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

Abstract

To provide an anti-AS-SPIK antibody that blocks the binding between SPIK (serine protease inhibitor Kazal type) and granzyme A.SOLUTION: Anti-AS-SPIK antibodies are disclosed, along with methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to diagnose and / or treat disorders characterized by the expression of AS-SPIK (e.g., liver cancer). Diagnostic methods and kits comprising the anti-AS-SPIK antibodies are also disclosed.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 871,565, filed on July 8, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Statement of Government Rights 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.

[0003] Anti - AS - SPIK antibodies are disclosed, together with methods for making such antibodies, compositions including such antibodies and pharmaceutical compositions, and their use for diagnosing and / or treating disorders (such as liver cancer) characterized by the expression of AS - SPIK. Diagnostic methods and kits comprising such anti - AS - SPIK antibodies are also disclosed.

Background Art

[0004] The liver is one of the largest organs in the body. The liver has many functions, including the production of enzymes necessary for food digestion, bile production, regulation of glycogen storage, synthesis of plasma proteins, production of hormones, and detoxification of various metabolites. Liver disorders include liver cancers such as hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), viral infections, cirrhosis, and other inflammatory disorders of the liver, and millions of people worldwide suffer from liver disorders. For example, in the United States, more than 5 million people and worldwide more than 450 million people are infected with hepatitis B virus (HBV) and hepatitis C virus (HCV), and more than 30% of these infected people are at high risk of 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., Clinical gastroenterology and hepatology 2015;13(4):805-807. Despite advances in diagnosis and treatment, liver cancer remains a major cause of both morbidity and death. El-Serag, The New England journal of medicine 2011;365(12):1118-1127. The 5-year survival rate of primary liver cancer, that is, cancer that first occurs in the liver, is less than 10%. However, if liver cancer is detected early and is in the most treatable stage, the survival rate increases to almost 40%. El-Serag et al., Therapeutic advances in gastroenterology 2011;4(1): 5-10. Patients with early liver cancer may have few or no symptoms. Current detection methods such as serological methods, ultrasound, computed tomography (CT) scans, magnetic resonance imaging (MRI), and angiography may be unreliable due to low sensitivity and the possibility of technician error. Imaging diagnostic techniques can be costly and may be less accurate for detecting smaller early 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 differentiating between benign and malignant tumors, but it is invasive and requires surgical procedures. Lok et al., Gastroenterology 2001;122 (7):2092-2093. There is still a continuing need for new methods for diagnosing and treating liver cancer, especially for patients with cirrhosis, viral infections of the liver, and inflammatory disorders.

Summary of the Invention

[0005] Aspects of the present invention include (a) a heavy chain variable region comprising (i) CDRH1 having no more than 2 substitutions in any of the amino acid sequences of SEQ ID NOs: 21 to 30, and / or (ii) CDRH2 having no more than 2 substitutions in any of the amino acid sequences of SEQ ID NOs: 31 to 40, and / or (iii) CDRH3 having no more than 2 substitutions in any of the amino acid sequences of SEQ ID NOs: 41 to 50 and the above heavy chain variable region, (b) a light chain variable region comprising (i) CDRL1 having no more than 2 substitutions in any of the amino acid sequences of SEQ ID NOs: 51 to 60, and / or (ii) CDRL2 having no more than 2 substitutions in any of the amino acid sequences of SEQ ID NOs: 61 to 70, and / or (iii) CDRL3 having no more than 2 substitutions in any of the amino acid sequences of SEQ ID NOs: 71 to 80 and the above light chain variable region and an antibody that specifically binds to AS-SPIK, or an antigen-binding fragment thereof.

[0006] In some embodiments, the CDRH1 sequence, CDRH2 sequence, and CDRH3 sequence are present within a framework sequence, and the CDRL1 sequence, CDRL2 sequence, and CDRL3 sequence are present within a framework sequence. In some embodiments, at least a portion of the framework sequence comprises a human consensus framework sequence.

