Anti-serine protease inhibitor KAZAL (SPIK) antibodies, immunoconjugates and methods of use

Abstract

Disclosed are anti-AS-SPIK antibodies, as well as methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use for the diagnosis and / or treatment of diseases characterized by the expression of AS-SPIK, such as liver cancer. Also disclosed are diagnostic methods and kits comprising the anti-AS-SPIK antibodies.

Description

[0001] This application is a divisional, the application number of the parent application is 202080054708.9, the application date is July 8, 2020, and the name of the invention is “Anti-serine protease inhibitor KAZAL (SPIK) antibodies, immunoconjugates and methods of use”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to the filing date of U.S. Provisional Patent Application Serial No. 62 / 871,565, filed on July 8, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Government Rights

[0005] This invention was made with government support under Grant No. 2R44CA165314-02A1 and FAIN No. R44CA165314 awarded by the National Institutes of Health (NIH) under the Small Business Innovation Research (SBIR) program. The government has certain rights in this invention. Technical Field

[0006] Disclosed are anti-AS-SPIK antibodies and methods for preparing such antibodies, compositions (including pharmaceutical compositions) comprising such antibodies, and their use for diagnosing and / or treating diseases characterized by the expression of AS-SPIK (e.g., liver cancer). Also disclosed are diagnostic methods and kits comprising the anti-AS-SPIK antibodies. Background Art

[0007] The liver is one of the largest organs in the human body. The liver has many functions, including the production of enzymes and bile required for digestion of food, regulation of glycogen storage, plasma protein synthesis, hormone production, and detoxification of various metabolites. Liver disorders include liver cancer, such as hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), viral infections, cirrhosis, and other inflammatory disorders of the liver, which affect millions of people worldwide. For example, more than 5 million people in the United States and more than 450 million people worldwide have hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, and more than 30% of these infected individuals are at high risk for developing liver cancer. Kew et al., Pathologie-biologie 2010;58(4):273-277; Saraswat et al., J Viral Hepat. 2015;22 Suppl 1:6-25; El-Serag et al., Hepatology 2014;60(5):1767-1775; Kanwal et al., Clinicalgastroenterology and hepatology 2015;13(4):805-807. Despite advances in diagnosis and treatment, liver cancer remains a significant cause of morbidity and mortality. El-Serag, The New England journal of medicine 2011;365(12):1118-1127. The five-year survival rate for primary liver cancer, or cancer that originates in the liver, is less than 10%. However, if liver cancer is detected early and during its most treatable stage, the survival rate increases to nearly 40%. El-Serag et al., Therapeutic advances in gastroenterology 2011;4(1):5-10. Patients with early-stage liver cancer may have few symptoms. Current detection methods (such as serological methods, ultrasound, computed tomography (CT) scans, magnetic resonance imaging (MRI), and angiography) can be unreliable due to low sensitivity and the possibility of operator error. Costly imaging techniques may be less accurate for detecting smaller, early-stage tumors. Yu et al., Clinical gastroenterology and hepatology 2011;9(2):161-167; Bruix et al., Hepatology 2011;53(3):1020-1022. Liver biopsy is still considered the most reliable method for distinguishing benign from malignant tumors, is invasive and requires surgery. Lok et al., Gastroenterology 2001;122(7):2092-2093. There is a continuing need for new methods of diagnosing and treating liver cancer, especially for those affected by cirrhosis, viral infections, and inflammatory conditions of the liver. Summary of the invention

[0008] Aspects of the invention include antibodies or antigen-binding fragments thereof that specifically bind to AS-SPIK, wherein the antibodies or antigen-binding fragments comprise: (a) a heavy chain variable region comprising: (i) CDRH1 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 21 to 30; and / or (ii) CDRH2 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 31 to 40; and / or (iii) CDRH3 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 41 to 50; and (b) a light chain variable region comprising: (i) CDRL1 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 51 to 60; and / or (ii) CDRL2 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 51 to 60. and / or (iii) a CDRL3 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 61 to 70; and / or (iii) a CDRL3 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 71 to 80.

[0009] In some embodiments, the CDRH1, CDRH2 and CDRH3 sequences are present within framework sequences, and wherein the CDRL1, CDRL2 and CDRL3 sequences are present within framework sequences. In some embodiments, at least a portion of the framework sequences comprises a human consensus framework sequence.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a CDRH1 sequence selected from the group consisting of SEQ ID NOs: 21 to 30; and / or (b) a CDRH2 sequence selected from the group consisting of SEQ ID NOs: 31 to 40; and / or (c) a CDRH3 sequence selected from the group consisting of SEQ ID NOs: 41 to 50; and / or (d) a CDRL1 sequence selected from the group consisting of SEQ ID NOs: 51 to 60; and / or (e) a CDRL2 sequence selected from the group consisting of SEQ ID NOs: 61 to 70; and / or (f) a CDRL3 sequence selected from the group consisting of SEQ ID NOs: 71 to 80.

[0011] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a CDRH1 sequence selected from the group consisting of SEQ ID NOs: 21 to 30; (b) a CDRH2 sequence selected from the group consisting of SEQ ID NOs: 31 to 40; (c) a CDRH3 sequence selected from the group consisting of SEQ ID NOs: 41 to 50; (d) a CDRL1 sequence selected from the group consisting of SEQ ID NOs: 51 to 60; (e) a CDRL2 sequence selected from the group consisting of SEQ ID NOs: 61 to 70; and (f) a CDRL3 sequence selected from the group consisting of SEQ ID NOs: 71 to 80.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a CDRH1 sequence of SEQ ID NO:21, a CDRH2 sequence of SEQ ID NO:31, a CDRH3 sequence of SEQ ID NO:41, a CDRL1 sequence of SEQ ID NO:51, a CDRL2 sequence of SEQ ID NO:61, and a CDRL3 sequence of SEQ ID NO:71; or (b) a CDRH1 sequence of SEQ ID NO:22, a CDRH2 sequence of SEQ ID NO:32, a CDRH3 sequence of SEQ ID NO:42, a CDRL1 sequence of SEQ ID NO:52, a CDRL2 sequence of SEQ ID NO:62, and a CDRL3 sequence of SEQ ID NO:72; or (c) a CDRH1 sequence of SEQ ID NO:23, a CDRH2 sequence of SEQ ID NO:33, a CDRH3 sequence of SEQ ID NO:43, a CDRL1 sequence of SEQ ID NO:53, a CDRL2 sequence of SEQ ID NO:63, and a CDRL3 sequence of SEQ ID NO:73; or (d) a CDRH1 sequence of SEQ ID NO:24, a CDRH2 sequence of SEQ ID NO:34, a CDRH3 sequence of SEQ ID NO:43, a CDRL1 sequence of SEQ ID NO:53, a CDRL2 sequence of SEQ ID NO:63, and a CDRL3 sequence of SEQ ID NO:73. NO:24, the CDRH1 sequence of SEQ ID NO:34, the CDRH2 sequence of SEQ ID NO:44, the CDRL1 sequence of SEQ ID NO:54, the CDRL2 sequence of SEQ ID NO:64, and the CDRL3 sequence of SEQ ID NO:74; or (e) the CDRH1 sequence of SEQ ID NO:25, the CDRH2 sequence of SEQ ID NO:35, the CDRH3 sequence of SEQ ID NO:45, the CDRL1 sequence of SEQ ID NO:55, the CDRL2 sequence of SEQ ID NO:65, and the CDRL3 sequence of SEQ ID NO:75; or (f) the CDRH1 sequence of SEQ ID NO:26, the CDRH2 sequence of SEQ ID NO:36, the CDRH3 sequence of SEQ ID NO:46, the CDRL1 sequence of SEQ ID NO:56, the CDRL2 sequence of SEQ ID NO:66, and the CDRL3 sequence of SEQ ID NO:76; or (g) the CDRH1 sequence of SEQ ID NO:27, the CDRH2 sequence of SEQ ID NO:37, the CDRH3 sequence of SEQ ID NO:46, the CDRL1 sequence of SEQ ID NO:56, the CDRL2 sequence of SEQ ID NO:66, and the CDRL3 sequence of SEQ ID NO:76. the CDRH2 sequence of SEQ ID NO:37, the CDRH3 sequence of SEQ ID NO:47, the CDRL1 sequence of SEQ ID NO:57, the CDRL2 sequence of SEQ ID NO:67, and the CDRL3 sequence of SEQ ID NO:77;or (h) the CDRH1 sequence of SEQ ID NO:28, the CDRH2 sequence of SEQ ID NO:38, the CDRH3 sequence of SEQ ID NO:48, the CDRL1 sequence of SEQ ID NO:58, the CDRL2 sequence of SEQ ID NO:68, and the CDRL3 sequence of SEQ ID NO:78; or (i) the CDRH1 sequence of SEQ ID NO:29, the CDRH2 sequence of SEQ ID NO:39, the CDRH3 sequence of SEQ ID NO:49, the CDRL1 sequence of SEQ ID NO:59, the CDRL2 sequence of SEQ ID NO:69, and the CDRL3 sequence of SEQ ID NO:79; or (j) the CDRH1 sequence of SEQ ID NO:30, the CDRH2 sequence of SEQ ID NO:40, the CDRH3 sequence of SEQ ID NO:50, the CDRL1 sequence of SEQ ID NO:60, the CDRL2 sequence of SEQ ID NO:70, and the CDRL3 sequence of SEQ ID NO:80.;

[0013] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 1 to 10 and / or a light chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 11 to 20. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 1 to 10 and / or a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 11 to 20. In some embodiments, the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region sequence of SEQ ID NO: 1 and a light chain variable region sequence of SEQ ID NO: 11; or (b) a heavy chain variable region sequence of SEQ ID NO: 2 and a light chain variable region sequence of SEQ ID NO: 12; or (c) a heavy chain variable region sequence of SEQ ID NO: 3 and a light chain variable region sequence of SEQ ID NO: 13; or (d) a heavy chain variable region sequence of SEQ ID NO: 4 and a light chain variable region sequence of SEQ ID NO: 14; or (e) a heavy chain variable region sequence of SEQ ID NO: 5 and a light chain variable region sequence of SEQ ID NO: 15; or (f) a heavy chain variable region sequence of SEQ ID NO: 6 and a light chain variable region sequence of SEQ ID NO: 16; or (g) a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 17; or (h) a heavy chain variable region sequence of SEQ ID NO: 8 and a light chain variable region sequence of SEQ ID NO: or (i) the heavy chain variable region sequence of SEQ ID NO: 9 and the light chain variable region sequence of SEQ ID NO: 19; or (j) the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 20.

[0014] Aspects of the invention include an isolated anti-AS-SPIK antibody or antigen-binding fragment thereof that specifically binds to AS-SPIK but not to NS-SPIK, the isolated anti-AS-SPIK antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 sequences in a human VH framework, wherein the CDRH sequence is a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 21-50; and (b) a light chain variable region comprising CDRL1, CDRL2, and CDRL3 sequences in a human VL framework, wherein the CDRL sequence is a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 51-80. In some embodiments, the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 sequences in a human VH framework, wherein the CDRH sequence is selected from the group consisting of SEQ ID NOs: 21 to 50; and (b) a light chain variable region comprising CDRL1, CDRL2 and CDRL3 sequences in a human VL framework, wherein the CDRL sequence is selected from the group consisting of SEQ ID NOs: 51 to 80.

[0015] Aspects of the invention include an isolated anti-AS-SPIK antibody or antigen-binding fragment thereof that specifically binds to AS-SPIK but not to NS-SPIK, the isolated anti-AS-SPIK antibody or antigen-binding fragment thereof comprising: (a) a CDRH1 sequence of SEQ ID NO:21, a CDRH2 sequence of SEQ ID NO:31, and a CDRH3 sequence of SEQ ID NO:41 in a human VH framework, and a CDRL1 sequence of SEQ ID NO:51, a CDRL2 sequence of SEQ ID NO:61, and a CDRL3 sequence of SEQ ID NO:71 in a human VL framework; or (b) a CDRH1 sequence of SEQ ID NO:22, a CDRH2 sequence of SEQ ID NO:32, and a CDRH3 sequence of SEQ ID NO:42 in a human VH framework, and a CDRL1 sequence of SEQ ID NO:52, a CDRL2 sequence of SEQ ID NO:62, and a CDRL3 sequence of SEQ ID NO:72 in a human VL framework; or (c) a CDRH1 sequence of SEQ ID NO:23, a CDRH2 sequence of SEQ ID NO:31, and a CDRH3 sequence of SEQ ID NO:41 in a human VH framework. NO:33 CDRH2 sequence and SEQ ID NO:43 CDRH3 sequence and SEQ ID NO:53 CDRL1 sequence, SEQ ID NO:63 CDRL2 sequence and SEQ ID NO:73 CDRL3 sequence in human VL framework; or (d) SEQ ID NO:24 CDRH1 sequence, SEQ ID NO:34 CDRH2 sequence and SEQ ID NO:44 CDRH3 sequence in human VH framework and SEQ ID NO:54 CDRL1 sequence, SEQ ID NO:64 CDRL2 sequence and SEQ ID NO:74 CDRL3 sequence in human VL framework; or (e) SEQ ID NO:25 CDRH1 sequence, SEQ ID NO:35 CDRH2 sequence and SEQ ID NO:45 CDRH3 sequence in human VH framework and SEQ ID NO:55 CDRL1 sequence, SEQ ID NO:65 CDRL2 sequence and SEQ ID NO:75 CDRL3 sequence in human VL framework; or (f) SEQ ID NO:24 CDRH1 sequence, SEQ ID NO:34 CDRH2 sequence and SEQ ID NO:44 CDRH3 sequence in human VH framework and SEQ ID NO:54 CDRL1 sequence, SEQ ID NO:64 CDRL2 sequence and SEQ ID NO:74 CDRL3 sequence in human VL framework; The CDRH1 sequence of SEQ ID NO:26, the CDRH2 sequence of SEQ ID NO:36, and the CDRH3 sequence of SEQ ID NO:46, and the CDRL1 sequence of SEQ ID NO:56, the CDRL2 sequence of SEQ ID NO:66, and the CDRL3 sequence of SEQ ID NO:76 in a human VL framework;or (g) the CDRH1 sequence of SEQ ID NO:27, the CDRH2 sequence of SEQ ID NO:37, and the CDRH3 sequence of SEQ ID NO:47 in a human VH framework, and the CDRL1 sequence of SEQ ID NO:57, the CDRL2 sequence of SEQ ID NO:67, and the CDRL3 sequence of SEQ ID NO:77; or (h) the CDRH1 sequence of SEQ ID NO:28, the CDRH2 sequence of SEQ ID NO:38, and the CDRH3 sequence of SEQ ID NO:48 in a human VH framework, and the CDRL1 sequence of SEQ ID NO:58, the CDRL2 sequence of SEQ ID NO:68, and the CDRL3 sequence of SEQ ID NO:78 in a human VL framework; or (i) the CDRH1 sequence of SEQ ID NO:29, the CDRH2 sequence of SEQ ID NO:39, and the CDRH3 sequence of SEQ ID NO:49 in a human VH framework, and the CDRL1 sequence of SEQ ID NO:59, the CDRL2 sequence of SEQ ID NO:69, and the CDRL3 sequence of SEQ ID NO:78 in a human VL framework. or (j) the CDRH1 sequence of SEQ ID NO:30, the CDRH2 sequence of SEQ ID NO:40, and the CDRH3 sequence of SEQ ID NO:50 in the human VH framework and the CDRL1 sequence of SEQ ID NO:60, the CDRL2 sequence of SEQ ID NO:70, and the CDRL3 sequence of SEQ ID NO:80 in the human VL framework. ;

[0016] In some embodiments, the antibody or antigen binding fragment is multispecific. In some embodiments, the antibody or antigen binding fragment is bispecific. In some embodiments, the antibody or antigen binding fragment has binding affinity for effector cells. In some embodiments, the antibody or antigen binding fragment has binding affinity for T cell antigens. In some embodiments, the antibody or antigen binding fragment has binding affinity for CD3.

[0017] In some embodiments, the antibody or antigen-binding fragment is monoclonal. In some embodiments, the antibody or antigen-binding fragment is in the form of a CAR-T.

[0018] Aspects of the invention include an immunoconjugate comprising an antibody as described herein covalently attached to a cytotoxic agent. In some embodiments, the cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, a radioisotope, and a nucleolytic enzyme.

[0019] Aspects of the invention include an immunoconjugate having the formula Ab-(LD)p, wherein: (a) Ab is an antibody as described herein; (b) L is a linker; (c) D is a drug moiety; and (d) p is an integer ranging from 1 to 8. In some embodiments, D is selected from the group consisting of: maytansinoids, auristatins, and dolastatins. In some embodiments, L comprises one or more linkers selected from the group consisting of: 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 4-(2-pyridylthio)pentanoic acid N-succinimidyl ester (SPP), 4-(N-maleimidomethyl)cyclohexane-1carboxylic acid N-succinimidyl ester (SMCC), 4-(2-pyridyldithio)butyric acid-N-hydroxysuccinimidyl ester (SPDB), and (4-iodo-acetyl)aminobenzoic acid N-succinimidyl ester (SIAB).

[0020] Aspects of the invention include a pharmaceutical composition comprising an antibody, antigen-binding fragment, or immunoconjugate as described herein.

[0021] Aspects of the invention include a method of treating a disorder characterized by expression of AS-SPIK, the method comprising administering to a subject suffering from the disorder an antibody, antigen-binding fragment or immunoconjugate as described herein or a pharmaceutical composition as described herein.

[0022] Aspects of the invention include use of an antibody, antigen binding fragment or immunoconjugate as described herein in the preparation of a medicament for treating a disorder characterized by expression of AS-SPIK. Aspects of the invention include an antibody, antigen binding fragment or immunoconjugate as described herein for use in treating a disorder characterized by expression of AS-SPIK.

[0023] In some embodiments, the disorder is a liver disorder. In some embodiments, the liver disorder is hepatocellular carcinoma. In some embodiments, the liver disorder is intrahepatic bile duct carcinoma. In some embodiments, the liver disorder is a viral infection. In some embodiments, the liver disorder is an inflammatory liver disorder. In some embodiments, the inflammatory liver disorder is cirrhosis.

[0024] Aspects of the invention include a polynucleotide encoding an antibody or antigen-binding fragment as described herein; a vector comprising the polynucleotide; or a host cell comprising the vector.

[0025] Aspects of the invention include a method of producing an antibody, antigen-binding fragment or immunoconjugate as described herein, the method comprising growing a host cell as described herein under conditions that permit expression of the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the cell.

[0026] Aspects of the invention include a diagnostic method for determining whether a subject has a disorder characterized by expression of AS-SPIK or is at risk of developing the disorder, the method comprising: (a) contacting a biological test sample from the subject with an AS-SPIK antibody or antigen-binding fragment as described herein to produce an AS-SPIK-antibody complex; (b) detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; and (c) comparing the concentration of the AS-SPIK-antibody complex to a reference value to determine whether the subject has the disorder or is at risk of developing the disorder.

