., Immunological reviews 230, 97-113 (2009); Yue, T. et al. PLoS One 6, e29180 (2011); Yue, T. et al. Proteomics 11, 3665-3674 (2011); Hirao, Y. et al., Journal of Molecular Biomarkers & Diagnosis 03, (2012); all of which are incorporated herein by reference in their entirety). The human PDAC cell line MiaPaCa-2 is CA19-9 negative and was used as a control to identify human CA19-9 core protein in CA19-9 positive cell lines Capan2, Suite2, and hM1-2D ( Figure 2 B). We compared mouse and human CA19-9 protein loading and found that an average of 72.3% (95% CI 60.3-84.3%) of CA19-9 modified proteins identified in three of three human PDAC cell lines were also found in the FB PDAC cell line, indicating that expression of FUT3 and β3GALT5 in mouse cells largely recapitulates the profile of human CA19-9 loading ( Figure 2C ). Identification of sialyl Lewis in three mouse pancreatic ductal adenocarcinoma (PDAC) cell models a A total of 4260 non-redundant proteins were identified in the vector; sialyl Lewis aA total of 1521 common proteins in the carrier; as sialyl Lewis in three model human PDAC cells a A total of 3017 non-redundant proteins were identified in the vectors, as well as sialyl Lewis proteins in three human PDAC cell models. a A total of 926 common proteins were identified in the vector.

[0432] CA19-9 expression causes pancreatitis:

[0433] We generated mouse embryonic stem (mES) cells that multigenetically express FUT3, β3GALT5, and eGFP via inducible and conditional promoters regulated by LoxP-flanked STOP boxes (LSLs) and tetracycline-responsive promoter elements (TREs). Figure 3 A-3D) (Beard, C. et al., Genesis 44, 23-28 (2006), which is hereby incorporated by reference in its entirety). Whole body (R; F), whole pancreas (C PDX ; R LSL ; F) or focal pancreatic (C PDX或P48 ; R LSL ; F LSL ) expression in several types of mouse models. In all CA19-9 mouse models, non-pancreatic tissues showed no...

Claims

1. A sialyl Lewis a The bound antibody or its functional fragment (anti-sLe a ) in the manufacture of a medicament for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, wherein the anti-sLe a The antibody or its functional fragment comprises: (a) a variable heavy chain VH domain comprising three VH complementarity determining regions (CDRs) present in (i) the amino acid sequence of SEQ ID NO: 2, or (ii) the amino acid sequence of an antibody produced by a hybridoma deposited with American Type Culture Collection (ATCC) Accession No. HB-8059; and (b) a variable light chain VL domain comprising three VL complementarity determining regions (CDRs) present in (i) the amino acid sequence of SEQ ID NO: 4, or (ii) the amino acid sequence of an antibody produced by a hybridoma deposited with American Type Culture Collection (ATCC) Accession No. HB-8059.

2. A sialyl Lewis a The bound antibody or its functional fragment (anti-sLe a ) in the manufacture of a medicament for use in a method for preventing, treating, alleviating or managing pancreatitis in a subject in need thereof, wherein the method comprises: (1) administering the drug to the subject at a first dose, (2) determining the severity of pancreatitis in the subject, and (3) administering a second dose of the drug in an amount that is the same as or lower than the first dose if the severity of pancreatitis in the subject is reduced compared to the severity of pancreatitis in the subject before administration of the drug; Alternatively, if the severity of pancreatitis in the subject has not decreased, administering a second dose of the drug in an amount higher than the first dose, wherein the anti-sLe a The antibody or its functional fragment comprises: (a) a variable heavy chain VH domain comprising three VH complementarity determining regions (CDRs) present in (i) the amino acid sequence of SEQ ID NO: 2, or (ii) the amino acid sequence of an antibody produced by a hybridoma deposited with American Type Culture Collection (ATCC) Accession No. HB-8059; and (b) a variable light chain VL domain comprising three VL complementarity determining regions (CDRs) present in (i) the amino acid sequence of SEQ ID NO: 4, or (ii) the amino acid sequence of an antibody produced by a hybridoma deposited with American Type Culture Collection (ATCC) Accession No. HB-8059.

3. The use of claim 1 or 2, wherein the VH domain comprises residues 20-142 of SEQ ID NO:

2.

4. The use of claim 1 or 2, wherein the VL domain comprises residues 20-130 of SEQ ID NO:

4.

5. The use of claim 1 or 2, wherein the VH domain comprises residues 20-142 of SEQ ID NO: 2, and the VL domain comprises residues 20-130 of SEQ ID NO:

4.

6. The use of claim 1 or 2, wherein the VH domain comprises the amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited with American Type Culture Collection (ATCC) Accession No. HB-8059; and the VL domain comprises the amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited with ATCC Accession No. HB-8059.

7. The use according to claim 1 or 2, wherein the anti-sLe a The antibody or its functional fragment comprises a heavy chain consisting of the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited with ATCC Accession No. HB-8059; and a light chain consisting of the light chain amino acid sequence of the antibody produced by the hybridoma deposited with ATCC Accession No. HB-8059.