[0007] In some embodiments, the antibody or antigen-binding fragment (a) a CDRH1 sequence selected from the group consisting of SEQ ID NOs: 21-30, and / or (b) a CDRH2 sequence selected from the group consisting of SEQ ID NOs: 31-40, and / or (c) a CDRH3 sequence selected from the group consisting of SEQ ID NOs: 41-50, and / or (d) a CDRL1 sequence selected from the group consisting of SEQ ID NOs: 51-60, and / or (e) a CDRL2 sequence selected from the group consisting of SEQ ID NOs: 61-70, and / or (f) a CDRL3 sequence selected from the group consisting of SEQ ID NOs: 71-80 comprises.

[0008] In some embodiments, the antibody or antigen-binding fragment (a) a CDRH1 sequence selected from the group consisting of SEQ ID NOs: 21-30, and (b) a CDRH2 sequence selected from the group consisting of SEQ ID NOs: 31-40, and (c) a CDRH3 sequence selected from the group consisting of SEQ ID NOs: 41-50, and (d) a CDRL1 sequence selected from the group consisting of SEQ ID NOs: 51-60, and (e) a CDRL2 sequence selected from the group consisting of SEQ ID NOs: 61-70, and (f) a CDRL3 sequence selected from the group consisting of SEQ ID NOs: 71-80 and comprises.

[0009] In some embodiments, the antibody or antigen-binding fragment (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 comprising.

[0010] 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: 1-10 and / or a light-chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 11-20. 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: 1-10 and / or a light-chain variable region sequence selected from the group consisting of SEQ ID NOs: 11-20. In some embodiments, the antibody or antigen-binding fragment is (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) 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 comprising.

[0011] Aspects of the present invention are (a) A heavy chain variable region comprising a CDRH1 sequence, a CDRH2 sequence, and a CDRH3 sequence in a human VH framework, wherein the CDRH sequences have no more than 2 substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 21 to 50, and (b) A light chain variable region comprising a CDRL1 sequence, a CDRL2 sequence, and a CDRL3 sequence in a human VL framework, wherein the CDRL sequences have no more than 2 substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 51 to 80, and An isolated anti-AS-SPIK antibody that specifically binds to AS-SPIK and does not bind to NS-SPIK, or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is (a) A heavy chain variable region comprising a CDRH1 sequence, a CDRH2 sequence, and a CDRH3 sequence in a human VH framework, wherein the CDRH sequences are selected from the group consisting of SEQ ID NOs: 21 to 50, and (b) A light chain variable region comprising a CDRL1 sequence, a CDRL2 sequence, and a CDRL3 sequence in a human VL framework, wherein the CDRL sequences are selected from the group consisting of SEQ ID NOs: 51 to 80, and Comprising.

[0012] Aspects of the present invention are (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 (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 (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 the 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 the human VL framework, or (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 (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 (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 (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 (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 (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 the 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 the 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 Comprising an isolated anti-AS-SPIK antibody that specifically binds to AS-SPIK and does not bind to NS-SPIK, or an antigen-binding fragment thereof.

[0013] 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.

[0014] In some embodiments, the antibody or antigen-binding fragment is monoclonal. In some embodiments, the antibody or antigen-binding fragment is in a CAR-T format.

[0015] Aspects of the invention include an immunocomplex comprising an antibody described herein covalently linked to a cytotoxic agent. In some embodiments, the cytotoxic agent is selected from the group consisting of toxins, chemotherapeutic agents, drug moieties, antibiotics, radioisotopes, and nuclease enzymes.

[0016] Aspects of the invention are of the formula Ab-(L-D)p, wherein (a) Ab is an antibody described herein, (b) L is a linker, (c) D is a drug moiety, (d) p is an integer in the range of 1 to 8) It includes immune complexes having []. In some embodiments, D is selected from the group consisting of maytansinoids, auristatins, and dolostatins. In some embodiments, L includes one or more linkers selected from the group consisting of 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyl oxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimide ester of 4-(2-pyridyldithio)butyric acid (SPDB), and N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB).