[0027] Aspects of the invention include a diagnostic method for determining whether a subject suffers from a disorder characterized by expression of AS-SPIK or is at risk of developing the disorder, the method comprising: (a) contacting a biological test sample from the subject with a first antibody or antigen-binding fragment that specifically binds to SPIKE to form a SPIKE-antibody complex; (b) contacting the SPIKE-antibody complex with an AS-SPIK antibody or antigen-binding fragment as described herein to produce an AS-SPIK-antibody complex; (c) detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; and (d) comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject suffers from the disorder or is at risk of developing the disorder.

[0028] In some embodiments, the antibody or antigen-binding fragment comprises a detectable label. In some embodiments, the disorder is a liver disorder. In some embodiments, the liver disorder is selected from the group consisting of hepatocellular carcinoma, intrahepatic bile duct carcinoma, viral infection of the liver, inflammatory disorder of the liver, and cirrhosis of the liver.

[0029] Aspects of the invention include a kit comprising an antibody, antigen binding fragment, or immunoconjugate as described herein. In some embodiments, the kit further comprises an antibody or antigen binding fragment that specifically binds to SPIKE. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown are the results of ELISA showing the relative binding activities of various monoclonal antibodies to AS-SPIK and NS-SPIK.

[0031] Figure 2 The results of detecting the antibody-antigen complex in the sample using the antibody IM-E2 are shown.

[0032] Figure 3 An alignment of the VH regions of 10 antibodies is provided: IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6 and IM-G7. The consensus sequences of all antibodies are listed. Antibodies as provided herein may be named by their long names including the letters "IM", such as IM-A1, IM-A6, etc., or may be referred to in abbreviated form by dropping "IM", such as A1 or A6, etc.

[0033] Figure 4 Provides an alignment of the VL regions of 10 antibodies A1, A6, B10, C6, D3, D5, E2, F5, G6 and G7. Lists the consensus sequence of all antibodies.

[0034] Figure 5 Alignment of the VH CDRs of 10 antibodies A1, A6, B10, C6, D3, D5, E2, F5, D3 and D5 is provided. The consensus sequence of each CDR is listed.

[0035] Figure 6 An alignment of the VL CDRs of 10 antibodies A1, A6, B10, C6, D3, D5, E2, F5, G6 and G7 is provided. The consensus sequence of each CDR is listed.

[0036] Figure 7 Provides amino acid and nucleic acid sequence information. DETAILED DESCRIPTION

[0037] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the capabilities of a skilled artisan. Such techniques are fully explained in the following literature, such as: "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel et al., ed., 1987 and periodically updated); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0038] When providing a range of values, it should be understood that each intermediate value to the tenth unit of the lower limit (unless the context clearly indicates otherwise), between the upper and lower limits of the range, and any other intermediate values ​​stated or within the stated range are all included in the present invention. The upper and lower limits of the smaller ranges that may be independently included in these smaller ranges are also included in the present invention, subject to any specific excluded limits in the stated range. When the stated range includes one or both of the limits, the present invention also includes ranges excluding either or both of those included limits.

[0039] Unless otherwise indicated, antibody residues herein are numbered according to the Kabat numbering system (eg, Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0040] In the following description, numerous specific details are set forth in order to provide a more detailed understanding of the present invention. However, it will be clear to those skilled in the art that the present invention may be practiced without one or more of these specific details. In other cases, features and well-known procedures well known to those skilled in the art are not described to avoid obscuring the present invention.

[0041] All references cited throughout this disclosure, including patent applications and publications, are incorporated by reference in their entirety.

[0042] definition

[0043] For the purpose of interpreting this specification, the following definitions will apply and where appropriate, terms used in the singular will also include the plural and vice versa. In the event of any conflict between any definition set forth and any document incorporated herein by reference, the definitions set forth below shall prevail.

[0044] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Typically, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes. The term includes any molecular determinant that can specifically bind to an antibody. In certain embodiments, epitope determinants include chemically active surface collections of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural features and / or specific charge characteristics. An epitope is a region in an antigen that is bound by an antibody. A "binding region" is a region on a binding target that is bound by a binding molecule.

[0045] "Epitope mapping" is the process of identifying the binding site, or epitope, of an antibody on its target antigen. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed by contiguous sequences of amino acids in a protein. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence, but come together when the protein folds into its three-dimensional structure.

[0046] As used herein, "epitope partitioning" is the process of grouping antibodies based on the epitopes they recognize. More particularly, epitope partitioning includes methods and systems for distinguishing the epitope recognition properties of different antibodies in combination with computational methods for clustering antibodies based on their epitope recognition properties and identifying antibodies with different binding specificities.

[0047] When two antibodies recognize the same or spatially overlapping epitopes, the antibody binds "substantially the same epitope" as a reference antibody. The most widely used and rapid method for determining whether two epitopes bind to the same or spatially overlapping epitopes is a competition assay, which can be configured in any number of different formats using labeled antigens or labeled antibodies. Typically, the antigen is immobilized on a 96-well plate and the ability of the unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive or enzyme label.

[0048] As used herein, "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue / position. For example, a typical modification includes replacing a residue (or at the position) with another amino acid (e.g., conservative or non-conservative substitution), inserting one or more (e.g., usually less than 5 or 3) amino acids next to the residue / position and / or deleting the residue / position. "Amino acid substitution" or its variant refers to replacing an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. Typically and preferably, compared to a polypeptide comprising an initial (or "wild type") amino acid sequence, modification results in a change in at least one physical or biochemical activity of a variant polypeptide. For example, in the case of an antibody, the physical or biochemical activity of the change can be binding affinity, binding capacity and / or binding effect to a target molecule.

[0049] The term "antibody" includes monoclonal antibodies (including full-length antibodies with an immunoglobulin Fc region), single-chain molecules, and antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Unless otherwise indicated, the term "antibody" is used in the broadest sense herein, and specifically includes all isotypes, subclasses, and forms of antibodies, including IgG, IgM, IgA, IgD, and IgE antibodies and fragments thereof, preferably antigen-binding fragments.

[0050] Unless otherwise indicated, the term "antibody" specifically includes native human and non-human IgGl, IgG2 (IgG2a, IgG2b), IgG3, IgG4, IgE, IgA, IgD and IgM antibodies, including naturally occurring variants.

[0051] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible naturally occurring mutations that may exist in a small amount, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, contrary to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" represents the characteristics of the antibody obtained from a substantially homogeneous antibody population, and is not to be construed as requiring antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by the hybridoma method described by Kohler et al. (1975) Nature 256:495, or can be prepared by a recombinant DNA method (see, for example, U.S. Patent number 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597.

[0052] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species; and fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0053] "Humanized" forms of non-human (e.g., murine) antibodies are antibodies containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from receptor hypervariable regions are replaced by residues from hypervariable regions with desired specificity, affinity and ability from non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates. In some cases, the Fv framework region (FR) residues of human immunoglobulins are also replaced by corresponding non-human residues. In addition, humanized antibodies may be included in residues that are not present in receptor antibodies or donor antibodies. These modifications are made so that antibody performance is further improved. In general, humanized antibodies will include substantially all at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FRs are FR regions of human immunoglobulin sequences. Humanized antibodies will optionally also include at least a portion of an immunoglobulin constant region (Fc), usually at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[0054] " Amino acid sequence identity percentage (%) " relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence after aligning the sequences and, if necessary, introducing spaces (achieving maximum sequence identity percentage) and not treating any conservative substitutions as part of sequence identity. Alignment for purposes of determining amino acid sequence identity percentage can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequence. However, for purposes herein, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity percentage values.

[0055] As used herein, the term "percent sequence homology" refers to the degree of homology between any given query sequence and a subject sequence. For example, a naturally occurring AS-SPIK polypeptide or NS-SPIK polypeptide can be a query sequence, and a fragment of the AS-SPIK polypeptide or NS-SPIK polypeptide can be a subject sequence. Similarly, a fragment of an AS-SPIK polypeptide or NS-SPIK polypeptide can be a query sequence and a biologically active variant thereof can be a subject sequence.

[0056] The term "consensus sequence" as used herein refers to a sequence of amino acid or nucleotide residues that represents the most frequent residues found at each position in an alignment of sequences, after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence matching and not considering any conservative substitutions as part of the sequence identity.

[0057] "Separated" antibodies herein are antibodies that have been identified and separated from components of their natural environment in a recombinant host cell and / or recovered from components of their natural environment. The contaminating components of their natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteins or non-protein solutes and unwanted production byproducts. In preferred embodiments, the separated antibodies herein will (1) be purified to greater than 95% by weight, or greater than 98% by weight, or greater than 99% by weight as determined by SDS-PAGE or SEC-HPLC methods, (2) be purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using an amino acid sequencer, or (3) be purified to homogeneity by using Coomassie blue or preferably silver staining under reducing or non-reducing conditions by SDS-PAGE. Typically, the separated antibodies will be prepared by at least one purification step.

[0058] In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also have a regularly spaced intrachain disulfide bridge. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of the α and γ chains and four CH domains of the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain at its other end. VL is compared with VH, and CL is compared with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain variable domain and the heavy chain variable domain. The pairing of VH and VL forms a single antigen binding site together.

[0059] The term "polypeptide" is used herein in the broadest sense and includes peptide sequences.The term "peptide" generally describes a linear molecular chain of amino acids containing up to about 60, preferably up to about 30 amino acids covalently linked by peptide bonds.

[0060] The term "specific binding" or "specifically binds to" or "is specific for" refers to the binding of an antibody to a target antigen, such as an epitope on a specific polypeptide, peptide, or other target, such as a glycoprotein target, and means binding that is measurably different from non-specific interactions, such as binding to bovine serum albumin or casein. Specific binding can be measured, for example, by measuring the binding of an antibody to a target molecule compared to binding to a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, such as an excess of unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target can be exhibited, for example, by a molecule having a Kd of at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM or more for the target. In some cases, the term "specific binding" refers to the binding of a molecule to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0061] "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity of 1:1 interactions between members (e.g., antibodies and antigens) that reflect binding. The affinity of molecule X to its partner Y can generally be represented by a dissociation constant (Kd). For example, Kd can be about 200nM, 150nM, 100nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 8nM, 6nM, 4nM, 2nM, 1nM or stronger. Affinity can be measured by common methods known in the art (including those described herein). Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens faster and tend to keep binding longer. The various methods for measuring binding affinity are known in the art.

[0062] As used herein, "Kd" or "Kd value" refers to the Kd value determined by a technique applicable to the antibody and target pair, such as using BIAcore TM -2000 or BIAcore TM The dissociation constant is measured, for example, using surface plasmon resonance assays using a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU).

[0063] As used herein, the term "valent" indicates the presence of a specified number of binding sites in an antibody. Thus, the term "bivalent" indicates the presence of two binding sites.

[0064] "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. "Monospecificity" refers to the ability to bind to only one epitope. In some embodiments, the antibody binds to each epitope with an affinity of at least 10-7M or 10-8M or better.

[0065] The term "target" or "binding target" is used in the broadest sense and specifically includes, but is not limited to, polypeptides, nucleic acids, carbohydrates, lipid cells and other molecules with or without biological function as they occur in nature.

[0066] The term "antigen" refers to an entity or fragment thereof that can bind to an antibody or trigger a cellular immune response. The term immunogen refers to an antigen that can elicit an immune response in an organism, particularly an animal, more particularly a mammal (including a human). The term antigen includes regions known as antigenic determinants or epitopes, as defined above.

[0067] As used herein, the term "immunogenic" refers to a substance that elicits the production of antibodies and / or activates T cells and / or other reactive immune cells against the antigen of the immunogen.

[0068] The "antigen binding site" or "antigen binding region" of the antibodies of the present invention generally contains six hypervariable regions (HVRs), which contribute to the affinity of the binding site for the antigen to varying degrees. The term "complementarity determining region" or "CDR" is used interchangeably with the term "hypervariable region" or "HVR" herein. There are three heavy chain variable domain HVRs (HVR-H1, HVR-H2, and HVR-H3) and three light chain variable domain HVRs (HVR-L1, HVR-L2, and HVR-L3). The extent of the HVRs and framework regions (FRs) is determined by comparing compiled amino acid sequence databases, in which the regions have been defined based on the variability between sequences and / or structural information of the antibody / antigen complex. Also included within the scope of the present invention are functional antigen binding sites consisting of fewer HVRs (i.e., binding specificity is determined by three, four, or five HVRs). Less than a complete set of 6 HVRs may be sufficient to bind to some binding targets. Therefore, in some cases, the HVRs of a single VH or VL domain will be sufficient. Furthermore, certain antibodies may have non-HVR associated binding sites for antigen. Such binding sites are specifically included in this definition.

[0069] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or a radiolabel.

[0070] By "antibody-drug conjugate" (ADC) or immunoconjugate is meant an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent (such as a chemotherapeutic agent), a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[0071] The term "host cell" as used herein refers to any kind of cell system that can be engineered to produce the antibodies according to the invention. In one embodiment, Chinese Hamster Ovary (CHO) cells are used as host cells.

[0072] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be identical in DNA content, due to intentional or unintentional mutations. Variant progeny that have the same function or biological activity as the screened original transformed cell are included.

[0073] A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA of a polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably linked to a coding sequence; or if a ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and in reading frame. However, enhancers do not have to be continuous. Connection is achieved by connecting to a suitable restriction site. If the site does not exist, a synthetic oligonucleotide adapter or joint is used according to conventional practice.

[0074] The terms "anti-AS-SPIK antibody," "AS-SPIK antibody," or "antibody that binds to AS-SPIK" all refer to an antibody that is capable of binding AS-SPIK with sufficient affinity to render the antibody useful as a diagnostic and / or therapeutic agent in targeting AS-SPIK.

[0075] In one embodiment, "AS-SPIK antibody" is used herein to specifically refer to an anti-AS-SPIK monoclonal antibody that (i) comprises a heavy chain variable binding domain sequence as provided in any one of SEQ ID NOs: 1-10 and / or a light chain variable domain sequence as provided in any one of SEQ ID NOs: 11-20; or (ii) comprises 1, 2, 3, 4, 5 or 6 of the CDRs provided in SEQ ID NOs: 21-80.

[0076] The term "variable" refers to the fact that certain segments of the variable domain are widely different in sequence between antibodies. The "variable" or "V" domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is unevenly distributed over the 110 amino acid spans of the variable domain. In fact, the V region consists of a relatively constant elongated region called a framework region (FR) with 15-30 amino acids, which is separated by a shorter region called a "hypervariable region" that is each 9-12 amino acids long and highly variable. The variable domains of native heavy and light chains each include four FRs (mainly in a β-folded configuration) connected by three hypervariable regions, which form a loop connecting the β-folded structure, and in some cases form a part of the β-folded structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0077] A "complete" antibody is an antibody that contains an antigen binding site and a light chain constant domain (CL) and at least a heavy chain constant domain of a particular antibody class. For example, a complete IgG antibody contains an antigen binding site, a light chain constant domain CL, and at least heavy chain constant domains CH1 (Cγ1), CH2 (Cγ2), and CH3 (Cγ3). A complete IgM antibody contains an antigen binding site, a light chain constant domain CL, and at least heavy chain constant domains CM1 (Cμ1), CM2 (Cμ2), CM3 (Cμ3), and CM4 (Cμ4). A complete IgA antibody contains an antigen binding site, a light chain constant domain CL, and at least heavy chain constant domains CA1 (Cα1), CA2 (Cα2), and CA3 (Cα3). A complete IgD antibody contains an antigen binding site, a light chain constant domain CL, and at least heavy chain constant domains CD1 (Cδ1), CD2 (Cδ2), and CD3 (Cδ3). A complete IgE antibody comprises an antigen binding site, a light chain constant domain CL and at least a heavy chain constant domain CE1 (Cε1), CE2 (Cε2), CE3 (Cε3) and CE4 (Cε4). The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the complete antibody has one or more effector functions.

[0078] An "antibody fragment" or "antigen-binding fragment" of an antibody comprises a portion of an intact antibody, preferably the antigen-binding region or variable region of an intact antibody. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises the antigen-binding site of an intact antibody and thus retains the ability to bind to an antigen. One of ordinary skill in the art will appreciate that an antibody fragment can be produced by any intact antibody, such as an IgG, IgM, IgA, IgD, or IgE antibody, by separating at least one antigen-binding portion of an antibody from the rest of its light and heavy chains to create an antigen-binding fragment. In certain embodiments, an antibody fragment may comprise the antigen-binding region of an antibody, and one or more additional domains of the light and / or heavy chains of the antibody. For example, in some embodiments, the antibody fragment may comprise an antigen binding region comprising VH and VL domains, a light chain constant domain CL, and one or more heavy chain constant domains, such as a CH1 (Cγ1) domain, a CM1 (Cμ1) domain, a CA1 (Cα1) domain, a CD1 (Cδ1) domain, or a CE1 (Cε1) domain.

[0079] In the case of IgG antibody fragments, papain digestion produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment (a name that reflects the ability to crystallize easily). The Fab fragment consists of the entire L chain together with the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of IgG antibodies produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and still capable of cross-linking antigen. The Fab' fragment differs from the Fab fragment in having additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the name herein for Fab' in which one or more cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were initially produced in the form of Fab' fragment pairs, which had hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0080] The Fc fragment of an IgG antibody comprises the carboxyl terminal portions of two H chains held together by disulfide bonds. The effector functions of an antibody are determined by sequences in the Fc region, which is also the part recognized by Fc receptors (FcRs) present on certain types of cells.

[0081] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0082] A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region in at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, such as about one to about ten amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide, and preferably about one to about five amino acid substitutions. The variant Fc region herein preferably has at least about 80% homology with a native sequence Fc region and / or with the Fc region of a parent polypeptide, most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

[0083] The human IgG1 amino acid sequence is provided by UniProtKB No. P01857, which is incorporated herein by reference in its entirety. The human IgG2 amino acid sequence is provided by UniProtKB No. P01859, which is incorporated herein by reference in its entirety. The human IgG3 amino acid sequence is provided by UniProtKB No. P01860, which is incorporated herein by reference in its entirety. The human IgG4 amino acid sequence is provided by UniProtKB No. P01861, which is incorporated herein by reference in its entirety.

[0084] "Fv" is the smallest antibody fragment containing complete antigen recognition and binding sites. This fragment is composed of a dimer of a heavy chain variable domain and a light chain variable region domain in a tight, non-covalent association. In single-chain Fv (scFv) materials, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and heavy chain can associate with a "dimerization" structure similar to the "dimerization" structure in a double-chain Fv material. Six hypervariable loops (3 loops each from the H chain and the L chain) are produced from the folding of these two domains, and the hypervariable loops facilitate the amino acid residues for antigen binding and give antigen binding properties to antibodies. However, even a single variable domain (or only half Fv comprising three HVRs specific for antigens) has the ability to recognize and bind antigens, although compared with the entire binding site, it is at a lower affinity.