8. The use according to claim 1 or 2, wherein the anti-sLe a The antibody functional fragment is selected from the group consisting of Fab, Fab', F(ab')2, scFV, diabody, triabody, small molecule antibody and single domain antibody (sdAB).

9. The use according to claim 8, wherein the anti-sLe a The antibody or functional fragment thereof is a diabody.

10. The use according to claim 9, wherein the diabody comprises the amino acid sequence of SEQ ID NO: 18 or 20.

11. The use according to claim 1 or 2, wherein the anti-sLe a The antibody or functional fragment thereof is a monoclonal antibody.

12. The use according to claim 1 or 2, wherein the anti-sLe a The antibody or functional fragment thereof is of IgG or IgM isotype.

13. The use according to claim 12, wherein the IgG antibody is of IgG1, IgG2, IgG3 or IgG4 subclass.

14. The use according to claim 1 or 2, wherein the anti-sLe a The antibody or its functional fragment is formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

15. The use according to claim 1 or 2, wherein the drug is administered by a parenteral administration route.

16. The use according to claim 15, wherein the parenteral administration is selected from the group consisting of subcutaneous injection, intramuscular injection and intravenous injection.

17. The use of claim 1 or 2, wherein the drug is administered in a dose range of about 0.1 mg / kg / dose to about 10 mg / kg / dose.

18. The method of claim 1 or 2, wherein the drug is administered weekly, biweekly, monthly or bimonthly.

19. The use of claim 18, wherein the medicament is administered for about one, two, three, four, five or six months.

20. The use according to claim 1 or 2, wherein the anti-sLe a The antibody or functional fragment thereof is a mouse, chimeric, humanized or fully human monoclonal antibody.

21. The use of claim 1 or 2, wherein the pancreatitis is characterized by one or more markers.

22. The use of claim 2, wherein the severity of pancreatitis is determined by one or more markers.

23. The use of claim 1 or 2, wherein the subject has elevated levels of one or more markers of pancreatitis.

24. The use of claim 1 or 2, wherein administration of the medicament results in a decrease in the level of one or more markers of pancreatitis in the subject.

25. The use of claim 21, wherein the one or more markers are selected from the group consisting of: physiological markers, histological markers, serological markers and signaling markers.

26. The use of claim 25, wherein the histological marker is selected from the group consisting of: immune cell invasion, collagen deposition, fibrotic tissue, fat necrosis, interstitial edema, acinar and vascular destruction, interstitial hemorrhage, ductal dilatation, acinar cell homogenization and expression level of CA19-9.

27. The use according to claim 21, wherein the one or more markers comprise the expression level of CA19-9.

28. The use according to claim 26, wherein the one or more markers are determined by immunohistochemistry.

29. The use according to claim 25, wherein the serological marker is selected from the group consisting of: serum lipase level, serum amylase level and serum C-reactive protein level.

30. The use according to claim 29, wherein the serum lipase level exceeds 200 U / L.

31. The use of claim 29, wherein the serum amylase level exceeds 200 U / L.

32. The use of claim 29, wherein the serum amylase is three times higher than the upper limit of normal serum amylase levels in control subjects.

33. The use of claim 29, wherein the serum lipase level is three times higher than the upper limit of normal serum lipase level in control subjects.

34. The use of claim 25, wherein the physiological marker is selected from the group consisting of weight loss, food malabsorption, pancreatic atrophy, pancreatic enlargement, pancreatic inflammation, and bile reflux.

35. A method comprising: a The bound antibody or its functional fragment (anti-sLe a ) in the manufacture of a therapeutic agent for preventing, treating, alleviating or managing pancreatic sLe a Use of a medicament for treating pancreatitis in a subject with elevated sLe a The antibody or its functional fragment comprises: (a) a variable heavy chain VH domain comprising three VH complementarity determining regions (CDRs) present in (i) the amino acid sequence of SEQ ID NO: 2, or (ii) the amino acid sequence of an antibody produced by a hybridoma deposited with American Type Culture Collection (ATCC) Accession No. HB-8059; and (b) a variable light chain VL domain comprising three VL complementarity determining regions (CDRs) present in (i) the amino acid sequence of SEQ ID NO: 4, or (ii) the amino acid sequence of an antibody produced by a hybridoma deposited with American Type Culture Collection (ATCC) Accession No. HB-8059.

36. The use as described in any one of claims 1, 2 and 35, wherein the pancreatitis is pancreatitis selected from the group consisting of chronic pancreatitis and acute pancreatitis.

37. The use of claim 36, wherein the chronic pancreatitis is selected from the group consisting of mild chronic pancreatitis, moderate chronic pancreatitis and severe chronic pancreatitis.

38. The use of claim 36, wherein the acute pancreatitis is selected from the group consisting of mild acute pancreatitis, moderate acute pancreatitis and severe acute pancreatitis.

39. The use of any one of claims 1, 2, and 35, wherein the subject is a mammal.

40. The use according to claim 39, wherein the mammal is a human.

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