[0017] Aspects of the invention include pharmaceutical compositions comprising the antibodies, antigen-binding fragments, or immune complexes described herein.

[0018] Aspects of the invention include methods of treating disorders characterized by the expression of AS-SPIK, the methods including administering to a subject having the disorder an antibody, antigen-binding fragment, or immune complex described herein, or a pharmaceutical composition described herein.

[0019] Aspects of the invention include the use of an antibody, antigen-binding fragment, or immune complex described herein in the manufacture of a medicament for the treatment of a disorder characterized by the expression of AS-SPIK. Aspects of the invention include an antibody, antigen-binding fragment, or immune complex described herein for use in the treatment of a disorder characterized by the expression of AS-SPIK.

[0020] 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 cholangiocarcinoma. 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.

[0021] Aspects of the invention include a polynucleotide encoding an antibody or antigen-binding fragment described herein, a vector comprising the polynucleotide, or a host cell comprising the vector.

[0022] Aspects of the invention include a method for producing an antibody, antigen-binding fragment, or immune complex described herein, the method comprising culturing a host cell 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.

[0023] Aspects of the invention include a diagnostic method for determining whether a subject has a disorder characterized by AS-SPIK expression or is at risk of developing the disorder, (a) contacting a biological test sample from the subject with an AS-SPIK antibody or antigen-binding fragment described herein to generate an AS-SPIK-antibody complex; (b) detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; (c) comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject has the disorder or is at risk of developing the disorder; and including the above method.

[0024] Aspects of the invention include a diagnostic method for determining whether a subject has a disorder characterized by AS-SPIK expression or is at risk of developing the disorder, (a) Contacting a biological test sample derived from the subject with a first antibody or antigen-binding fragment that specifically binds to SPIK to form an SPIK-antibody complex; (b) Contacting the SPIK-antibody complex with the AS-SPIK antibody or antigen-binding fragment described herein to generate an AS-SPIK-antibody complex; (c) Detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; (d) Comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject has the disorder or is at risk of developing the disorder. Including the above method.

[0025] 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 cholangiocarcinoma, viral liver infection, inflammatory liver disorder, and cirrhosis.

[0026] Aspects of the invention include kits comprising the antibodies, antigen-binding fragments, or immunocomplexes described herein. In some embodiments, the kit further comprises an antibody or antigen-binding fragment that specifically binds to SPIK.

Brief Description of the Drawings

[0027]

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Modes for Carrying Out the Invention

[0028] The practice of the present invention, unless otherwise indicated, uses conventional techniques within the skill of the art in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology. Such techniques are described in detail in the literature such as “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “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).

[0029] Where a range of values is given, each value between the upper and lower limits of that range, to the tenth of the lowest order, and any other recited or entrapped value in the recited range, is to be understood as being embraced within the invention, unless a clear contrary indication by context is given. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges above, and be embraced within the invention, provided that any limit in the recited range is specifically excluded. Where the recited range includes one or both of the limits, ranges excluding either or both of those included limits are also embraced within the invention.

[0030] Unless otherwise expressly indicated, antibody residues in this specification are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0031] In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0032] All references cited through this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0033] Definitions of Terms For the purposes of understanding this specification, the following definitions apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. Where any definition set forth herein conflicts with any document incorporated by reference herein, the definition set forth below shall control.

[0034] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. This term encompasses in detail linear epitopes and conformational epitopes. This term encompasses any molecular determinant that can specifically bind to an antibody. In certain embodiments, an epitope determinant includes chemically active surface groups of a molecule such as an amino acid, sugar side chain, phosphoryl, or sulfonyl, and in certain embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of an antigen to which an antibody binds. A "binding region" is the region on a binding target that is bound by a binding molecule.