[0085] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains linked into a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

[0086] The term "chimeric antigen receptor" or "CAR" is used herein in the broadest sense to refer to an engineered receptor that transplants the desired binding specificity (e.g., the antigen binding region of a monoclonal antibody or other ligand) to a transmembrane and intracellular signaling domain. Typically, the receptor is used to transplant the specificity of a monoclonal antibody onto a T cell to produce a chimeric antigen receptor (CAR). (Dai et al., J Natl Cancer Inst, 2016; 108(7): djv439; and Jackson et al., Nature Reviews Clinical Oncology, 2016; 13: 370–383).

[0087] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells such as natural killer cells can induce antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages expressing FcR participate in the specific killing of target cells and present antigens to other components of the immune system, or bind to cells presenting antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0088] "Human effector cells" are leukocytes that express receptors such as T cell receptors or FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among which NK cells are preferred. The effector cells may be isolated from their natural sources, e.g., from blood or PBMCs as described herein.

[0089] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, for example, lymphocytes (such as B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0090] Antibody "effector functions" refer to those biological activities attributable to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

[0091] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells and subsequently cause target cell lysis. Primary cells NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). In order to assess the ADCC activity of target molecules, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the relevant molecules can be assessed in vivo, for example in an animal model such as disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0092] "Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed.

[0093] A "blocking" antibody or "antagonist" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. Preferred blocking antibodies or antagonist antibodies are capable of substantially or completely inhibiting the biological activity of the antigen.

[0094] An antibody that "binds to an antigen of interest" (e.g., an AS-SPIK or NS-SPIK polypeptide) is an antibody that binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent targeting cells or tissues expressing the antigen, and does not significantly cross-react with other proteins. With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target means a measurable different binding from non-specific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of a similar structure that does not have binding activity. For example, specific binding can be measured by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, it is indicated as specific binding. In one embodiment, the term "specific binding" refers to the binding of a molecule to a specific polypeptide or an epitope on a specific polypeptide, without substantially binding to any other polypeptide or polypeptide epitope.

[0095] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" includes one or more cancer cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), liver cancer (e.g., hepatocellular carcinoma (HCC), intrahepatic bile duct carcinoma (ICC)), bladder cancer (e.g., urothelial bladder carcinoma), testicular cancer (germ cell tumors), liver cancer, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney or renal cancer (e.g., renal cell carcinoma, nephroblastoma or Wilms' tumor), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. Additional examples of cancer include, but are not limited to, retinoblastoma, thecoma, androblastoma, liver cancer, hematologic malignancies (including non-Hodgkin lymphoma (NHL), multiple myeloma, and acute hematologic malignancies), endometrial or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, liver cancer, anal cancer, penile cancer, nasopharyngeal cancer, laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, schwannoma, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, and urinary tract cancer.

[0096] The term "metastatic cancer" refers to a cancer state in which cancer cells of a tissue of origin metastasize from the original site to one or more sites elsewhere in the body through blood vessels or lymphatic vessels to form one or more secondary tumors in one or more organs other than the tissue of origin.

[0097] As used herein, an "AS-SPIK-associated disorder" of a "disorder characterized by AS-SPIK expression" is a disorder associated with the expression or overexpression of the AS-SPIK gene or gene product (AS-SPIK polypeptide), which can be any disorder characterized by cells expressing normal or elevated levels of AS-SPIK relative to suitable control cells. Suitable control cells can be cells from an individual not affected by a cancer that expresses or overexpresses AS-SPIK, or they can be non-cancerous cells from a subject in need, or they can be non-cancerous cells from another individual affected by a cancer that expresses or overexpresses AS-SPIK. A prominent example of an AS-SPIK-associated disorder is liver cancer.

[0098] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0099] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0100] As used herein, the terms "predict" and "prognosis" are also interchangeable in the sense that methods for prediction or prognosis allow a person practicing the method to select cells considered (usually prior to treatment, but not necessarily) more likely to respond to treatment with an anti-cancer agent, including an anti-AS-SPIK antibody.

[0101] As used herein, "treat," "treatment," or "treating" refers to both therapeutic treatment and prophylactic or preventative measures aimed at preventing or slowing down (alleviating) the targeted pathological disorder or condition. Subjects in need of treatment include those already suffering from a particular disorder or condition as well as those prone to suffering from the disorder or those in whom the disorder is to be prevented. DETAILED DESCRIPTION

[0103] The present invention is based at least in part on the discovery that certain conditions are characterized by the expression of a unique form or serine protease inhibitor Kazal (SPIK). A prominent example is liver cancer, which includes but is not limited to hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). More specifically, the inventors have found that certain cancers (such as liver cancer) express a form of SPIKE that contains an additional 23 amino acids at the N-terminus of the secreted SPIKE polypeptide. This 23 amino acid segment (SEQ ID NO: 81) is not found in the SPIKE polypeptide secreted by normal cells (such as pancreatic cells). This is consistent with previous reports that the first 9 amino acids of this 23 amino acid segment can be present in unprocessed SPIKE secreted by liver cancer cell lines. Lu et al., Immunology 2011; 134 (4): 398-408. The longer form of SPIKE can be referred to as AS-SPIK or abnormally secreted SPIKE. AS-SPIK produced by liver cancer cells can also be referred to as LC-SPIK or liver cancer secreted SPIKE. The terms AS-SPIK and LC-SPIK can be used interchangeably herein. An exemplary AS-SPIK polypeptide can have the amino acid sequence of SEQ ID NO: 82. The form of SPIKE secreted by normal cells (such as pancreatic cells) can be referred to as NS-SPIK or normally secreted SPIKE. An exemplary NS-SPIK polypeptide can have the amino acid sequence of SEQ ID NO: 83. It has also been found that the conformation (e.g., 3D structure) of AS-SPIK is different from that of NS-SPIK.

[0104] Therefore, aspects of the invention include compositions, such as antibodies, that specifically or preferentially bind to AS-SPIK and do not bind to NS-SPIK. An AS-SPIK complex is also provided. The AS-SPIK complex according to an embodiment of the invention comprises an antibody that specifically or preferentially binds to AS-SPIK and an AS-SPIK polypeptide or a fragment thereof.

[0105] Aspects of the invention include antibody-drug conjugates (ADCs) comprising an antibody (Ab), a linker (L), and a drug moiety (D) as described herein. In some embodiments, the ADC has the formula Ab-(LD)p, wherein p is an integer ranging from 1 to 8.

[0106] Aspects of the invention also include methods of using the subject antibodies to detect a disorder characterized by expression of AS-SPIK (eg, a liver disorder, such as liver cancer, eg, HCC or ICC).

[0107] While it is believed that certain events that occur during AS-SPIK expression are understood, the compositions and methods of the invention are not limited to those that act by affecting any particular cellular mechanism. Without being bound by theory, the inventors hypothesize that because SPIKE is a protease inhibitor, overexpression of SPIKE in cancer cells may inhibit the activity of signal peptide peptidase (a protease), resulting in the secretion of the full-length protein by the cancer cells without attenuation.

[0108] Composition

[0109] Compositions provided herein include antibodies that specifically or preferentially bind to AS-SPIK and do not bind to NS-SPIK.

[0110] Serine protease inhibitor Kazal (SPIK), also known as SPINK1, PSTI and TATI, is a small protein that has been shown to broadly regulate the activity of many cellular proteases, such as trypsin-like proteases and chymotrypsin-like proteases. Greene, LJ, J Surg Oncol. 1975; 7(2): 151-154; Horii et al., Biochemical and biophysical research communications 1987; 149(2): 635-641; Stenman, UH, Clin Chem. 2002; 48(8): 1206-1209. SPIK may also play a role in inhibiting apoptosis. Lu et al., Immunology 2011; 134(4): 398-408. Exemplary human SPIKE amino acid sequences include GenBank Accession No.: M11949, GI No.: 190687; GenBank Accession No.: NM003122, GI: 657940887; and GenBank Accession No.: BC025790, GI: 19343607.

[0111] Antibody

[0112] The antibodies provided herein may include antibodies that specifically or preferentially bind to an epitope within amino acids 1-23 of SEQ ID NO: 82 or an epitope containing at least one amino acid within this region. The epitope may be a conformational epitope (conformation-specific epitope) or a linear epitope. In some embodiments, the antibody specifically or preferentially binds to an epitope in the AS-SPIK protein sequence shown in SEQ ID NO: 81. In some embodiments, the antibody specifically or preferentially binds to a conformation-specific epitope comprising at least one amino acid of SEQ ID NO: 81.

[0113] The antibodies according to embodiments of the present invention may be polyclonal or monoclonal, particularly monoclonal, and may be produced by human, mouse, rabbit, sheep or goat cells, or by hybridomas derived from these cells. In some embodiments, the antibodies may be humanized or chimeric antibodies.

[0114] The antibody according to the embodiment of the present invention can present various configurations and encompass proteins consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Any of a variety of antibody structures can be used, including complete antibodies, antibody polymers or antibody fragments or other variants thereof comprising the functional antigen binding region of antibodies. The term "immunoglobulin" can be used synonymously with "antibody". The antibody can be monoclonal or polyclonal in origin. Regardless of the source of the antibody, suitable antibodies include complete antibodies (e.g., IgG tetramers with two heavy (H) chains and two light (L) chains), single-chain antibodies, chimeric antibodies, humanized antibodies, complementary determining regions (CDR)-grafted antibodies and antibody fragments, such as Fab, Fab', F(ab')2, scFv, Fv, and recombinant antibodies derived from such fragments, such as camel antibodies, mini antibodies, double antibodies and bispecific antibodies.

[0115] A complete antibody is an antibody comprising an antigen-binding variable region (VH and VL) and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. As is well known in the art, the VH and VL regions are further subdivided into hypervariable regions, referred to as "complementarity determining regions" (CDRs), interspersed with more conservative framework regions (FRs). The CDRs of an antibody typically comprise amino acid sequences that together define the binding affinity and specificity of the native Fv region of a natural immunoglobulin binding site.

[0116] Anti-AS-SPIK antibodies can be from any class of immunoglobulins, such as IgA, IgG, IgE, IgD, IgM (and subtypes thereof (e.g., IgG1, IgG2, IgG3, and IgG4)), and the light chain of the immunoglobulin can be of the κ type or the λ type. Recognized human immunoglobulin genes include κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes.

[0117] The term "antigen binding portion" of an immunoglobulin or antibody generally refers to a portion of an immunoglobulin that specifically or preferentially binds to a target, in this case, an epitope comprising amino acid residues on AS-SPIK (SEQ ID NO: 81) rather than NS-SPIK. Thus, an antigen binding portion of an immunoglobulin is a molecule in which one or more immunoglobulin chains are not full-length, but specifically or preferentially bind to a cellular target. Examples of antigen binding portions or fragments include: (i) Fab fragments, which are monovalent fragments consisting of VLC, VHC, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragments consisting of the VLC and VHC domains of a single arm of an antibody; and (v) isolated CDRs of antigen binding portions having sufficient framework to specifically or preferentially bind, for example, to variable regions. The antigen binding portion of the light chain variable region and the antigen binding portion of the heavy chain variable region (e.g., the two domains of the Fv fragment, VLC and VHC) can be connected by a synthetic linker using recombinant methods so that they can be made into a single protein chain, wherein the VLC and VHC regions are paired to form a monovalent molecule (called a single-chain Fv (scFv)). The term "antigen binding portion" of an antibody encompasses such scFv.

[0118] The "Fv" fragment is the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight non-covalent association. It is the configuration in which the three hypervariable regions of each variable domain interact that defines the antigen binding site on the surface of the VH-VL dimer. Although the six hypervariable regions confer antigen binding specificity, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the complete binding site. In order to improve stability, the VH-VL domains can be connected by a flexible peptide linker such as (Gly4Ser)3 to form a single-chain Fv or scFV antibody fragment, or can be engineered by introducing two cysteine ​​residues in the framework region to form a disulfide bond, thereby generating a disulfide-stabilized Fv (dsFv).

[0119] Fragments of antibodies are suitable for use in the provided methods so long as they retain the desired specificity of the full-length antibody and / or bind AS-SPIK but not NS-SPIK with sufficient specificity.

[0120] The antigen binding domains of the antibodies described herein can be used to generate T cell engagement molecules (e.g., bispecific T cell engagement molecules, also known as BiTE molecules) and CAR-T structures. T cell engagement molecules are described in, for example, Huehls et al., Bispecific T cell engagers for cancer immunotherapy, Immunol Cell Biol. 2015 March; 93(3): 290-296. CAR-T structures comprising single domain antibodies as binding (targeting) domains are described in, for example, Iri-Sofla et al., 2011, Experimental Cell Research 317: 2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840: 378-386.

[0121] Methods for preparing antibody fragments include biochemical methods (e.g., proteolytic digestion of intact antibodies, which can then be chemically cross-linked) and recombinant DNA-based methods in which immunoglobulin sequences are genetically engineered to direct the synthesis of the desired fragments. Antibody fragments can be obtained by proteolysis of whole immunoglobulins by the nonspecific thiol protease papain. Papain digestion produces two identical antigen-binding fragments, called "Fab fragments," each with a single antigen-binding site; and a residual "Fc fragment." The various fractions can be separated by protein A-Sepharose or ion exchange chromatography. A common procedure for preparing F(ab')2 fragments from IgG of rabbit and human origin is limited proteolysis by pepsin. Pepsin treatment of intact antibodies produces F(ab')2 fragments that have two antigen-binding sites and are still able to cross-link antigens. Fab fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a small number of residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them.

[0122] Methods for preparing anti-AS-SPIK antibodies are also within the scope of the invention. For example, variable regions can be constructed using PCR mutagenesis methods to alter the DNA sequence encoding the immunoglobulin chain (eg, using methods for producing humanized immunoglobulins).

[0123] Monoclonal antibodies are homologous antibodies with the same antigen specificity produced by a single clone of antibody-producing cells, and polyclonal antibodies generally recognize different epitopes on the same antigen and are produced by more than one clone of antibody-producing cells. Each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring the antibody to be produced by any particular method.

[0124] The monoclonal antibodies herein may include chimeric antibodies, i.e. antibodies generally have a portion of a heavy chain and / or a light chain that is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the one or more chains is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity. Target chimeric antibodies include primatized antibodies comprising variable domain antigen-binding sequences derived from non-human primates (e.g., apes, old world monkeys, new world monkeys, prosimians) and human constant region sequences.

[0125] Murine and rabbit monoclonal antibodies are produced by immunizing mice or rabbits with specially designed recombinant proteins that have an additional 23 amino acid sequence found in AS-SPIK (SEQ ID NO: 81) that is not present in NS-SPIK, except for a common region (SEQ ID NO: 83), which is an amino acid sequence found in both NS-SPIK and AS-SPIK. In some embodiments, the recombinant protein may not need to have the complete 23 amino acid sequence (SEQ ID NO: 81) to produce antibodies that effectively bind only to AS-SPIK and not to NS-SPIK. Methods for screening and selecting antibodies that specifically or preferentially bind to AS-SPIK are described in Example 1.

[0126] Figure 1 The results of the monoclonal antibody tests are provided in Example 1, which show that the antibodies of the present invention can specifically or preferentially recognize AS-SPIK, but not NS-SPIK; these include but are not limited to IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6 and IM-G7. For a description of the tests, see Example 1.

[0127] The sequences of the light chain (VL) and heavy chain (VH) variable regions of monoclonal antibodies that specifically or preferentially bind to AS-SPIK (but not to NS-SPIK) were determined (SEQ ID NOs: 1-20). All CDRs of these antibodies were also determined (SEQ ID NOs: 21-80). Figure 3-6The consensus amino acid sequences for these regions are provided.Tables 1-3 provide the SEQ ID NOs: for various VH and VL regions of ten exemplary antibodies of the invention.

[0128] For example, the IM-A1 antibody has a VH region shown in SEQ ID NO: 1 and has a VL region shown in SEQ ID NO: 11. The VH region has three CDR regions corresponding to SEQ ID NOs: 21, 31, and 41. The VL region has three CDRs corresponding to SEQ ID NOs: 51, 61, and 71.

[0129] The IM-A6 antibody has the VH region shown in SEQ ID NO: 2 and has the VL region shown in SEQ ID NO: 12. The VH region has three CDR regions corresponding to SEQ ID NOs: 22, 32, and 42. The VL region has three CDR regions corresponding to SEQ ID NOs: 52, 62, and 72.

[0130] The IM-B10 antibody has the VH region shown in SEQ ID NO: 3 and has the VL region shown in SEQ ID NO: 13. The VH region has three CDR regions corresponding to SEQ ID NOs: 23, 33, and 43. The VL region has three CDR regions corresponding to SEQ ID NOs: 53, 63, and 73.

[0131] The IM-C6 antibody has the VH region shown in SEQ ID NO: 4 and has the VL region shown in SEQ ID NO: 14. The VH region has three CDR regions corresponding to SEQ ID NOs: 24, 34, and 44. The VL region has three CDR regions corresponding to SEQ ID NOs: 54, 64, and 74.

[0132] The IM-D3 antibody has a VH region shown in SEQ ID NO: 5 and has a VL region shown in SEQ ID NO: 15. The VH region has three CDR regions corresponding to SEQ ID NOs: 25, 35, and 45. The VL region has three CDR regions corresponding to SEQ ID NOs: 55, 65, and 75.

[0133] The IM-D5 antibody has the VH region shown in SEQ ID NO: 6 and has the VL region shown in SEQ ID NO: 16. The VH region has three CDR regions corresponding to SEQ ID NOs: 26, 36, and 46. The VL region has three CDR regions corresponding to SEQ ID NOs: 56, 66, and 76.

[0134] The IM-E2 antibody has the VH region shown in SEQ ID NO: 7 and has the VL region shown in SEQ ID NO: 17. The VH region has three CDR regions corresponding to SEQ ID NOs: 27, 37, and 47. The VL region has three CDR regions corresponding to SEQ ID NOs: 57, 67, and 77.

[0135] The IM-F5 antibody has the VH region shown in SEQ ID NO: 8 and has the VL region shown in SEQ ID NO: 18. The VH region has three CDR regions corresponding to SEQ ID NOs: 28, 38, and 48. The VL region has three CDR regions corresponding to SEQ ID NOs: 58, 68, and 78.

[0136] The IM-G6 antibody has the VH region shown in SEQ ID NO: 9 and has the VL region shown in SEQ ID NO: 19. The VH region has three CDR regions corresponding to SEQ ID NOs: 29, 39, and 49. The VL region has three CDR regions corresponding to SEQ ID NOs: 59, 69, and 79.

[0137] The IM-G7 antibody has a VH region shown in SEQ ID NO: 10 and has a VL region shown in SEQ ID NO: 20. The VH region has three CDR regions corresponding to SEQ ID NOs: 30, 40, and 50. The VL region has three CDR regions corresponding to SEQ ID NOs: 60, 70, and 80.

[0138] Furthermore, the antibody according to an embodiment of the present invention may be any non-naturally occurring (artificial) antibody that specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK.

[0139] In some embodiments, the antibody comprises a VH region having a sequence provided in any one of SEQ ID NOs: 1-10, or a VH region comprising a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to a heavy chain variable region sequence in any one of SEQ ID NOs: 1-10, provided that the resulting antibody or antigen-binding fragment specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK.