[0035] "Epitope mapping" is the process of identifying the binding site of an antibody on a target antigen, i.e., the epitope. An antibody epitope may be a linear epitope or a conformational epitope. A linear epitope is formed by a continuous sequence of amino acids in a protein. A conformational epitope is formed from amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0036] As defined herein, "epitope binning" is a process of classifying antibodies, which is based on the epitopes recognized by the antibodies. More specifically, epitope binning includes methods and systems for distinguishing the epitope recognition characteristics of different antibodies, combined with a computational process for clustering antibodies based on their epitope recognition characteristics and identifying antibodies with distinct binding specificities.

[0037] If two antibodies recognize the same or sterically overlapping epitopes, the antibody binds to the reference antibody as an "essentially identical epitope." The most widely used and rapid method for determining whether two antibodies bind to the same or sterically overlapping epitopes is a competition assay that can be configured in any number of different ways using either a labeled antigen or a labeled antibody. Typically, the antigen is immobilized on a 96-well plate, and a radiolabel or enzyme label is used to measure the ability of an unlabeled antibody to block the binding of the labeled antibody.

[0038] As used herein, "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence as compared to the original amino acid sequence, which change is due to a sequence change in which the amino acid residue / position is involved. For example, common modifications include substitution of the residue (or at the position) by another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more (generally 5 or less than 3) amino acids adjacent to the residue / position, and deletion of the residue / position. "Amino acid substitution" or variations thereof refers to substituting an existing amino acid residue in a given (original) amino acid sequence with a different amino acid residue. Generally and preferably, the modification results in a change in at least one physical or biochemical activity of the mutant polypeptide as compared to the polypeptide comprising the original (or "wild-type") amino acid sequence. For example, in the case of an antibody, the physical or biochemical activity that changes may be the binding affinity, binding ability, and / or binding effect for the target molecule.

[0039] The term "antibody" encompasses monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), single-chain molecules, and antibody fragments (e.g., Fab, F(ab’)2, and Fv). As used herein, the term "immunoglobulin" (Ig) is used synonymously with "antibody". The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Unless otherwise specified, the term "antibody" is used herein in its broadest sense and includes, in particular, all isotypes, subclasses, and forms of antibodies, including IgG, IgM, IgA, IgD, and IgE antibodies and their fragments, preferably antigen-binding fragments.

[0040] Unless otherwise expressly stated, the term "antibody" includes, in particular, native human and non-human IgG1, IgG2 (IgG2a, IgG2b), IgG3, IgG4, IgE, IgA, IgD, and IgM antibodies, including naturally occurring variants.

[0041] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Further, in contrast to conventional (polyclonal) antibody preparations that typically include various antibodies targeting various determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed to require that the antibody be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first reported by Kohler et al. (1975) Nature 256:495, or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The above "monoclonal antibodies" may 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.

[0042] Monoclonal antibodies as used herein include, in particular, antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species, while the remaining portion of the chain(s) is derived from a different species, and "chimeric" antibodies (immunoglobulins) (U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855) in which the corresponding sequence in a fragment of such an antibody is identical or homologous (as long as the desired biological activity is exhibited).

[0043] The "humanized" form of a non-human (e.g., mouse) antibody is an antibody that contains minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues derived from the hypervariable regions of the recipient in the human immunoglobulin are replaced by residues derived from the hypervariable regions of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate that have the desired specificity, affinity, and capacity. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are also replaced with corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are not present in the recipient antibody or donor antibody. These modifications are made to further improve the performance of the antibody. Generally, the humanized antibody will comprise substantially all of at least one, and generally two, variable domains, in which all or substantially all of the hypervariable loops correspond to loops of non-human immunoglobulins and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. Optionally, the humanized antibody will also comprise at least a portion of the immunoglobulin constant region (Fc), generally the constant region 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.

[0044] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignments for determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this specification, the value of percent amino acid sequence identity is obtained using the sequence comparison computer program ALIGN-2.

[0045] 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 may be used as the query sequence, and a fragment of an AS-SPIK polypeptide or NS-SPIK polypeptide may be used as the subject sequence. Similarly, a fragment of an AS-SPIK polypeptide or NS-SPIK polypeptide may be used as the query sequence, and a biologically active variant thereof may be used as the subject sequence.

[0046] As used herein, the term "consensus sequence" refers to an amino acid residue sequence or nucleotide residue sequence that represents the most frequently occurring residue at each position in a sequence alignment after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence match, without considering any conservative substitutions as part of the sequence identity.

[0047] As used herein, an "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment in a recombinant host cell. Contaminant components of the natural environment of the antibody are substances that would interfere with diagnostic or therapeutic uses for the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes, as well as undesirable by-products of production. In preferred embodiments, an isolated antibody herein is purified to (1) 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, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of an amino acid sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue staining, preferably silver staining. Ordinarily, an isolated antibody will be prepared by at least one purification step.

[0048] In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H chain and L chain also have regularly spaced intrachain disulfide cross-links. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) in each of the α and γ chains, and four CH domains in the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain at the other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form the interface between the light and heavy chain variable domains. The pairing of VH and VL with each other forms a single antigen-binding site.

[0049] As used herein, the term "polypeptide" is used in its broadest sense and encompasses peptide sequences. The term "peptide" generally refers to a linear molecular chain of amino acids containing up to about 60, preferably up to about 30, amino acids covalently linked by peptide bonds.

[0050] The term "specific binding" or "binds specifically to" or "is specific for" refers to the binding of an antibody to an epitope on a target antigen, e.g., a specific polypeptide, peptide, or other target (e.g., a glycoprotein target), and a non-specific interaction means a binding that is measurably different (e.g., a non-specific interaction may be 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 as compared to binding to a control molecule. For example, specific binding can be determined 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 the excess unlabeled target, it is shown to be specific binding. For example, a molecule having a Kd for a target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or more can indicate "specific binding" to the above terms used herein, a specific polypeptide or an epitope on a specific polypeptide target, or "binds specifically to" or "is specific for" the above. In certain cases, the term "specific binding" refers to a binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0051] "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 that reflects a 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be represented by the dissociation constant (Kd). For example, the Kd may be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, 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 more quickly and tend to remain bound for longer. Various methods for measuring binding affinity are known in the art.

[0052] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured at 25°C using an immobilized antigen CM5 chip at about 10 response units (RU) by a technique suitable for the pair of an antibody and a target, for example, using a surface plasmon resonance assay, for example, using BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ).

[0053] As used herein, the term "valence" means the presence of a specific number of binding sites in an antibody. Thus, the term "bivalent" means the presence of two binding sites.

[0054] "Polyepitope specificity" refers to the ability to specifically bind two or more different epitopes on the same or different target(s). "Monospecific" refers to the ability to bind only one epitope. In some embodiments, the antibody binds each epitope with an affinity of at least 10 -7 M, or 10 -8 M or better.

[0055] The terms "target" or "binding target" are used in the broadest sense and, in particular, include polypeptides, nucleic acids, carbohydrates, lipids, cells, and other molecules, whether or not such molecules are naturally occurring and whether or not they are associated with a biological function, but are not limited thereto.

[0056] The term "antigen" refers to an entity or a fragment thereof that can bind to an antibody or elicit a cellular immune response. An 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 a region known as an antigenic determinant or epitope, as defined above.

[0057] As used herein, the term "immunogenicity" refers to a substance that induces the production of antibodies and / or activates T cells and / or other reactive immune cells directed against the antigen of the immunogen.

[0058] The "antigen-binding site" or "antigen-binding region" of an antibody of the present invention typically includes six hypervariable regions (HVRs) that contribute to varying degrees to the affinity of the binding site for an antigen. As used herein, the term "complementary determining region" or "CDR" is used synonymously with the term "hypervariable region" or "HVR". 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 ranges of the HVRs and framework regions (FRs) are determined by comparison to a compiled database of amino acid sequences in which these regions are defined according to variability between sequences and / or structural information from antibody / antigen complexes. Also included within the scope of the present invention are functional antigen-binding sites composed of fewer HVRs (i.e., where the binding specificity is determined by three, four, or five HVRs). For binding to some binding targets, less than a complete set of six HVRs may be sufficient. Thus, in some cases, only the HVRs of the VH or VL domain may be sufficient. Additionally, a particular antibody may have a binding site that is non-HVR related to the antigen. Such binding sites are clearly encompassed within this definition.