[0140] In some embodiments, the antibody comprises a VL region having a sequence provided in any one of SEQ ID NOs: 11-20, or a VL region comprising a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to a heavy chain variable region sequence in any one of SEQ ID NOs: 11-20, provided that the resulting antibody or antigen-binding fragment specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK.

[0141] In some embodiments, the antibody comprises a VH CDR1 region having a sequence provided in any one of SEQ ID NOs: 21-30 or comprising two or fewer amino acid mutations in any one of SEQ ID NOs: 21-30, as long as the resulting antibody specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK. In some embodiments, the VH CDR1 region may comprise the following structural formula, wherein X represents a variable amino acid, which may be any of the identified amino acids: S X1X2X3X4S (SEQ ID NO: 107), wherein X1 is S or A; X2 is Y or N; X3 is A, G, P or V; and X4 is I or V.

[0142] In some embodiments, the antibody comprises a VH CDR2 region having a sequence provided in any one of SEQ ID NOs: 31-40 or comprising two or fewer amino acid mutations in any one of SEQ ID NOs: 31-40, so long as the resulting antibody specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK. In some embodiments, the VH CDR2 region may comprise the following structural formula, wherein X represents a variable amino acid, which may be any of the identified amino acids: X5I X6X7X8G X9X 10 X 11 YA SWAKS (SEQ ID NO: 108), wherein X8 is S, G or Y; and X5, X6, X7, X9, X 10 and X 11 is any amino acid.

[0143] In some embodiments, the antibody comprises a VH CDR3 region having a sequence provided in any one of SEQ ID NOs: 41-50 or comprising two or fewer amino acid mutations in any one of SEQ ID NOs: 41-50, so long as the resulting antibody specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK. In some embodiments, the VH CDR3 region may comprise the following structural formula, wherein X represents a variable amino acid, which may be any of the identified amino acids: RX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 (SEQ ID NO: 109), wherein X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 and X 21 is any amino acid.

[0144] In some embodiments, the antibody comprises a VL CDR1 region having a sequence provided in any one of SEQ ID NOs: 51-60 or comprising two or fewer amino acid mutations in any one of SEQ ID NOs: 51-60, so long as the resulting antibody specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK. In some embodiments, the VL CDR1 region may comprise the following structural formula, wherein X represents a variable amino acid, which may be any of the identified amino acids: QASX 22 X 23 IX 24 X 25 X 26 LX 27 (SEQ ID NO: 110), wherein X 22 It is Q or E; X 23 is S, G, D or N; X 24 is S, N, E or G; X 25 is any amino acid; X 26 is Y or A; and X 27 is any amino acid.

[0145] In some embodiments, the antibody comprises a VL CDR2 region having a sequence provided in any one of SEQ ID NOs: 61-70 or comprising two or fewer amino acid mutations in any one of SEQ ID NOs: 61-70, so long as the resulting antibody specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK. In some embodiments, the VL CDR2 region may comprise the following structural formula, wherein X represents a variable amino acid, which may be any of the identified amino acids: X 28 AX 29 X 30 LX 31 S (SEQ ID NO: 111), wherein X 28 is any amino acid; X 29 is S or T; X 30 is T, Y or K; and X 31 It is A, P or V.

[0146] In some embodiments, the antibody comprises a VL CDR3 region having a sequence provided in any one of SEQ ID NOs: 71-80 or comprising two or fewer amino acid mutations in any one of SEQ ID NOs: 71-80, so long as the resulting antibody specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK. In some embodiments, the VL CDR3 region may comprise the following structural formula, wherein X represents a variable amino acid, which may be any of the identified amino acids: X 32 QX 33 YX 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 (SEQ ID NO: 112), wherein X 32 is Q or H; X 33 is G or D; X 34 is any amino acid; X 35 is any amino acid; X 36 is S or N; X 37 is N, D, V or G; X 38 is V, D or I; X 39 is D, G or N; X 40 is N or V; and X 41 is any amino acid.

[0147] Antibodies according to embodiments of the invention may comprise any VH, VL, VH CDR1, VH CDR2, or VH CDR3, or VL CDR1, VL CDR2, or VL CDR3 regions described herein, or any combination of these regions, including having the recited homology or percent identity provided above, so long as the antibody binds to AS-SPIK and does not bind to NS-SPIK.

[0148] The structural formulas and consensus sequences described herein were determined using the software program "BioEdit" developed by North Carolina State University.

[0149] All of these antibodies were investigated for their high binding activity to AS-SPIK. The results are described in Example 1.

[0150] Methods for producing monoclonal antibodies may include purification steps. For example, the antibody can be further purified, for example, using filtration, centrifugation and various chromatographic methods, such as HPLC or affinity chromatography, all of which are well known to those of ordinary skill in the art. These purification techniques all involve fractionation to separate the desired antibody from the other components of the mixture. Analytical methods particularly suitable for prepared antibodies include, for example, protein A-Sepharose and / or protein G-Sepharose chromatography.

[0151] The anti-AS-SPIK antibodies of the present invention may include CDRs from human or non-human sources. "Humanized" antibodies are typically chimeric or mutant monoclonal antibodies from mice, rats, hamsters, rabbits or other species, with human constant and / or variable region domains or specific changes. The framework of an immunoglobulin can be human, humanized or non-human (e.g., a mouse framework modified to reduce antigenicity in humans) or a synthetic framework (e.g., a consensus sequence). Humanized immunoglobulins are those in which framework residues correspond to human germline sequences and CDRs are produced by V (D) J recombination and somatic mutations. However, humanized immunoglobulins may also include amino acid residues that are not encoded in human germline immunoglobulin nucleic acid sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro). Antibody variable domain genes based on germline sequences but with framework mutations introduced by, for example, a somatic mutation process in vivo are referred to as "people".

[0152] Humanized antibodies can be engineered by a variety of methods known in the art, including, for example: (1) non-human complementary determining regions (CDRs) are transplanted onto human frameworks and constant regions (a process known in the art as humanization), or alternatively, (2) entire non-human variable domains are transplanted, but they are provided with a human-like surface by replacing surface residues (a process known in the art as veneer). Humanized antibodies may include humanized and veneered antibodies. Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon stimulation, human antibody production is observed, which is very similar to that observed in humans in all aspects, including gene rearrangement, assembly, and antibody pedigree.

[0153] In addition to chimeric antibodies and humanized antibodies, fully human antibodies can be derived from transgenic mice with human immunoglobulin genes. In some embodiments, antibodies can be produced and identified by scFv-phage display libraries.

[0154] Anti-AS-SPIK antibodies can be modified to modulate their antigen binding affinity, their effector function or their pharmacokinetics. In particular, random mutations can be performed in the CDRs and the products screened to identify antibodies with higher affinity and / or higher specificity. Typically, the CDRs may differ in 1 or 2 amino acids.

[0155] For example, CDR reorganization and implantation techniques can be used together with antibodies provided herein. CDR reorganization inserts CDR sequences into specific framework regions. CDR implantation techniques allow CDR sequences to be randomly combined into a single main framework. Using such techniques, for example, the CDR sequences of anti-AS-SPIK antibodies can be mutated to produce multiple different sequences, which can be incorporated into the scaffold sequence and the resulting antibody variants can be screened for desired features (e.g., higher affinity).

[0156] Studies on the function of SPIKE have shown that SPIKE can bind to granzyme A (GzmA) and inhibit its induction of apoptosis. Lu et al., Immunology 2011; 134(4): 398-408. GzmA is a cytotoxic serine protease secreted by activated CTL and NK cells to kill target cells during immune surveillance. The role of GzmA-induced apoptosis in removing malignant cells (such as tumor precursors / tumor germ cells) has been confirmed. Pardo et al., Eur J Immunol 2002; 32(10): 2881-2887. Therefore, overexpression of AS-SPIK in liver cancer cells may cause cells to be resistant to GzmA-induced apoptosis during immune clearance. This causes these cancer cells to evade killing by immune pathways. Lu et al., Immunology 2011; 134(4): 398-408. Based on this hypothesis and without being limited by theory, it is concluded that inhibiting the overexpression of AS-SPIK or inhibiting the activity of overexpressed AS-SPIK can restore GzmA-induced immune killing of cancer cells during human immune clearance.

[0157] The anti-AS-SPIK antibodies according to embodiments of the present invention can inhibit the activity of AS-SPIK, as demonstrated by the disclosure of PCT Application No. PCT / US19 / 20999, the disclosure of which is incorporated herein by reference in its entirety. Therefore, it is possible to use anti-AS-SPIK antibodies to block the binding of AS-SPIK to GzmA, release GzmA, and restore apoptotic killing of these cancer cells by immune clearance. To this end, anti-SPIK antibodies can be used to treat conditions characterized by the expression of AS-SPIK, including but not limited to cancer, viral infection, and inflammation.

[0158] One therapeutic use of antibodies is through humanization. Therapeutic use of humanized monoclonal antibodies is a rapidly developing field, and their specificity and efficiency have been well studied. Rothernberg, ME, Cell 2016; 165(3):509. The subject anti-AS-SPIK monoclonal antibodies, including but not limited to IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6 and IM-G7, and other antibodies of the invention capable of inhibiting SPIKE activity can also be humanized and used to treat diseases.

[0159] Recombinant techniques using, for example, phagemid technology allow the preparation of antibodies with desired specificity from recombinant genes encoding a range of antibodies. Certain recombinant techniques involve the isolation of antibody genes by immunoscreening a combinatorial immunoglobulin phage expression library prepared from RNA isolated from the spleen of an immunized animal. For such methods, a combinatorial immunoglobulin phagemid library can be prepared from RNA isolated from the spleen of an immunized animal, and phagemids expressing appropriate antibodies can be selected by panning using cells expressing the antigen and control cells.

[0160] In addition to the combinatorial immunoglobulin phage expression libraries disclosed above, a molecular cloning method is used to prepare antibodies from transgenic mice containing human antibody libraries. Such transgenic animals can be used to produce human antibodies of a single isotype, more specifically, isotypes required for B cell maturation, such as IgM and possibly IgD.

[0161] Anti-AS-SPIK immunoglobulins can be modified to reduce or eliminate glycosylation. An immunoglobulin lacking glycosylation can be an immunoglobulin that is not glycosylated at all; an immunoglobulin that is not fully glycosylated; or an immunoglobulin that is atypically glycosylated (i.e., the glycosylation pattern of the mutant is different from the glycosylation pattern of the corresponding wild-type immunoglobulin). The IgG polypeptide comprises one or more (e.g., 1, 2, or 3 or more) mutations that reduce glycosylation, i.e., mutations that produce an IgG CH2 domain that lacks glycosylation, or is not fully glycosylated or atypically glycosylated. The oligosaccharide structure can also be modified, for example, by removing fucose moieties from N-linked glycans.

[0162] The antibody can also be modified by conjugation with a non-protein polymer (eg, polyethylene glycol) to increase its stability and / or solubility in vivo. Any pegylation method can be used as long as the anti-AS-SPIK antibody retains the ability to selectively bind AS-SPIK and not NS-SPIK.

[0163] A variety of antibody / immunoglobulin frameworks or scaffolds can be used, so long as the resulting polypeptide comprises at least one binding region that is specific for the target (i.e., AS-SPIK). Such frameworks or scaffolds include the five major idiotypes of human immunoglobulins or fragments thereof (such as those disclosed elsewhere herein), and include immunoglobulins of other animal species, preferably with humanized aspects. Of particular interest in this regard are single heavy chain antibodies, such as those identified in camelids.

[0164] The anti-AS-SPIK antibodies of the present invention specifically or preferably bind to an epitope on AS-SPIK and do not bind to an epitope on NS-SPIK. An epitope refers to an antigenic determinant on a target to which a paratope specifically binds, i.e., the binding site of an antibody. An epitope determinant is usually composed of a chemically active surface group of a molecule (such as an amino acid or a sugar side chain), and usually has specific three-dimensional structural characteristics and specific charge characteristics. An epitope usually has between about 4 to about 10, preferably 4 to 8 consecutive amino acids (linear or continuous epitopes), or alternatively may be a group of non-continuous amino acids that define a specific structure (e.g., a conformational epitope). Thus, an epitope may be composed of at least 4, at least 6, at least 8, at least 10, and at least 12 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, and peptide arrays using CLIPS (chemically linked peptides on a scaffold) (Timmerman, Puijk et al., J Mol Recognit, 20 (5), 283-299, (2007).

[0165] Methods for predicting other potential epitopes to which an antibody may bind may include, but are not limited to, Kyte-Doolittle analysis (Kyte and Dolittle, J. Mol. Biol. 157: 105-132 (1982)), Hopp and Woods analysis (Hopp and Woods, Proc. Natl. Acad. Sci. USA 78: 3824-3828 (1981); Hopp and Woods, Mol. Immunol. 20: 483-489 (1983); Hopp, J. Immunol. Methods 88: 1-18 (1986)), Jameson-Wolf analysis (Jameson and Wolf, Comput. Appl. Biosci. 4: 181-186 (1988)), and Emini analysis (Emini et al., Virology 140:13-20 (1985)), Chou and Fasman analysis (Ponomarenko & Regenmortel, Structural Bioinformatics, 2009), Karplus and Schulz analysis (Kolaskar and Tongaonkar Analysis Kolaskar & Tongaonkar, FEBS Letters, 172-174 (1990)) and Parker analysis. In some embodiments, potential epitopes are determined by correlation with known antigenic sites from other studies, and these prediction techniques can be combined with structural data such as X-ray crystallography data. Epitope prediction can also include techniques for predicting continuous and discontinuous epitopes. Methods for predicting discontinuous epitopes include, but are not limited to, the following methods: DiscoTope, BEpro, ElliPro, SEPPA, EPITOPIA, EPCES, Bpredictor, and EPMeta (Yao et al., PLOS ONE, (2013)). In some embodiments, potential epitopes are identified by determining theoretical extracellular domains. Analysis algorithms such as TMpred (see Hofmann and Stoffel, Biol. Chem. 374: 166 (1993)) or TMHMM (Krogh et al., J. Mol. Biol., 305 (3): 567-580 (2001)) can be used to make such predictions. Other algorithms, such as SignalP 3.0 (Bednsten et al., J. Mol. Biol. 340 (4): 783-795 (2004)) can be used to predict the presence of signal peptides and predict the position where those peptides will be cleaved from the full-length protein. Portions of the protein on the outside of the cell can serve as targets for antibody interaction.

[0166] The compositions of the present invention include antibodies described herein that (1) exhibit a threshold level of binding activity (2) do not significantly cross-react with known related polypeptide molecules; (3) bind to AS-SPIK and (4) do not bind to NS-SPIK. One of ordinary skill in the art can easily determine the binding affinity of an antibody, for example, by Scatchard analysis (Scatchard, Ann. NY Acad, Sci. 51: 660-672 (1949)).

[0167] In some embodiments, anti-AS-SPIK antibodies may bind to their target epitope or mimetic decoy that is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, 106-fold or greater for the target AS-SPIK compared to other proteins predicted to have some homology with AS-SPIK (e.g., NS-SPIK).

[0168] In some embodiments, the anti-AS-SPIK antibody is present in an amount of 10-4 M or less, 10 -7 M or smaller, 10 -9 In some embodiments, the binding affinity of an anti-AS-SPIK antibody to its corresponding target is at least 1 x 10 6 Ka. In some embodiments, the binding affinity of the anti-AS-SPIK antibody to AS-SPIK is at least 5x10 6 Ka, at least 1x10 7 Ka, at least 2x10 7 Ka, at least 1x10 8 Ka or greater. Antibodies can also be described or specified based on their binding affinity for AS-SPIK. In some embodiments, the binding affinity includes having a binding affinity of less than 5x10 -2 M, 10 -2 M, 5x10 -3 M, 10 -3 M, 5x10 -3 M, 10 -4 M, 5x10 -5 M, 10 -5 M, 5x10 -6 M, 10 -6 M, 5x10 -7 M, 10 -7 M, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 10 -9M, 5x10 -10 M, 10 -10 M, 5x10 -11 M, 10 -11 M, 5x10 -12 M, 10 -12 M, 5x10 -13 M, 10 -13 M, 5x10 -14 M, 10 -14 M, 5x10 -15 M or 10 -15 M or less Kd. That is, the terms such as "specific binding", "specific binding" or "specific binding" to AS-SPIK as used herein refer to antibodies that bind to AS-SPIK and do not bind to NS-SPIK. Although not bound by theory, it is believed that the antibodies of the present invention that specifically bind to AS-SPIK bind to an epitope within the first 1-23 amino acids that are present only in AS-SPIK but not in NS-SPIK (see SEQ ID NO:82). It is possible that the antibodies of the present invention bind to an epitope that spans 1-23 amino acids of AS-SPIK but also includes some amino acids in the common region (SEQ ID NO:83). Or it is possible that the antibody binds to at least one amino acid in the first 1-23 amino acids of AS-SPIK (SEQ ID NO:82), but also binds to at least one amino acid in the common region (SEQID NO:83). In these cases, the antibody may also bind to NS-SPIK, but the binding level is equal to or lower than the background level. This is referred to as "preferably binding" or "preferably binding" in this article. Antibodies that preferably bind AS-SPIK are still useful in diagnostic methods because assays can be developed to treat background levels of binding as "noise." Thus, the assay will show that only certain levels of binding (above a certain threshold level and above background noise) are acceptable to diagnose a patient with a disorder characterized by expression of AS-SPIK (e.g., a liver disorder such as liver cancer).

[0169] The antibodies of the present invention can be 10 -4 M or smaller, 10 -7 M or smaller, 10 -9 In some embodiments, the binding affinity of an anti-AS-SPIK antibody to its corresponding target is at least 1 x 10 6 Ka. In some embodiments, the binding affinity of the anti-AS-SPIK antibody to AS-SPIK is at least 5x10 6 Ka, at least 1x10 7Ka, at least 2x10 7 Ka, at least 1x10 8 Ka or greater. In some embodiments, the binding affinity comprises a binding affinity of less than 5x10 -2 M, 10 -2 M, 5x10 -3 M, 10 -3 M, 5x10 -3 M, 10 -4 M, 5x10 -5 M, 10 -5 M, 5x10 -6 M, 10 -6 M, 5x10 -7 M, 10 -7 M, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11 M, 10 -11 M, 5x10 -12 M, 10 -12 M, 5x10 -13 M, 10 -13 M, 5x10 -14 M, 10 -14 M, 5x10 -15 M or 10 -15 In contrast, the term "non-specific binding" such as to NS-SPIK as used herein refers to a binding affinity that is at least 1.5, 2, 5, 10, 100, 10 less than the binding affinity determined for "specific binding" such as to AS-SPIK. 3 , 10 4 , 10 5 , 10 6 or more times. Affinity (such as Kd) can be measured by radiolabeled antigen binding assay (radioimmunoassay, RIA) performed with a Fab version of the target antibody and its antigen. According to another embodiment, Kd can be measured using a surface plasmon resonance assay using an immobilized antigen. In a preferred embodiment, the antibody of the present invention specifically or preferentially binds to AS-SPIK and does not specifically bind to NS-SPIK, wherein the affinity of the antibody for AS-SPIK is at least 1.5 times, 2 times, 5 times, 10 times, 100 times, 10 times higher than the affinity for NS-SPIK. 3 times, 10 4 times, 10 5 times or 10 6 times.

[0170] In some embodiments, the antibodies do not bind to known related polypeptide molecules; for example, they bind to AS-SPIK but do not bind to known related polypeptides, such as NS-SPIK. Antibodies can be screened for known related polypeptides to separate antibody populations that specifically or preferentially bind to AS-SPIK. For example, antibodies specific for AS-SPIK will flow through a column containing NS-SPIK adhered to an insoluble matrix under appropriate buffer conditions. This screening allows the separation of polyclonal and monoclonal antibodies that are not cross-reactive with closely related polypeptides. Other methods for screening and isolating specific antibodies include, but are not limited to, for example, parallel immunoelectrophoresis, radioimmunoassay (RIA), radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or protein blot assay, inhibition or competition assay, and sandwich assay.

[0171] Antibodies according to embodiments of the present invention may include detectable labels, which may also be referred to as reporter factors (e.g., detectable reporter factors). In some embodiments, detectable labels may be any molecule covalently linked to an antibody (e.g., an anti-AS-SPIK antibody) or a biologically active fragment thereof, which allows qualitative and / or quantitative assessment of the expression or activity of the labeled peptide. Activity may include biological activity, physicochemical activity, or a combination thereof. As long as the labeled antibody remains biologically active, the form and position of the detectable label may vary. Many different labels may be used, and the selection of a specific label will depend on the desired application. Labeled anti-AS-SPIK antibodies may be used, for example, to assess the level of AS-SPIK in a biological sample, such as urine, saliva, cerebrospinal fluid, blood, or a biopsy sample.

[0172] Detectable labels may include enzymes, photoaffinity ligands, radioisotopes, and fluorescent or chemiluminescent compounds. Exemplary enzyme labels may include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and urease. The covalent attachment of anti-AS-SPIK antibodies to enzymes may be performed by different methods, such as coupling with glutaraldehyde via free amino groups. Alternatively, anti-AS-SPIK antibodies may be coupled to enzymes via sugar residues. Other carbohydrate-containing enzymes may also be coupled to antibodies in this manner. Enzyme coupling may also be performed by interconnecting the amino groups of antibodies with free thiol groups of enzymes (such as β-galactosidase) using heterobifunctional linkers (such as 6-(N-maleimido) hexanoic acid succinimidyl ester). Horseradish peroxidase detection systems may be used with, for example, the chromogenic substrate tetramethylbenzidine (TMB), which produces a soluble product detectable at 450 nm in the presence of hydrogen peroxide. For example, an alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, which produces a soluble product that can be easily detected at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-PD-galactopyranoside (ONPG), which produces a soluble product that can be detected at 410 nm. A urease detection system can be used with a substrate such as urea-bromocresol purple.

[0173] The detectable label can be a fluorescent label, including but not limited to fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine; a chemiluminescent compound selected from the group consisting of luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalate esters; liposomes or dextran; or a bioluminescent compound such as luciferin, luciferase and aequorin. Alternatively or in addition, detectable markers include, but are not limited to, radiopaque materials or contrast agents such as barium, diatrizoate, ethiodized oil, gallium citrate, iodamide, iodine ...

[0174] The label can be added during or after synthesis. The recombinant anti-AS-SPIK antibody or biologically active variant thereof can also be labeled by adding a labeled precursor (e.g., a radiolabeled amino acid) to the culture medium in which the transformed cells are grown. In some embodiments, analogs or variants of the peptide can be used to facilitate the incorporation of a detectable label. For example, any N-terminal phenylalanine residue can be easily labeled with 125In some embodiments, additional functional groups that support effective labeling can be added to fragments of anti-AS-SPIK antibodies or biologically active variants thereof. For example, a 3-tributyltinbenzoyl group can be added to the N-terminus of the native structure; 125 Displacement of the tributyltin group by I will generate a radiolabeled iodobenzoyl group.

[0175] Antibody Drug Conjugate (ADC)

[0176] Aspects of the invention include immunoconjugates or antibody-drug conjugates (ADCs) comprising antibodies conjugated to cytotoxic agents (such as chemotherapeutic agents), drugs, growth inhibitors, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof) or radioactive isotopes (i.e., radioconjugates). In another aspect, the invention further provides methods of using the immunoconjugates. In one aspect, the immunoconjugate comprises any of the above-mentioned anti-AS-SPIK antibodies covalently linked to a cytotoxic agent or a detectable agent. For example, ADCs are described in U.S. Patent No. 8,362,213, the disclosure of which is incorporated herein by reference in its entirety.

[0177] ADCs are used in the treatment of cancer for local delivery of cytotoxic or cytostatic agents (i.e., drugs) to kill or inhibit tumor cells (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9):1137-1146; Payne, G. (2003) Cancer Cell 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26: 151-172; U.S. Pat. No. 4,975,278) allow targeted delivery of drug moieties to tumors and intracellular accumulation therein, where systemic administration of these unconjugated agents may cause unacceptable levels of toxicity to normal cells as well as the tumor cells being eliminated (Baldwin et al. (1986) Lancet pp. (Mar. 15, 1986): 603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). Efforts to improve the therapeutic index of ADCs, i.e., maximum efficacy and minimum toxicity, have focused on the selectivity of polyclonal antibodies (Rowland et al. (1986) Cancer Immunol. Immunother., 21: 183-87) and monoclonal antibodies (mAbs) as well as drug-linking and drug-release properties (Lambert, J. (2005) Curr. Opinion in Pharmacology 5: 543-549).Drug moieties used in ADCs include bacterial protein toxins (e.g., diphtheria toxin), plant protein toxins (e.g., ricin), small molecules such as auristatins, geldanamycin (Mandler et al. (2000) J. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 59:2929). Res. 53:3336-3342), daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al. (1986) supra). The drug moiety can affect cytotoxic and cytostatic mechanisms, including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactivated or have reduced activity when conjugated to large antibodies or protein receptor ligands.

[0178] The auristatin peptides, auristatin E (AE) and monomethyl auristatin (MMAE), are synthetic analogs of dolastatin (WO 02 / 088172) that have been conjugated as drug moieties to: (i) the chimeric monoclonal antibody cBR96 (specific for Lewis Y on carcinomas); (ii) cAC10, which is specific for CD30 on hematological malignancies (Klussman et al. (2004), Bioconjugate Chemistry 15(4):765-773; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; Francisco et al. (2003) Blood 102(4):1458-1465; US 2004 / 0018194); (iii) anti-CD20 antibodies such as rituximab (WO 02 / 088172); and (iv) anti-CD20 antibodies such as rituximab (WO 02 / 088172). 04 / 032828), for the treatment of cancers and immune disorders expressing CD20; (iv) anti-EphB2R antibody 2H9, for the treatment of colorectal cancer (Mao et al. (2004) Cancer Research 64(3):781-788); (v) E-selectin antibody (Bhaskar et al. (2003) Cancer Res. 63:6387-6394); (vi) trastuzumab ( US 2005 / 0238649); and (vi) anti-CD30 antibodies (WO 03 / 043583). Variants of auristatin E are disclosed in U.S. Pat. No. 5,767,237 and U.S. Pat. No. 6,124,431. Monomethyl auristatin E conjugated to a monoclonal antibody is disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, filed March 28, 2004. The auristatin analogs MMAE and MMAF have been coupled to various antibodies (US 2005 / 0238649).

[0179] Conventional means of connecting the drug moiety (i.e., by covalent bonding) to the antibody usually form a heterogeneous mixture of molecules at many sites where the drug moiety is connected to the antibody. For example, cytotoxic drugs are usually conjugated to the antibody through a large amount of lysine or cysteine ​​residues in the antibody, thereby producing a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, the heterogeneous mixture usually contains a distribution of antibodies with 0 to about 8 or more connected drug moieties. In addition, in each subgroup of the conjugate with a specific integer ratio of the drug moiety to the antibody, there may be a heterogeneous mixture in which the drug moiety is connected to different sites on the antibody. Analysis and preparation methods may not be enough to separate and characterize the antibody-drug conjugate species molecules in the heterogeneous mixture produced by the conjugation reaction. Antibodies are large, complex and structurally diverse biomolecules, usually with many reactive functional groups. Their reactivity with linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration and cosolvents. In addition, due to the difficulty in controlling reaction conditions and characterizing reactants and intermediates, multi-step conjugation processes may be non-reproducible.

[0180] Cysteine ​​thiols are reactive at neutral pH values, unlike most amines which are protonated near pH 7 and have low nucleophilicity. Because free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine ​​residues usually exist in their oxidized form as disulfide-linked oligomers or have internal bridged disulfide bonds. Extracellular proteins generally do not have free thiols (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London, page 55). Antibody cysteine ​​thiol groups are generally more reactive, i.e., more nucleophilic, to electrophilic conjugation reagents than antibody amino or hydroxyl groups. Cysteine ​​residues have been introduced into proteins by genetic engineering techniques to form covalent linkages with ligands or to form new intramolecular disulfide bonds (Better et al. (1994) J. Biol. Chem. 13:9644-9650; Bernhard et al. (1994) Bioconjugate Chem. 5:126-132; Greenwood et al. (1994) Therapeutic Immunology 1:247-255; Tu et al. (1999) Proc. Natl. Acad. Sci USA 96:4862-4867; Kanno et al. (2000) J. of Biotechnology, 76:207-214; Chmura et al. (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484; U.S. Pat. No. 6,248,564). However, engineering cysteine ​​thiol groups by mutating various amino acid residues of a protein to cysteine ​​amino acids can be problematic, especially in the case of unpaired (free Cys) residues or in the case of relatively easy to react or oxidize. In concentrated protein solutions, whether in the periplasm of E. coli, culture supernatant, or in partially or completely purified proteins, unpaired Cys residues on the surface of proteins can pair and oxidize to form intermolecular disulfide bonds, and thus form protein dimers or polymers. The formation of disulfide-bonded dimers renders the new Cys unresponsive to coupling with drugs, ligands, or other labels. In addition, if a protein is oxidized to form intramolecular disulfide bonds between a newly engineered Cys and an existing Cys residue, both Cys thiol groups are unavailable to participate in active sites and interactions. In addition, proteins may be inactivated or nonspecific by misfolding or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311: 1-9).

[0181] Cysteine ​​engineered antibodies have been designed as FAB antibody fragments (thioFab) and expressed as full-length IgG monoclonal (thioMab) antibodies (Junutula, JR et al. (2008) J Immunol Methods 332:41-52; US 2007 / 0092940, the contents of which are incorporated by reference). ThioFab and thioMab antibodies have been conjugated to thiol-reactive linker reagents and drug-linker reagents via linkers at the newly introduced cysteine ​​thiols to prepare antibody drug conjugates (thioADCs).

[0182] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0183] Peptides

[0184] Aspects of the present invention include compositions comprising a SPIKE polypeptide, such as an AS-SPIK polypeptide encoded by any of the nucleic acid sequences of SEQ ID NOs: 104 and 105. The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, although they generally refer to peptide sequences of different sizes. The amino acid-based compositions of the present invention may be referred to as "polypeptides" to indicate that they are linear polymers of amino acid residues and to help distinguish them from full-length proteins. Polypeptides according to embodiments of the present invention may "constitute" or "comprise" fragments of AS-SPIK polypeptides or NS-SPIK polypeptides, and the present invention encompasses polypeptides that constitute or comprise biologically active variants of AS-SPIK polypeptides or NS-SPIK polypeptides. It should be understood that the polypeptide may therefore only contain fragments of AS-SPIK polypeptides or NS-SPIK polypeptides (or biologically active variants thereof), but may also contain additional residues. The biologically active variant will retain sufficient activity to inhibit proteases.

[0185] The bond between the amino acid residues can be a conventional peptide bond or another covalent bond (such as an ester bond or an ether bond), and the polypeptide can be modified by amidation, phosphorylation or glycosylation. The modification can affect the polypeptide backbone and / or one or more side chains. The chemical modification can be a naturally occurring modification (e.g., glycosylation in a bacterial host) performed in vivo after translation of the mRNA encoding the polypeptide or a synthetic modification performed in vitro. The biologically active variant of the AS-SPIK polypeptide or NS-SPIK polypeptide may comprise one or more structural modifications produced by any combination of naturally occurring modifications (i.e., naturally occurring in vivo) and synthetic modifications (i.e., naturally occurring or non-naturally occurring modifications performed in vitro). Examples of modifications include, but are not limited to, amidation (e.g., replacement of a free carboxyl group at the C-terminus with an amino group); biotinylation (e.g., acylation of lysine or other reactive amino acid residues with a biotin molecule); glycosylation (e.g., addition of a sugar group to asparagine, hydroxylysine, serine, or threonine residues to produce glycoproteins or glycopeptides); acetylation (e.g., addition of an acetyl group, typically at the N-terminus of a polypeptide); alkylation (e.g., addition of an alkyl group); prenylation (e.g., addition of an isoprenoid group); fatty acylation (e.g., attachment of a lipoic acid moiety); and phosphorylation (e.g., addition of a phosphate group to serine, tyrosine, threonine, or histidine).

[0186] One or more amino acid residues in the biologically active variant can be non-natural amino acid residues. Naturally occurring amino acid residues include those naturally encoded by the genetic code and non-standard amino acids (e.g., amino acids with D- configuration rather than L- configuration). The peptides of the present invention may also include amino acid residues of modified forms of standard residues (e.g., pyrrolysine can be used instead of lysine, and selenocysteine ​​can be used instead of cysteine). Non-natural amino acid residues are those amino acid residues that have not been found in nature but conform to the basic formula of amino acids and can be incorporated into peptides. These include D- alloisoleucine (2R, 3S) -2-amino-3-methylpentanoic acid and L- cyclopentylglycine (S) -2-amino-2-cyclopentyl acetic acid. For other examples, textbooks or the World Wide Web (currently maintained by the California Institute of Technology, and showing the structure of non-natural amino acids that have been successfully incorporated into functional proteins) can be consulted.

[0187] Alternatively or in addition, one or more amino acid residues in the biologically active variant may be naturally occurring residues that are different from the naturally occurring residues found in the corresponding position in the wild-type sequence. In other words, the biologically active variant may contain one or more, particularly one or two amino acid substitutions. The substitution, addition or deletion of an amino acid residue may be referred to as a mutation of the wild-type sequence. As described above, substitutions may replace naturally occurring amino acid residues with non-naturally occurring residues or simply different naturally occurring residues. In addition, substitutions may constitute conservative or non-conservative substitutions. Conservative amino acid substitutions generally include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

[0188] A polypeptide that is a biologically active variant of AS-SPIK can be characterized according to the degree to which its sequence is similar or homologous to the corresponding wild-type polypeptide. For example, the sequence of the biologically active variant can be at least or about 80% homologous (or identical) to the corresponding residue in the wild-type polypeptide. For example, a biologically active variant of an AS-SPIK polypeptide or NS-SPIK polypeptide can have an amino acid sequence that has at least or about 80% sequence homology (e.g., at least or about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology) (or such percentage identity) with an AS-SPIK or NS-SPIK polypeptide (SEQ ID NO: 81, 82, 83) or a homolog or ortholog thereof.

[0189] The biologically active variants of the AS-SPIK polypeptide or NS-SPIK polypeptide will retain sufficient biological activity to be used in the methods of the present invention. The biologically active variants will retain sufficient activity to act as inhibitors of protease activity. Biological activity can be assessed in a manner known to those of ordinary skill in the art and include, but are not limited to, in vitro cleavage assays or functional assays.

[0190] Polypeptide can be produced by several methods, and described method comprises for example recombinant technology or chemical synthesis.Once produced, polypeptide just can be separated and purified to any desired degree.For example, can be in for example reverse phase (preferred) or normal phase HPLC, or after size exclusion or distribution chromatography on polysaccharide gel medium such as Sephadex G-25, use freeze drying.The composition of final polypeptide can be confirmed by amino acid analysis by standard method, amino acid sequencing or FAB-MS technology after peptide degradation.Salt (comprising acid salt), ester, amide and N-acyl derivative of the amino group of polypeptide can be prepared using methods known in the art, and this type of peptide can be used in the context of the present invention.

[0191] Also provided is an AS-SPIK complex. The AS-SPIK complex according to an embodiment of the present invention comprises an antibody of the present invention as described herein, which specifically or preferably binds to AS-SPIK and an AS-SPIK polypeptide or a fragment thereof. The fragment has in particular a length of at least 23 amino acids (SEQ ID NO: 81), preferably at least 10 amino acids, more preferably at least the 7th to (and including) the 23rd amino acid of SEQ ID NO: 81, and even more preferably at least the 8th to (and including) the 17th amino acid of SEQ ID NO: 81. The antibody may be any of the above-mentioned anti-AS-SPIK antibodies. The AS-SPIK polypeptide or a fragment thereof may be the above-mentioned AS-SPIK polypeptide or a fragment thereof. In some embodiments, the antibody is the anti-AS-SPIK monoclonal antibodies IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6 and IM-G7 (VL and VH sequences listed in the sequence data). In some embodiments, the AS-SPIK polypeptide is a polypeptide having an amino acid sequence that is at least 98% homologous (or identical) to the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 81. In some embodiments, the AS-SPIK polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 82.

[0192] The specific binding of anti-AS-SPIK antibodies such as IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6 and IM-G7 can form immune complexes with AS-SPIK or AS-SPIK peptides under certain conditions. The complex can be precipitated from the solution for further analysis, for example, using a sandwich ELISA test. Using a 96-well plate fixed with a second anti-SPIK antibody as a carrier, the immune complex can be captured by the plate. If the antibody in the complex is labeled with a reporter gene such as horseradish peroxidase (HPR), the amount of the AS-SPIK immune complex formed can be determined. The AS-SPIK immune complex can also be captured by agarose beads connected to protein A or G for Western blot analysis.

[0193] Nucleic Acids

[0194] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs, any of which can encode the polypeptide of the present invention, and all of which are encompassed in the present invention. Polynucleotides can have substantially any three-dimensional structure. Nucleic acids can be double-stranded or single-stranded (i.e., sense strand or antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA) and parts thereof, transfer RNA, ribosomal RNA, siRNA, microRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers, and nucleic acid analogs. In the context of the present invention, nucleic acids can encode fragments of naturally occurring AS-SPIK polypeptides or NS-SPIK polypeptides or their biologically active variants. Non-limiting examples of nucleic acid sequences according to embodiments of the present invention include SEQ ID NO: 104, 105, and 106, or biologically active fragments or variants thereof. In some embodiments, the fragment may have a length of at least about 66 nucleotides, preferably at least about 54 nucleotides. In a preferred embodiment, the nucleic acid sequence comprises nucleotide residues 28-105 of SEQ ID NO: 105. In a preferred embodiment, the nucleic acid sequence comprises nucleotide residues 49-105 of SEQ ID NO: 105. Lu et al., Immunology 2011; 134(4): 398-408.