[0059] For the purposes of this specification, a "naked antibody" is an antibody that is not complexed with a cytotoxic moiety or radiolabel.

[0060] An "antibody-drug conjugate" (ADC) or immunoconjugate means an antibody or an antigen-binding fragment thereof that is conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin from bacteria, fungi, plants, or animals, or fragments thereof), or a radioisotope (i.e., a radiolabeled conjugate).

[0061] As used herein, the term "host cell" means any type of cell line that can be engineered to produce an antibody according to the present invention. In one embodiment, Chinese hamster ovary (CHO) cells are used as the host cell.

[0062] In this specification, the expressions "cell", "cell line", and "cell culture" are used synonymously, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the original target cell and cultures derived therefrom, regardless of the number of transfers. It will also be understood that due to deliberate or inadvertent mutations, the DNA content of all progeny may not be exactly the same. Progeny of mutants having the same function or biological activity as screened in the originally transformed cells are included.

[0063] A nucleic acid is "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a protein precursor that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking can occur by ligation at convenient restriction enzyme recognition sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0064] The terms "anti-AS-SPIK antibody", "AS-SPIK antibody", or "antibody that binds to AS-SPIK" all refer to an antibody that can bind to AS-SPIK with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting AS-SPIK.

[0065] In one embodiment, as used herein, the term "AS-SPIK antibody" specifically refers to an anti-AS-SPIK monoclonal antibody that (i) includes a heavy chain variable domain sequence shown in any one of SEQ ID NOs: 1 to 10 and / or a light chain variable domain sequence shown in any one of SEQ ID NOs: 11 to 20; or (ii) includes one, two, three, four, five, or six of the CDRs shown in SEQ ID NOs: 21 to 80.

[0066] The term "variable" refers to the fact that the sequences of certain segments of the variable domains vary widely among antibodies. The "variable" or "V" domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, this variability is not evenly distributed throughout the entire 110 amino acid span of the variable domains. Instead, the V regions are composed of relatively invariant stretches called framework regions (FRs) of 15 to 30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9 to 12 amino acids in length. The variable domains of the native heavy and light chains each have a predominantly β-sheet structure and include four FRs connected by three hypervariable regions that form loops connecting the β-sheet structures and in some cases forming part of the β-sheet structures. The hypervariable regions in each chain are held in close proximity by the FRs and together with the hypervariable regions of the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0067] An "intact" antibody is an antibody that includes an antigen-binding site of a particular antibody class, as well as a light chain constant domain (CL) and at least a heavy chain constant domain. For example, an intact IgG antibody includes an antigen-binding site, a light chain constant domain CL, and at least heavy chain constant domains CH1 (Cγ1), CH2 (Cγ2), and CH3 (Cγ3). An intact IgM antibody includes 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). An intact IgA antibody includes an antigen-binding site, a light chain constant domain CL, and at least heavy chain constant domains CA1 (Cα1), CA2 (Cα2), and CA3 (Cα3). An intact IgD antibody includes an antigen-binding site, a light chain constant domain CL, and at least heavy chain constant domains CD1 (Cδ1), CD2 (Cδ2), and CD3 (Cδ3). An intact IgE antibody includes an antigen-binding site, a light chain constant domain CL, and at least heavy chain constant domains CE1 (Cε1), CE2 (Cε2), CE3 (Cε3), and CE4 (Cε4). The constant domains may be constant domains of a native sequence (e.g., a constant domain of a human native sequence) or amino acid sequence variants thereof. It is preferred that the intact antibody has one or more effector functions.