[0195] "Isolated" nucleic acid can be, for example, a naturally occurring DNA molecule or a fragment thereof, provided that at least one of the nucleic acid sequences generally found in the naturally occurring genome immediately flanking the DNA molecule is removed or absent. Therefore, isolated nucleic acids include, but are not limited to, DNA molecules present as separate molecules, independent of other sequences (e.g., chemically synthesized nucleic acids, or cDNA or genomic DNA fragments produced by polymerase chain reaction (PCR) or restriction endonuclease treatment). Isolated nucleic acids also refer to DNA molecules incorporated into vectors, autonomously replicating plasmids, viruses, or into the genomic DNA of prokaryotes or eukaryotes. In addition, isolated nucleic acids can include engineered nucleic acids, such as DNA molecules as a part of a hybrid or fusion nucleic acid. Nucleic acids present in, for example, many (e.g., tens, hundreds to millions) other nucleic acids in a cDNA library or genomic library or in a gel slice containing a restriction digest of genomic DNA are not isolated nucleic acids.

[0196] The nucleic acid molecules of separation can be produced, for example, by polymerase chain reaction (PCR) technology, which can be used to obtain the nucleic acid of separation containing nucleotide sequences as described herein, including nucleotide sequences encoding polypeptides as described herein. PCR can be used to increase specific sequences from DNA and RNA, including sequences from total genomic DNA or total cell RNA. In general, oligonucleotide primers are designed using sequence information from the end of the target region or further regions, which are identical or similar in sequence to the opposite strand of the template to be amplified. Various PCR strategies can also be used, by which site-specific nucleotide sequence modifications can be introduced into the template nucleic acid.

[0197] The isolated nucleic acid can also be chemically synthesized as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology), or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >50-100 nucleotides) can be synthesized, the oligonucleotides containing the desired sequence, wherein each pair contains a short complementary segment (e.g., about 15 nucleotides) so that a duplex is formed when the oligonucleotides are annealed. DNA polymerase is used to extend the oligonucleotides, thereby each oligonucleotide pair produces a single double-stranded nucleic acid molecule, which can then be connected to a vector. The isolated nucleic acid of the present invention can also be obtained by mutagenesis of a naturally occurring portion of, for example, an AS-SPIK- or NS-SPIK-encoding DNA (according to, for example, the above formula).

[0198] Two nucleic acids or the polypeptides encoded by them can be described as having a certain degree of homology or identity to each other. For example, AS-SPIK polypeptides or NS-SPIK polypeptides and biologically active variants thereof can be described as exhibiting a certain degree of homology or identity. Alignment can be assembled in the following manner: short AS-SPIK polypeptides or NS-SPIK polypeptide sequences are located in the Protein Information Research (PIR) website (http: / / pir.georgetown.edu), and then analyzed using the Basic Local Alignment Search Tool (BLAST) algorithm on the NCBI website (http: / / www.ncbi.nlm.nih.gov / blast) using "almost identical short sequences".

[0199] To determine sequence homology or identity, a query nucleic acid or amino acid sequence can be aligned with one or more subject nucleic acid or amino acid sequences, respectively, using a computer program such as BioEdit (Version 4.8.5, North Carolina State University), which allows alignment of nucleic acid or protein sequences over their entire length (global alignment), or ALIGN-2, as described above.

[0200] BioEdit calculates the best match between the query sequence and one or more subject sequences and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more residues can be inserted into the query sequence, the subject sequence, or both to maximize sequence alignments. For fast pairwise alignments of nucleic acid sequences, the following default parameters are used: Font size: 2; Window size: 4; Scoring method: Percent; Number of top diagonals: 4; and Gap penalty: 5. For multiple alignments of nucleic acid sequences, the following parameters are used: Gap opening penalty: 10.0; Gap extension penalty: 5.0; and Weight shift: Yes. For fast pairwise alignments of protein sequences, the following parameters are used: Font size: 1; Window size: 5; Scoring method: Percent; Number of top diagonals: 5; Gap penalty: 3. For multiple alignments of protein sequences, the following parameters were used: Weight matrix: blosum; Gap opening penalty: 10.0; Gap extension penalty: 0.05; Hydrophilic gaps: On; Hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; Residue-specific gap penalties: On. The output is a sequence alignment reflecting the relationship between the sequences.

[0201] To determine the percent homology between the query sequence and the subject sequence, BioEdit divides the number of identities in the best alignment by the number of residues compared (excluding gap positions) and multiplies the result by 100. The output is the percent homology of the subject sequence relative to the query sequence. It should be noted that the percent homology values ​​are rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0202] Nucleic acids and polypeptides described herein may be referred to as "exogenous". The term "exogenous" means that the nucleic acid or polypeptide is part of or encoded by a recombinant nucleic acid construct, or is not in its natural environment. For example, an exogenous nucleic acid may be a sequence introduced from one species into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into another species by a recombinant nucleic acid construct. An exogenous nucleic acid may also be a sequence that is native to an organism and has been reintroduced into the cells of the organism. An exogenous nucleic acid comprising a native sequence may be distinguished from a naturally occurring sequence by the presence of a non-native sequence (e.g., a non-native regulatory sequence flanking a native sequence) connected to the exogenous nucleic acid in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids are typically integrated at a position other than the position of the visible native sequence.

[0203] Recombinant constructs are also provided herein and can be used to transform cells to express AS-SPIK. The recombinant nucleic acid construct comprises a nucleic acid encoding an AS-SPIK or NS-SPIK sequence, which is operably connected to a regulatory region suitable for expressing AS-SPIK or NS-SPIK in a specific cell. It should be understood that many nucleic acids can encode polypeptides with specific amino acid sequences. The degeneracy of the genetic code is well known in the art. For many amino acids, there are more than one nucleotide triplet that serves as a codon for an amino acid. For example, the codons in the coding sequence of AS-SPIK or NS-SPIK can be modified so that the optimal expression in the organism is obtained using the appropriate codon bias table for a specific organism.

[0204] Also provided are vectors containing nucleic acids, such as those described herein. A "vector" is a replicon, such as a plasmid, phage or cosmid, into which another DNA segment can be inserted to cause replication of the inserted segment. Typically, a vector is capable of replication when associated with appropriate control elements. Suitable vector backbones include, for example, those conventionally used in the art, such as plasmids, viruses, artificial chromosomes, BACs, YACs or PACs. The term "vector" includes cloning and expression vectors, as well as viral vectors and integration vectors. An "expression vector" is a vector comprising a regulatory region. A variety of host / expression vector combinations can be used to express nucleic acid sequences described herein. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, phages, baculoviruses and retroviruses.

[0205] The vectors provided herein may also include, for example, an origin of replication, a scaffold attachment region (SAR) and / or a marker. The marker gene can confer a selective phenotype on the host cell. For example, the marker can confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin or hygromycin). As described above, the expression vector may include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin or Flag TM Tag (Kodak, New Haven, CT) sequences are typically expressed as fusions to the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including the carboxyl or amino termini.

[0206] Additional expression vectors may also include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, such as E. coli plasmid col E1, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 and derivatives thereof, plasmids such as RP4; phage DNA (e.g., numerous derivatives of phage 1, such as NM989) and other phage DNA (e.g., M13 and filamentous single-stranded phage DNA); yeast plasmids (e.g., 2μ plasmids or derivatives thereof); vectors suitable for use in eukaryotic cells, such as vectors suitable for use in insect or mammalian cells; vectors derived from a combination of plasmids and phage DNA, such as plasmids that have been modified to employ phage DNA or other expression control sequences.

[0207] The vector may also include a regulatory region. The term "regulatory region" refers to a nucleotide sequence that affects the transcription or translation initiation and rate and stability and / or activity of a transcription or translation product. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, terminal sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0208] As used herein, the term "operably connected" refers to the positioning of the regulatory region and the sequence to be transcribed in the nucleic acid to affect the transcription or translation of such sequence. For example, in order to place the coding sequence under the control of the promoter, the translation start site of the translation reading frame of the polypeptide is usually positioned between one and about fifty nucleotides downstream of the promoter. However, the promoter can be positioned up to about 5,000 nucleotides upstream of the translation start site or about 2,000 nucleotides upstream of the transcription start site. The promoter usually comprises at least one core (basic) promoter. The promoter may also include at least one control element, such as an enhancer sequence, an upstream element, or an upstream activation region (UAR). The selection of the promoter to be included depends on several factors, including but not limited to efficiency, selectivity, inducibility, desired expression level, and cell or tissue preferential expression. The conventional subject of those skilled in the art is to regulate the expression of the coding sequence by appropriately selecting promoters and other regulatory regions and positioning promoters and other regulatory regions relative to the coding sequence.

[0209] The vector comprising the AS-SPIK or NS-SPIK nucleic acid sequence can be formulated in a manner that promotes cell (i.e., prokaryotic or eukaryotic cells, such as mammalian cells) uptake. Useful vector systems and preparations are as described above. In some embodiments, the vector can deliver the composition to a specific cell type. However, the present invention is not limited thereto, and other DNA delivery methods are also contemplated, such as chemical transfection, using, for example, calcium phosphate, DEAE dextran, liposomes, lipid complexes, surfactants, and perfluorochemical liquids; and physical delivery methods, such as electroporation, microinjection, ballistic particles, and "gene gun" systems. In some embodiments, the polynucleotides of the present invention can also be used together with micro-delivery vehicles (such as cationic liposomes, other lipid-containing complexes, and other macromolecular complexes that can mediate polynucleotide delivery to host cells). Another delivery method is to use a single-stranded DNA production vector that can produce an expression product in a cell.

[0210] How to use

[0211] The compositions disclosed herein can be used generally and multifacetedly for diagnosing and / or treating disorders characterized by the expression of AS-SPIK. Such disorders include, but are not limited to, cancer, viral infections, and inflammatory disorders. A prominent example is liver cancer. Other non-limiting examples include those cancers associated with the definition of the term "cancer" described herein. Therefore, aspects of the present invention relate to methods for diagnosing and / or treating cancer (e.g., liver cancer) in a subject, the subject suffering from the cancer or having a risk of developing the cancer. The terms "subject," "patient," and "individual" are used interchangeably herein.

[0212] In some embodiments, the method includes contacting a biological test sample from a subject with an AS-SPIK antibody or antigen binding fragment to produce an AS-SPIK-antibody complex; detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; and comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject suffers from the condition or is at risk of developing the condition. In certain embodiments, the method includes contacting a biological test sample with a first antibody or antigen binding fragment that binds to SPIKE to produce a SPIKE-antibody complex; contacting the SPIKE-antibody complex with an AS-SPIK antibody or antigen binding fragment to produce an AS-SPIK-antibody complex in the biological test sample; and comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject suffers from the condition or is at risk of developing the condition. Several non-limiting examples of antibodies that can be used in such methods are described herein.

[0213] In some embodiments, the method comprises administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof or antibody-drug conjugate as described herein to a patient suffering from a disease or disorder characterized by expression of AS-SPIK.

[0214] Liver cancer

[0215] A prominent example of a disorder characterized by the expression of AS-SPIK is liver cancer. Liver cancer encompasses a variety of disorders that lead to liver damage or impaired liver function. For example, liver cancer can be caused by infectious agents, disease, trauma or genetic disorders or a combination of infectious agents, disease, trauma and genetic disorders.

[0216] Liver cancer can include diseases involving abnormal cell growth, such as primary liver cancer, for example, hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma and hepatoblastoma. Such cancers can include cancers at any stage of disease progression, such as from very early (Barcelona Clinical Liver Cancer System (BCLC) 0 phase and tumor size <2 cm), early (BCLC A phase, tumor size between 2 cm and 5 cm), mid-term (BCLC B phase, intermediate tumor size>5 cm), late (BCLC C and D phase, late) or metastatic (Pons et al., HPB 2005; 7 (1): 35-41) HCC and from ICC early (I, II and IIIa phase, tumor size <2 cm), mid-term (IIIb and IIIc phase, tumor size>2 cm) and late (IV phase) ICC (Farges et al., Cancer 2011; 117 (10): 2170-2177).

[0217] Liver cancer can also be induced by infectious diseases caused by viruses, such as hepatitis B, hepatitis C, and hepatitis D. Regardless of the specific hepatitis virus, such infections can be acute or chronic.

[0218] Liver cancer can also be caused by liver damage, such as cirrhosis. Cirrhosis (advanced scarring or fibrosis of the liver) can be caused by many forms of liver disease and disorders. Cirrhosis can be caused by genetic conditions, such as hemochromatosis, cystic fibrosis, Wilson's disease, and autoimmune disorders. Cirrhosis can also be caused by hepatitis virus infection and alcohol consumption.

[0219] Liver cancer can also be caused by other diseases, including but not limited to alcoholic liver disease, conditions related to abnormal amounts of fat in the liver such as fatty liver, nonalcoholic fatty liver disease, nonalcoholic steatosis, and liver fibrosis.

[0220] Biological samples

[0221] "Biological sample", "test sample" or "sample" refers to a sample obtained or derived from a patient. The sample can be, for example, a body fluid sample. Exemplary body fluid samples include blood, serum, plasma, urine, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, or any combination thereof. In some embodiments, the biological sample can be a tissue sample. Exemplary tissue samples include a biopsy specimen, such as a liver biopsy specimen, or a primary cell culture specimen prepared from a patient's cells, or a supernatant from a primary culture.

[0222] Immunoassay

[0223] Aspects of the invention include diagnostic assays, such as diagnostic immunoassays, which can be used to detect the presence of AS-SPIK in a test sample. Immunoassay formats for detecting AS-SPIK can be configured in a variety of ways. Immunoassays can include homogeneous and heterogeneous assays, competitive and non-competitive assays, direct and indirect assays, and "sandwich" assays. Useful formats include, but are not limited to, enzyme immunoassays, such as enzyme-linked immunosorbent assays (ELISA), chemiluminescent immunoassays (CLIA), electrochemiluminescent assays, radioimmunoassays, immunofluorescence, fluorescence anisotropy, immunoprecipitation, equilibrium dialysis, immunodiffusion, immunoblotting, agglutination, luminescent proximity assays, and turbidimetry.

[0224] Regardless of the form, the biological sample is contacted with the anti-AS-SPIK antibody of the present invention. In some embodiments, the biological sample may be fixed on a solid support. In some embodiments, the biological sample is contacted with the anti-SPIK antibody of the present invention fixed on a solid support. The solid support can be, for example, a plastic surface, a glass surface, a paper or fiber surface or a particle surface. More specifically, the support may include a microplate, a bead, a polyvinylidene fluoride (PVDF) membrane, a nitrocellulose membrane, a nylon membrane, a porous membrane, a non-porous membrane. The composition of the substrate can vary. For example, the substrate or support may include glass, a cellulose-based material, a thermoplastic polymer (such as polyethylene, polypropylene or polyester), a sintered structure composed of a particulate material (e.g., glass or various thermoplastic polymers), or a cast film composed of nitrocellulose, nylon or polysulfone. In general embodiments, the substrate can be any surface or support to which the antibody or polypeptide can be immobilized, including one or more of a solid support (e.g., glass, such as a slide or coated plate, silica, plastic or derivatized plastic, paramagnetic or non-magnetic metal), a semi-solid support (e.g., a polymeric material, gel, agarose or other matrix), and / or a porous support (e.g., a filter, nylon or nitrocellulose membrane or other membrane). In some embodiments, synthetic polymers can be used as substrates, including, for example, polystyrene, polypropylene, polyglycidyl methacrylate, aminated or carboxylated polystyrene, polyacrylamide, polyamide, and polyvinyl chloride.

[0225] In some embodiments, the immunoassay format can be a two-antibody "sandwich" assay. The biological sample is contacted with an anti-SPIK antibody of the present invention fixed on a solid support such as a microtiter plate. The sample and the first antibody are incubated under conditions that are conducive to specific binding and the formation of a SPIKE antibody complex. After the contact step, the unbound components of the biological sample are removed. Then, the complex is contacted with a second anti-SPIK antibody. The second antibody binds to a SPIKE epitope different from the epitope bound by the first antibody. Therefore, the first and second antibodies do not competitively inhibit each other's binding to SPIKE. In some embodiments, the first antibody can recognize an epitope, i.e., an antigenic determinant, present on both AS-SPIK and NS-SPIK. This antibody can be referred to as a "pan-SPIK" antibody. Alternatively, the first antibody can recognize an epitope that is present only on AS-SPIK. In some embodiments, the second antibody can recognize an epitope, i.e., an antigenic determinant, present on both AS-SPIK and NS-SPIK. Alternatively, the second antibody can recognize an epitope that is present only on AS-SPIK or NS-SPIK. Thus, the sandwich assay can be configured such that the first antibody is a pan-SPIK antibody and the second antibody specifically or preferentially binds to AS-SPIK and does not specifically bind to NS-SPIK. Alternatively, the sandwich assay can be configured such that the first and second antibodies specifically or preferentially bind to AS-SPIK and do not specifically bind to NS-SPIK.

[0226] Antibody binding can be measured in a variety of ways. For example, the signal generated by the detectable label can be analyzed and quantified (if applicable) using an optical scanner or other image acquisition device and software that allows measurement of the signal, such as a fluorescent signal, luminescent signal, or phosphorescent signal or radioactive signal associated with complex formation. Exemplary instruments for measuring detectable signals may include, but are not limited to, microplate readers, fluorometers, spectrophotometers, and gamma counters.

[0227] Reference samples

[0228] The level of AS-SPIK in a biological sample can be compared to the level of a reference sample. The standard reference level typically represents the average AS-SPIK level derived from a population of individuals. The reference population can include individuals of similar age, body type, ethnic background, or overall health to the individual in question. Thus, the level of AS-SPIK in a patient sample can be compared to values ​​derived from: 1) individuals known to have liver cancer and express AS-SPIK and whose body fluids contain AS-SPIK; 2) individuals who do not have liver cancer and whose body fluids have lower levels of AS-SPIK.

[0229] In general, an elevated AS-SPIK level can be any AS-SPIK level that is higher than the level of AS-SPIK found in a control sample or the average level of AS-SPIK found in a sample from a population of normal healthy individuals who do not suffer from liver cancer (reference value), preferably at least 1%, 2%, 3%, 4% or 5%, more preferably at least 5%. A reduced AS-SPIK level can be any AS-SPIK level that is lower than the level of AS-SPIK found in a control sample or lower than the average level of AS-SPIK found in a sample from a population of individuals suffering from liver cancer. Any population size can be used to determine the average level of AS-SPIK found in a sample from a population of normal healthy individuals. For example, a population between 2 and 250, such as 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250 or more individuals can be used to determine the average level of AS-SPIK in a sample from a population of normal healthy individuals, with greater accuracy of measurement from a larger sample population.

[0230] In some embodiments, a reference chart can be used to determine whether a particular level of AS-SPIK in a sample is elevated relative to a control sample or a larger population. For example, a reference chart may contain a normal range of AS-SPIK found in healthy individuals of the same age, ethnic background, or overall health as the individual in question. Using this reference chart, any AS-SPIK level measured in a sample can be classified as low, normal, or elevated relative to a control sample or relative to an average value from a larger population. The term "elevated level" is defined as a level that is higher, preferably at least 2%, more preferably at least 5% higher than a reference level.

[0231] Alternatively or in addition, the level of AS-SPIK in a biological sample can be "normalized" to the level of one or more additional biomarkers (e.g., another marker whose expression is independent of AS-SPIK expression). That is, the level of the additional marker can be assessed in parallel with the level of AS-SPIK, either simultaneously or at a separate time. The additional marker can serve as an internal control for sample preparation, handling and storage, and daily assay variability. The value of the AS-SPIK level and the additional marker can be expressed as a ratio, and the ratio can be compared to a similar ratio obtained for a reference sample or population. A useful second marker can be alpha-fetoprotein.

[0232] In some embodiments, the method may include the use of a standard reference set. The reference set may include one or more samples of purified SPIKE polypeptides or fragments thereof. When multiple samples are used, the concentrations of these samples may be different. In one embodiment, the reference set may include 6 recombinant AS-SPIK samples with concentrations of 50 ng / ml, 30 ng / ml, 8 ng / ml, 3 ng / ml, 1 ng / ml and 0 ng / ml of AS-SPIK. Anti-AS-SPIK antibodies (such as IM-CA22) or anti-tag antibodies may be used to purify recombinant AS-SPIK by affinity chromatography (HPLC). The reference value in blood or other body fluids may vary. However, a technician is able to determine the average level of AS-SPIK in different body fluids of the corresponding population and determine the corresponding reference value, which ensures that the AS-SPIK level of the patient with liver cancer to be determined is much higher than the reference value, while the level of the patient or healthy individual who is not suffering from liver cancer to be detected is much lower than the corresponding reference value. In a preferred embodiment, the reference value is about 5%, more preferably about 7%, and even more preferably about 10% higher than the average level of AS-SPIK found in samples from a normal healthy individual population. It should be noted that the level of AS-SPIK in the biological sample and the control sample will be determined by the same method to give comparability. For example, the absolute value of the AS-SPIK level can be determined by using a calibration curve of recombinant AS-SPIK as described above.

[0233] Control samples

[0234] In some embodiments, the positive control may include a sample of AS-SPIK produced by a eukaryotic cell or cell line. For example, a useful control may be a culture medium containing 100 ng / ml of AS-SPIK from the stable cell line S2-3. This was generated by the inventors by inserting the DNA sequence of AS-SPIK into the chromosome of HCC cells under the control of an artificial promoter that overexpresses AS-SPIK.

[0235] The method disclosed herein can be used to detect liver cancer in patients suspected of having liver cancer or having a risk of suffering from liver cancer. The method can also be used to analyze samples from patients who have been treated for liver cancer, such as hepatocellular carcinoma, to determine whether the patient has a risk of experiencing hepatocellular carcinoma remission. The method can also be used to monitor the course of treatment, such as treatment with therapeutic agents such as small molecule drugs or therapeutic antibodies, chemotherapy, radiotherapy or surgery, to determine the efficacy of treatment and allow the managing clinician to change the treatment as needed. The method can also be used to detect, monitor or analyze any disease associated with the regulation (e.g., increase) of AS-SPIK levels in biological samples (e.g., blood or serum samples obtained from patients) or patients at risk of suffering from the disease.

[0236] The methods disclosed herein can be used in conjunction with other standard diagnostic methods, such as serological analysis of liver enzymes or alpha-fetoprotein, ultrasound (sonography), computed tomography (CT scan), magnetic resonance imaging (MRI), angiography, laparoscopy, or biopsy.

[0237] Products

[0238] The compositions described herein may be packaged in suitable containers, which are labeled, for example, for detecting, identifying and quantifying AS-SPIK in biological samples. The product (also referred to as a "kit") may include antibodies of the present invention, antigen-binding fragments of antibodies and / or antibody-drug conjugates, culture media, purified antigen samples used as positive controls, or any combination thereof. The container included in the kit may include a composition comprising an antibody of the present invention, which specifically or preferably binds to AS-SPIK but does not bind to NS-SPIK. The kit may also include antibodies that bind to both AS-SPIK and NS-SPIK. Suitable buffers for diluting or reconstructing test samples and antibodies may also be provided. Some components may be provided in dry form and may require reconstruction. Anti-SPIK antibodies may be pre-bound to an assay device, such as a microplate. Therefore, in one embodiment, a kit for detecting, identifying and quantifying AS-SPIK includes anti-AS-SPIK antibodies and pan-SPIK antibodies. The kit may optionally include a detectable label.

[0239] Therefore, packaged products (e.g., sterile containers containing one or more compositions described herein and packaged in concentrated or ready-to-use concentrations for storage, transportation or sale) and kits (including at least one composition of the invention (e.g., anti-AS-SPIK antibodies)) are also within the scope of the invention. Products may include containers (e.g., vials, jars, bottles, bags, etc.) containing one or more compositions of the invention. In addition, articles of manufacture may also include, for example, packaging materials, instructions for use, syringes, delivery devices, buffers or other control reagents for treating or monitoring a condition requiring diagnosis or treatment.

[0240] Reagents for specific types of assays can also be provided in the kit of the present invention. Thus, the kit can include a bead population (e.g., suitable for agglutination assays or lateral flow assays) or a plate (e.g., a plate suitable for ELISA assays). In other embodiments, the kit includes a device, such as a lateral flow immunoassay device, an analytical rotor, or an electrochemical, optical, or photoelectric sensor. Bead populations, plates, and devices can be used to perform immunoassays. For example, they can be used to detect the formation of a first reagent-analyte-second reagent complex.

[0241] In addition, the kit may include various diluents and buffers, labeled conjugates or other reagents for detecting specifically bound antigens or antibodies, and other signal generating reagents such as enzyme substrates, cofactors and chromogens. The kit may include one or more reference samples of different concentrations, such as purified recombinant AS-SPIK. The kit may also include a positive control, such as a cell supernatant from a cell line overexpressing AS-SPIK. Other components of the kit may include coating reagents, polyclonal or monoclonal capture antibodies specific to the antigen or analyte to be tested, or a mixture of two or more antibodies, purified or semi-purified extracts of these antigens as standards, monoclonal antibody detection antibodies, anti-mouse, anti-dog, anti-chicken or anti-human antibodies and indicator molecules conjugated thereto, indicator charts for colorimetric comparison, disposable gloves, decontamination instructions, smear sticks or containers, sample preparation cups, etc. In one embodiment, the kit includes a buffer or other reagents suitable for constituting a reaction medium that allows the formation of a peptide-antibody complex.

[0242] Such kits provide clinicians with a convenient and effective way to determine whether a subject has liver cancer or is at risk of liver cancer. Therefore, in certain embodiments, the kit also includes instructions for use. The product may also include a legend (e.g., a printed label or insert or other media (e.g., audio or video tape) describing the use of the product). The legend may be associated with the container (e.g., fixed to the container) and may describe the manner in which the assay should be performed, its indications, and other uses.

[0243] The following examples are provided only for illustrative purposes, and are not intended to limit the scope of the invention in any way. Although several embodiments have been provided in the present disclosure, it should be understood that the disclosed compositions and methods can be embodied in many other specific forms without departing from the spirit or scope of the present disclosure. The embodiments of the present invention should be considered to be illustrative and not restrictive, and the present invention is not limited to the details given herein. Various examples of variation, substitution and change can be determined by those skilled in the art and can be carried out without departing from the spirit and scope disclosed herein.

[0244] Example

[0245] Example 1: Preparation of rabbit monoclonal antibodies specific for AS-SPIK

[0246] Ten monoclonal antibodies described herein are produced from rabbits. In short, rabbits are immunized with recombinant AS-SPIK of different subgroups containing 23 additional amino acids (SEQ ID NO:81). Blood is tested by ELISA after three or four injections. In short, blood from immunized rabbits is reacted with plates coated with recombinant AS-SPIK. After washing, the plates are incubated with anti-rabbit antibodies labeled with HRP (horseradish peroxidase) to develop color, and the optical density is measured after reacting with substrate TMB. Rabbits that are positive in the test (producing antibodies) are killed. Monoclonal antibodies are then established, and the plates of partially purified AS-SPIK and NS-SPIK from pancreatic cells are further screened by ELISA using fixed plates from S2-3 cells (a cell line expressing a large amount of AS-SPIK through the integrated whole SPIKE gene, Lu et al. Tumor-associated protein SPIK / TATI suppresses serine protease dependent cell apoptosis, Apoptosis, 2008 13 (4), 483-494). Finally, 10 monoclonal antibodies that strongly bound AS-SPIK but not NS-SPIK were selected. Figure 1 It was shown that 10 clones named IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6, and IM-G7 exhibited high binding activity to AS-SPIK, whereas their binding activity to NS-SPIK was only at the background level as a negative control ( Figure 1 Negative control). This data indicates that the rabbit monoclonal antibodies identified herein only recognize AS-SPIK. In addition, all 10 antibodies can strongly bind to AS-SPIK and have similar binding affinities to AS-SPIK.

[0247] Example 2: Detection of antibody-antigen complexes in samples using IM-E2

[0248] The complexes formed by antibodies IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6 and IM-G7 with AS-SPIK in the culture medium of S2-3, the culture medium of PanC1, the serum of HCC patients and healthy people were measured by sandwich ELISA. In brief, the immune complexes of antibodies with AS-SPIK in different samples were formed by incubating the antibodies with the samples at 37°C for 0.5 hours. The mixture was then reacted with a 96-well plate immobilized with polyclonal anti-SPIK antibodies at 37°C for 1 hour. The plate was then incubated with an anti-rabbit antibody labeled with HRP at 37°C for 45 minutes. After reaction with the substrate TMB, the amount of the antibody-antigen complex was determined by optical density. Figure 2 The test results from IM-E2 are shown. Similar results were obtained for other antibodies. The results clearly show that IM-E2 can form an antibody-antigen complex with AS-SPIK ( Figure 2 ; S2-3: OD (1.389)), but did not form an antibody-antigen complex with NS-SPIK ( Figure 2 , PanC1: OD(0.061)). A high OD was also found in the sera of HCC patients (1.477), but not in healthy subjects (healthy: OD(0.106)), indicating that the antibodies described herein, such as IM-E2, can be used to detect serum AS-SPIK.

[0249] Example 3: Sequences of variable heavy and light chains of antibodies and their CDRs

[0250] The sequences of the variable heavy chains (VH) and variable light chains (VL) of all 10 antibodies were determined. Then their CDRs were identified. Table 1 shows the SEQ ID NOs of the VH and VL of the 10 antibodies: IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6 and IM-G7. Table 2 shows the SEQ ID NOs of the VH CDRs of these 10 antibodies, and Table 3 shows the SEQ ID NOs of the VL CDRs of these 10 antibodies.

[0251] Table 1: VH and VL SEQ ID NOs

[0252]

[0253]

[0254] Table 2: VH and HC CDR SEQ ID NOs

[0255]

[0256] Table 3: VL and LC CDR SEQ ID NOs

[0257]

[0258] Example 4: Consensus sequences of VH and VL sequences

[0259] The consensus sequence was determined by aligning the entire sequences of VH and VL of the 10 antibodies using a program called BioEdit Sequence Alignment Editor from the University of North Carolina. Figure 3 and Figure 4 The bottom line in the figure shows the sequence identity of the 10 antibodies in VH or VL. Obviously, the sequences of VH and VL in the 10 antibodies have high similarity. The sequence identity of the 10 antibodies ranges from 85% to 95%.

[0260] Example 5: Consensus sequence of CDR sequences

[0261] The consensus sequence of the CDR sequences of the variable heavy chain was determined using the above software. Figure 5 All 10 antibodies showed that they had high VH CDR1 similarity, with their identities ranging from 66% to 100%. The VH CRD2 of the 10 antibodies had 66% similarity and their identities ranged from 50% to 66%. In contrast, the VH CDR3 of the 10 antibodies had almost no similarity. Only one amino acid in the VH CDR3 was the same in all 10 antibodies. The consensus sequence showed that among the 10 antibodies, the CDRs of the VL had more similarity than the CDRs of the VH. The VL CDR1 of the 10 antibodies had 82% similarity, with their identities ranging from 73% to 82% ( Figure 6 ). The VL CDR2 of the 10 antibodies has 86% similarity, and their identity is 72%-86%. For VL CDR 3, the similarity of the 10 antibodies is 75%, and the identity is 45%-75% ( Figure 6 ).

[0262] Exemplary embodiments:

[0263] 1. An antibody or an antigen-binding fragment thereof that specifically binds to AS-SPIK, the antibody or the antigen-binding fragment thereof comprising:

[0264] (a) a heavy chain variable region, the heavy chain variable region comprising:

[0265] (i) a CDRH1 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 21 to 30; and / or

[0266] (ii) a CDRH2 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 31 to 40; and / or

[0267] (iii) a CDRH3 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 41 to 50; and

[0268] (b) a light chain variable region, the light chain variable region comprising:

[0269] (i) CDRL1 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 51 to 60; and / or

[0270] (ii) CDRL2 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 61 to 70; and / or

[0271] (iii) CDRL3 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 71 to 80.

[0272] 2. The antibody or antigen-binding fragment of embodiment 1, wherein the CDRH1, CDRH2 and CDRH3 sequences are present within a framework sequence, and wherein the CDRL1, CDRL2 and CDRL3 sequences are present within a framework sequence.

[0273] 3. The antibody or antigen-binding fragment of embodiment 2, wherein at least a portion of the framework sequence comprises a human consensus framework sequence.

[0274] 4. The antibody or antigen-binding fragment of any one of embodiments 1-3, comprising:

[0275] (a) a CDRH1 sequence selected from the group consisting of SEQ ID NOs: 21 to 30; and / or

[0276] (b) a CDRH2 sequence selected from the group consisting of SEQ ID NOs: 31 to 40; and / or

[0277] (c) a CDRH3 sequence selected from the group consisting of SEQ ID NOs: 41 to 50; and / or

[0278] (d) a CDRL1 sequence selected from the group consisting of SEQ ID NOs: 51 to 60; and / or

[0279] (e) a CDRL2 sequence selected from the group consisting of SEQ ID NOs: 61 to 70; and / or

[0280] (f) a CDRL3 sequence selected from the group consisting of SEQ ID NOs: 71 to 80.

[0281] 5. The antibody or antigen-binding fragment of embodiment 4, comprising:

[0282] (a) a CDRH1 sequence selected from the group consisting of SEQ ID NOs: 21 to 30;

[0283] (b) a CDRH2 sequence selected from the group consisting of SEQ ID NOs: 31 to 40;

[0284] (c) a CDRH3 sequence selected from the group consisting of SEQ ID NOs: 41 to 50;

[0285] (d) a CDRL1 sequence selected from the group consisting of SEQ ID NOs: 51 to 60;

[0286] (e) a CDRL2 sequence selected from the group consisting of SEQ ID NOs: 61 to 70; and

[0287] (f) a CDRL3 sequence selected from the group consisting of SEQ ID NOs: 71 to 80.

[0288] 6. The antibody or antigen-binding fragment of embodiment 5, comprising:

[0289] (a) the CDRH1 sequence of SEQ ID NO:21, the CDRH2 sequence of SEQ ID NO:31, the CDRH3 sequence of SEQ ID NO:41, the CDRL1 sequence of SEQ ID NO:51, the CDRL2 sequence of SEQ ID NO:61, and the CDRL3 sequence of SEQ ID NO:71; or

[0290] (b) the CDRH1 sequence of SEQ ID NO:22, the CDRH2 sequence of SEQ ID NO:32, the CDRH3 sequence of SEQ ID NO:42, the CDRL1 sequence of SEQ ID NO:52, the CDRL2 sequence of SEQ ID NO:62, and the CDRL3 sequence of SEQ ID NO:72; or

[0291] (c) the CDRH1 sequence of SEQ ID NO:23, the CDRH2 sequence of SEQ ID NO:33, the CDRH3 sequence of SEQ ID NO:43, the CDRL1 sequence of SEQ ID NO:53, the CDRL2 sequence of SEQ ID NO:63, and the CDRL3 sequence of SEQ ID NO:73; or

[0292] (d) the CDRH1 sequence of SEQ ID NO:24, the CDRH2 sequence of SEQ ID NO:34, the CDRH3 sequence of SEQ ID NO:44, the CDRL1 sequence of SEQ ID NO:54, the CDRL2 sequence of SEQ ID NO:64, and the CDRL3 sequence of SEQ ID NO:74; or

[0293] (e) the CDRH1 sequence of SEQ ID NO:25, the CDRH2 sequence of SEQ ID NO:35, the CDRH3 sequence of SEQ ID NO:45, the CDRL1 sequence of SEQ ID NO:55, the CDRL2 sequence of SEQ ID NO:65, and the CDRL3 sequence of SEQ ID NO:75; or

[0294] (f) the CDRH1 sequence of SEQ ID NO:26, the CDRH2 sequence of SEQ ID NO:36, the CDRH3 sequence of SEQ ID NO:46, the CDRL1 sequence of SEQ ID NO:56, the CDRL2 sequence of SEQ ID NO:66, and the CDRL3 sequence of SEQ ID NO:76; or

[0295] (g) the CDRH1 sequence of SEQ ID NO:27, the CDRH2 sequence of SEQ ID NO:37, the CDRH3 sequence of SEQ ID NO:47, the CDRL1 sequence of SEQ ID NO:57, the CDRL2 sequence of SEQ ID NO:67, and the CDRL3 sequence of SEQ ID NO:77; or

[0296] (h) the CDRH1 sequence of SEQ ID NO:28, the CDRH2 sequence of SEQ ID NO:38, the CDRH3 sequence of SEQ ID NO:48, the CDRL1 sequence of SEQ ID NO:58, the CDRL2 sequence of SEQ ID NO:68, and the CDRL3 sequence of SEQ ID NO:78; or

[0297] (i) the CDRH1 sequence of SEQ ID NO:29, the CDRH2 sequence of SEQ ID NO:39, the CDRH3 sequence of SEQ ID NO:49, the CDRL1 sequence of SEQ ID NO:59, the CDRL2 sequence of SEQ ID NO:69, and the CDRL3 sequence of SEQ ID NO:79; or

[0298] (j) the CDRH1 sequence of SEQ ID NO:30, the CDRH2 sequence of SEQ ID NO:40, the CDRH3 sequence of SEQ ID NO:50, the CDRL1 sequence of SEQ ID NO:60, the CDRL2 sequence of SEQ ID NO:70, and the CDRL3 sequence of SEQ ID NO:80.

[0299] 7. The antibody or antigen-binding fragment of any one of embodiments 1-3, comprising a heavy chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 1 to 10 and / or a light chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 11 to 20.

[0300] 8. The antibody or antigen-binding fragment of embodiment 7, comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 1 to 10 and / or a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 11 to 20.

[0301] 9. The antibody or antigen-binding fragment of embodiment 8, comprising:

[0302] (a) the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 11; or

[0303] (b) the heavy chain variable region sequence of SEQ ID NO: 2 and the light chain variable region sequence of SEQ ID NO: 12; or

[0304] (c) the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 13; or

[0305] (d) the heavy chain variable region sequence of SEQ ID NO: 4 and the light chain variable region sequence of SEQ ID NO: 14; or

[0306] (e) the heavy chain variable region sequence of SEQ ID NO: 5 and the light chain variable region sequence of SEQ ID NO: 15; or

[0307] (f) the heavy chain variable region sequence of SEQ ID NO: 6 and the light chain variable region sequence of SEQ ID NO: 16; or

[0308] (g) the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 17; or

[0309] (h) the heavy chain variable region sequence of SEQ ID NO: 8 and the light chain variable region sequence of SEQ ID NO: 18; or

[0310] (i) the heavy chain variable region sequence of SEQ ID NO: 9 and the light chain variable region sequence of SEQ ID NO: 19; or

[0311] (j) the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 20.

[0312] 10. An isolated anti-AS-SPIK antibody or antigen-binding fragment thereof that specifically binds to AS-SPIK and does not bind to NS-SPIK, the isolated anti-AS-SPIK antibody or antigen-binding fragment thereof comprising:

[0313] (a) a heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 sequences in a human VH framework, wherein the CDRH sequence is a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 21-50; and

[0314] (b) a light chain variable region comprising CDRL1, CDRL2 and CDRL3 sequences in a human VL framework, wherein the CDRL sequence is a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 51-80.

[0315] 11. The antibody or antigen-binding fragment of embodiment 10, comprising:

[0316] (a) a heavy chain variable region comprising a CDRH1, CDRH2 and CDRH3 sequence in a human VH framework, wherein the CDRH sequence is selected from the group consisting of SEQ ID NOs: 21 to 50; and

[0317] (b) a light chain variable region comprising CDRL1, CDRL2 and CDRL3 sequences in a human VL framework, wherein the CDRL sequences are selected from the group consisting of SEQ ID NOs: 51 to 80.

[0318] 12. An isolated anti-AS-SPIK antibody or antigen-binding fragment thereof that specifically binds to AS-SPIK and does not bind to NS-SPIK, the isolated anti-AS-SPIK antibody or antigen-binding fragment thereof comprising:

[0319] (a) the CDRH1 sequence of SEQ ID NO:21, the CDRH2 sequence of SEQ ID NO:31, and the CDRH3 sequence of SEQ ID NO:41 in a human VH framework, and the CDRL1 sequence of SEQ ID NO:51, the CDRL2 sequence of SEQ ID NO:61, and the CDRL3 sequence of SEQ ID NO:71 in a human VL framework; or

[0320] (b) the CDRH1 sequence of SEQ ID NO:22, the CDRH2 sequence of SEQ ID NO:32, and the CDRH3 sequence of SEQ ID NO:42 in a human VH framework, and the CDRL1 sequence of SEQ ID NO:52, the CDRL2 sequence of SEQ ID NO:62, and the CDRL3 sequence of SEQ ID NO:72 in a human VL framework; or

[0321] (c) the CDRH1 sequence of SEQ ID NO:23, the CDRH2 sequence of SEQ ID NO:33, and the CDRH3 sequence of SEQ ID NO:43 in a human VH framework, and the CDRL1 sequence of SEQ ID NO:53, the CDRL2 sequence of SEQ ID NO:63, and the CDRL3 sequence of SEQ ID NO:73 in a human VL framework; or

[0322] (d) the CDRH1 sequence of SEQ ID NO:24, the CDRH2 sequence of SEQ ID NO:34, and the CDRH3 sequence of SEQ ID NO:44 in the human VH framework, and the CDRL1 sequence of SEQ ID NO:54, the CDRL2 sequence of SEQ ID NO:64, and the CDRL3 sequence of SEQ ID NO:74 in the human VL framework; or

[0323] (e) the CDRH1 sequence of SEQ ID NO:25, the CDRH2 sequence of SEQ ID NO:35, and the CDRH3 sequence of SEQ ID NO:45 in the human VH framework, and the CDRL1 sequence of SEQ ID NO:55, the CDRL2 sequence of SEQ ID NO:65, and the CDRL3 sequence of SEQ ID NO:75 in the human VL framework; or

[0324] (f) the CDRH1 sequence of SEQ ID NO:26, the CDRH2 sequence of SEQ ID NO:36, and the CDRH3 sequence of SEQ ID NO:46 in the human VH framework, and the CDRL1 sequence of SEQ ID NO:56, the CDRL2 sequence of SEQ ID NO:66, and the CDRL3 sequence of SEQ ID NO:76 in the human VL framework; or

[0325] (g) the CDRH1 sequence of SEQ ID NO:27, the CDRH2 sequence of SEQ ID NO:37, and the CDRH3 sequence of SEQ ID NO:47 in the human VH framework, and the CDRL1 sequence of SEQ ID NO:57, the CDRL2 sequence of SEQ ID NO:67, and the CDRL3 sequence of SEQ ID NO:77 in the human VL framework; or

[0326] (h) the CDRH1 sequence of SEQ ID NO:28, the CDRH2 sequence of SEQ ID NO:38, and the CDRH3 sequence of SEQ ID NO:48 in the human VH framework, and the CDRL1 sequence of SEQ ID NO:58, the CDRL2 sequence of SEQ ID NO:68, and the CDRL3 sequence of SEQ ID NO:78 in the human VL framework; or

[0327] (i) the CDRH1 sequence of SEQ ID NO:29, the CDRH2 sequence of SEQ ID NO:39, and the CDRH3 sequence of SEQ ID NO:49 in a human VH framework, and the CDRL1 sequence of SEQ ID NO:59, the CDRL2 sequence of SEQ ID NO:69, and the CDRL3 sequence of SEQ ID NO:79 in a human VL framework; or

[0328] (j) the CDRH1 sequence of SEQ ID NO:30, the CDRH2 sequence of SEQ ID NO:40, and the CDRH3 sequence of SEQ ID NO:50 in the human VH framework, and the CDRL1 sequence of SEQ ID NO:60, the CDRL2 sequence of SEQ ID NO:70, and the CDRL3 sequence of SEQ ID NO:80 in the human VL framework.

[0329] 13. The antibody or antigen-binding fragment of any one of embodiments 1 to 12, which is multispecific.

[0330] 14. The antibody or antigen-binding fragment of embodiment 13, which is bispecific.

[0331] 15. The antibody or antigen-binding fragment of embodiment 14, wherein the antibody or antigen-binding fragment has binding affinity for effector cells.

[0332] 16. The antibody or antigen-binding fragment of embodiment 14, wherein the antibody or antigen-binding fragment has binding affinity for a T cell antigen.

[0333] 17. The antibody or antigen-binding fragment of embodiment 16, wherein the antibody or antigen-binding fragment has binding affinity for CD3.

[0334] 18. The antibody or antigen-binding fragment of any one of embodiments 1-17, which is monoclonal.

[0335] 19. The antibody or antigen-binding fragment of any one of embodiments 1 to 12, wherein the antibody or antigen-binding fragment is in the form of a CAR-T.

[0336] 20. An immunoconjugate comprising the antibody of any one of embodiments 1-18 covalently linked to a cytotoxic agent.

[0337] 21. The immunoconjugate of embodiment 20, wherein the cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, a radioisotope, and a nucleolytic enzyme.

[0338] 22. An immunoconjugate having the formula Ab-(LD)p, wherein:

[0339] (a) Ab is an antibody according to any one of embodiments 1-18;

[0340] (b) L is a linker;

[0341] (c) D is a drug moiety; and

[0342] (d) p is an integer ranging from 1 to 8.

[0343] 23. The immunoconjugate of embodiment 22, wherein D is selected from the group consisting of maytansinoids, auristatins, and dolastatins.

[0344] 24. The immunoconjugate of embodiment 22, wherein L comprises one or more linkers selected from the group consisting of: 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 4-(2-pyridylthio)pentanoic acid N-succinimidyl ester (SPP), 4-(N-maleimidomethyl)cyclohexane-1carboxylic acid N-succinimidyl ester (SMCC), 4-(2-pyridyldithio)butyric acid-N-hydroxysuccinimidyl ester (SPDB), and (4-iodo-acetyl)aminobenzoic acid N-succinimidyl ester (SIAB).

[0345] 25. A pharmaceutical composition comprising the antibody, antigen-binding fragment or immunoconjugate of any one of embodiments 1 to 24.

[0346] 26. A method for treating a disorder characterized by expression of AS-SPIK, the method comprising administering the antibody, antigen-binding fragment or immunoconjugate of any one of embodiments 1 to 24 or the pharmaceutical composition of embodiment 25 to a subject suffering from the disorder.

[0347] 27. Use of the antibody, antigen-binding fragment or immunoconjugate of any one of embodiments 1 to 24 for the preparation of a medicament for treating a disorder characterized by expression of AS-SPIK.

[0348] 28. The antibody, antigen binding fragment or immunoconjugate of any one of embodiments 1 to 19 for use in treating a disorder characterized by expression of AS-SPIK.

[0349] 29. The method or use of any one of embodiments 26-28, wherein the disorder is a liver disorder.

[0350] 30. The method or use of embodiment 29, wherein the liver disorder is hepatocellular carcinoma.

[0351] 31. The method or use of embodiment 29, wherein the liver disorder is intrahepatic cholangiocarcinoma.

[0352] 32. The method or use of embodiment 29, wherein the liver disorder is a viral infection.

[0353] 33. The method or use of embodiment 29, wherein the liver disorder is an inflammatory liver disorder.

[0354] 34. The method or use of embodiment 33, wherein the inflammatory liver disorder is cirrhosis.

[0355] 35. A polynucleotide encoding the antibody or antigen-binding fragment of any one of embodiments 1 to 19.

[0356] 36. A vector comprising the polynucleotide of embodiment 35.

[0357] 37. A host cell comprising the vector of embodiment 36.

[0358] 38. A method of producing the antibody, antigen-binding fragment, or immunoconjugate of any one of embodiments 1 to 24, the method comprising growing the host cell of embodiment 37 under conditions that allow expression of the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the cell.

[0359] 39. A diagnostic method for determining whether a subject has, or is at risk of developing, a disorder characterized by expression of AS-SPIK, the method comprising:

[0360] (a) contacting a biological test sample from the subject with the AS-SPIK antibody or antigen-binding fragment according to any one of embodiments 1-19 to produce an AS-SPIK-antibody complex;

[0361] (b) detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; and

[0362] (c) comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject suffers from the disorder or is at risk of developing the disorder.

[0363] 40. A diagnostic method for determining whether a subject has, or is at risk of developing, a disorder characterized by expression of AS-SPIK, the method comprising:

[0364] (a) contacting a biological test sample from the subject with a first antibody or antigen-binding fragment that specifically binds to SPIKE to form a SPIKE-antibody complex;

[0365] (b) contacting the SPIKE-antibody complex with the AS-SPIK antibody or antigen-binding fragment according to any one of embodiments 1-19 to produce an AS-SPIK-antibody complex;

[0366] (c) detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; and

[0367] (d) comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject suffers from the disorder or is at risk of developing the disorder.

[0368] 41. The diagnostic method of embodiment 39 or 40, wherein the antibody or antigen-binding fragment comprises a detectable label.

[0369] 42. The diagnostic method of embodiment 39 or 40, wherein the disorder is a liver disorder.

[0370] 43. The diagnostic method of embodiment 42, wherein the liver disorder is selected from the group consisting of hepatocellular carcinoma, intrahepatic bile duct carcinoma, viral infection of the liver, inflammatory disorder of the liver, and cirrhosis.

[0371] 44. A kit comprising the antibody, antigen-binding fragment, or immunoconjugate of any one of embodiments 1-24.

[0372] 45. The kit of claim 44, further comprising an antibody or antigen-binding fragment that specifically binds to SPIKE.

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to AS-SPIK, the antibody or the antigen-binding fragment thereof comprising: (a) a heavy chain variable region, the heavy chain variable region comprising: (i) a CDRH1 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 21 to 30; and / or (ii) a CDRH2 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 31 to 40; and / or (iii) a CDRH3 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 41 to 50; and (b) a light chain variable region, the light chain variable region comprising: (i) CDRL1 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 51 to 60; and / or (ii) CDRL2 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 61 to 70; and / or (iii) CDRL3 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 71 to 80.

2. The antibody or antigen-binding fragment of claim 1, wherein the CDRH1, CDRH2 and CDRH3 sequences are present within a framework sequence, and wherein the CDRL1, CDRL2 and CDRL3 sequences are present within a framework sequence.

3. The antibody or antigen-binding fragment of claim 2, wherein at least a portion of the framework sequence comprises a human consensus framework sequence.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, comprising: (a) a CDRH1 sequence selected from the group consisting of SEQ ID NOs: 21 to 30; and / or (b) a CDRH2 sequence selected from the group consisting of SEQ ID NOs: 31 to 40; and / or (c) a CDRH3 sequence selected from the group consisting of SEQ ID NOs: 41 to 50; and / or (d) a CDRL1 sequence selected from the group consisting of SEQ ID NOs: 51 to 60; and / or (e) a CDRL2 sequence selected from the group consisting of SEQ ID NOs: 61 to 70; and / or (f) a CDRL3 sequence selected from the group consisting of SEQ ID NOs: 71 to 80.

5. The antibody or antigen-binding fragment of claim 4, comprising: (a) a CDRH1 sequence selected from the group consisting of SEQ ID NOs: 21 to 30; (b) a CDRH2 sequence selected from the group consisting of SEQ ID NOs: 31 to 40; (c) a CDRH3 sequence selected from the group consisting of SEQ ID NOs: 41 to 50; (d) a CDRL1 sequence selected from the group consisting of SEQ ID NOs: 51 to 60; (e) a CDRL2 sequence selected from the group consisting of SEQ ID NOs: 61 to 70; and (f) a CDRL3 sequence selected from the group consisting of SEQ ID NOs: 71 to 80.

6. The antibody or antigen-binding fragment of claim 5, comprising: (a) the CDRH1 sequence of SEQ ID NO:21, the CDRH2 sequence of SEQ ID NO:31, the CDRH3 sequence of SEQ ID NO:41, the CDRL1 sequence of SEQ ID NO:51, the CDRL2 sequence of SEQ ID NO:61, and the CDRL3 sequence of SEQ ID NO:71; or (b) the CDRH1 sequence of SEQ ID NO:22, the CDRH2 sequence of SEQ ID NO:32, the CDRH3 sequence of SEQ ID NO:42, the CDRL1 sequence of SEQ ID NO:52, the CDRL2 sequence of SEQ ID NO:62, and the CDRL3 sequence of SEQ ID NO:72; or (c) the CDRH1 sequence of SEQ ID NO:23, the CDRH2 sequence of SEQ ID NO:33, the CDRH3 sequence of SEQ ID NO:43, the CDRL1 sequence of SEQ ID NO:53, the CDRL2 sequence of SEQ ID NO:63, and the CDRL3 sequence of SEQ ID NO:73; or (d) the CDRH1 sequence of SEQ ID NO:24, the CDRH2 sequence of SEQ ID NO:34, the CDRH3 sequence of SEQ ID NO:44, the CDRL1 sequence of SEQ ID NO:54, the CDRL2 sequence of SEQ ID NO:64, and the CDRL3 sequence of SEQ ID NO:74; or (e) the CDRH1 sequence of SEQ ID NO:25, the CDRH2 sequence of SEQ ID NO:35, the CDRH3 sequence of SEQ ID NO:45, the CDRL1 sequence of SEQ ID NO:55, the CDRL2 sequence of SEQ ID NO:65, and the CDRL3 sequence of SEQ ID NO:75; or (f) the CDRH1 sequence of SEQ ID NO:26, the CDRH2 sequence of SEQ ID NO:36, the CDRH3 sequence of SEQ ID NO:46, the CDRL1 sequence of SEQ ID NO:56, the CDRL2 sequence of SEQ ID NO:66, and the CDRL3 sequence of SEQ ID NO:76; or (g) the CDRH1 sequence of SEQ ID NO:27, the CDRH2 sequence of SEQ ID NO:37, the CDRH3 sequence of SEQ ID NO:47, the CDRL1 sequence of SEQ ID NO:57, the CDRL2 sequence of SEQ ID NO:67, and the CDRL3 sequence of SEQ ID NO:77; or (h) the CDRH1 sequence of SEQ ID NO:28, the CDRH2 sequence of SEQ ID NO:38, the CDRH3 sequence of SEQ ID NO:48, the CDRL1 sequence of SEQ ID NO:58, the CDRL2 sequence of SEQ ID NO:68, and the CDRL3 sequence of SEQ ID NO:78; or (i) the CDRH1 sequence of SEQ ID NO:29, the CDRH2 sequence of SEQ ID NO:39, the CDRH3 sequence of SEQ ID NO:49, the CDRL1 sequence of SEQ ID NO:59, the CDRL2 sequence of SEQ ID NO:69, and the CDRL3 sequence of SEQ ID NO:79; or (j) the CDRH1 sequence of SEQ ID NO:30, the CDRH2 sequence of SEQ ID NO:40, the CDRH3 sequence of SEQ ID NO:50, the CDRL1 sequence of SEQ ID NO:60, the CDRL2 sequence of SEQ ID NO:70, and the CDRL3 sequence of SEQ ID NO:

80.

7. The antibody or antigen-binding fragment of any one of claims 1-3, comprising a heavy chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 1 to 10 and / or a light chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 11 to 20.

8. The antibody or antigen-binding fragment of claim 7, comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 1 to 10 and / or a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 11 to 20.

9. The antibody or antigen-binding fragment of claim 8, comprising: (a) the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 11; or (b) the heavy chain variable region sequence of SEQ ID NO: 2 and the light chain variable region sequence of SEQ ID NO: 12; or (c) the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 13; or (d) the heavy chain variable region sequence of SEQ ID NO: 4 and the light chain variable region sequence of SEQ ID NO: 14; or (e) the heavy chain variable region sequence of SEQ ID NO: 5 and the light chain variable region sequence of SEQ ID NO: 15; or (f) the heavy chain variable region sequence of SEQ ID NO: 6 and the light chain variable region sequence of SEQ ID NO: 16; or (g) the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 17; or (h) the heavy chain variable region sequence of SEQ ID NO: 8 and the light chain variable region sequence of SEQ ID NO: 18; or (i) a heavy chain variable region sequence of SEQ ID NO: 9 and a light chain variable region sequence of SEQ ID NO: 19; or (j) the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO:

20.

10. An isolated anti-AS-SPIK antibody or antigen-binding fragment thereof that specifically binds to AS-SPIK and does not bind to NS-SPIK, the isolated anti-AS-SPIK antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 sequences in a human VH framework, wherein the CDRH sequence is a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 21-50; and (b) a light chain variable region comprising CDRL1, CDRL2 and CDRL3 sequences in a human VL framework, wherein the CDRL sequence is a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 51-80.

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