[0068] An "antibody fragment" or "antigen-binding fragment" of an antibody comprises a portion of the intact antibody, preferably the antigen-binding region or variable region of the intact antibody. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (see, e.g., U.S. Patent 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, the antibody fragment comprises the antigen-binding site of the intact antibody and thus retains the ability to bind to an antigen. One of ordinary skill in the art will understand that an antibody fragment can be generated from any intact antibody, e.g., an IgG, IgM, IgA, IgD, or IgE antibody, by separating at least the antigen-binding portion of the antibody from the light and heavy chains of the antibody to produce an antigen-binding fragment. In certain embodiments, the antibody fragment may comprise the antigen-binding region of the antibody as well as 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 that includes the VH and VL domains, the light-chain constant domain CL, and one or more heavy-chain constant domains, e.g., the CH1 (Cγ1) domain, the CM1 (Cμ1) domain, the CA1 (Cα1) domain, the CD1 (Cδ1) domain, or the CE1 (Cε1) domain.

[0069] In the case of an IgG antibody fragment, papain digestion generates two identical antigen-binding fragments called "Fab" fragments and a "Fc" fragment of the remaining portion, the name of which reflects the ability to readily crystallize. The Fab fragment is composed of the variable region domain (VH) of the H chain, the first constant domain (CH1) of one heavy chain, and the entire L chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an IgG antibody generates a single large F(ab')2 fragment that has divalent antigen-binding activity and still has the ability to crosslink antigens, which is roughly equivalent to two disulfide-linked Fab fragments. The Fab' fragment differs from the Fab fragment in that it has several additional residues at the carboxy terminus of the CH1 domain that contain one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domain have free thiol groups. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments that had a hinge cysteine between the pair. Other chemical couplings of antibody fragments are also known.

[0070] The Fc fragment of an IgG antibody contains the carboxy-terminal portions of both H chains linked by disulfides. The effector function of an antibody is determined by the sequence of the Fc region, which is also the portion recognized by Fc receptors (FcRs) present on certain types of cells.

[0071] A "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of an Fc region that occurs in nature. Examples of native sequence human Fc regions include, for example, native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0072] The "variant Fc region" includes an amino acid sequence different from the amino acid sequence of the native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions (multiple possible). The variant Fc region preferably has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide, as compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology, most preferably at least about 90% homology, more preferably at least about 95% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide.

[0073] The human IgG1 amino acid sequence is shown by UniProtKB accession number P01857 (the entire sequence is incorporated herein by reference). The human IgG2 amino acid sequence is shown by UniProtKB accession number P01859 (the entire sequence is incorporated herein by reference). The human IgG3 amino acid sequence is provided by UniProtKB accession number P01860 (the entire sequence is incorporated herein by reference). The human IgG4 amino acid sequence is shown by UniProtKB accession number P01861 (the entire sequence is incorporated herein by reference).

[0074] "Fv" is the smallest antibody fragment that contains a complete antigen recognition site and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain that are tightly associated non-covalently. In single-chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker, such that the light and heavy chains can associate into a "dimer" structure similar to that in the double-chain Fv species. From the folding of these two domains, six hypervariable loops (three loops each from the H chain and L chain) emerge that contribute amino acid residues for antigen binding and confer antigen-binding specificity to this antibody. However, even a single variable domain (or half of the Fv containing only the three HVRs specific for the antigen), although having a lower affinity than the entire binding site, has the ability to recognize and bind the antigen.

[0075] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment that contains VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide further contains a polypeptide linker between the VH domain and the VL domain, whereby the sFv can form a structure desirable 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.

[0076] The term "chimeric antigen receptor" or "CAR" is used herein in the broadest sense and refers to an engineered receptor that fuses a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) to a transmembrane domain and an intracellular signaling domain. Typically, the receptor is used to fuse the specificity of a monoclonal antibody onto a T cell to create 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.).

[0077] As used herein, the term "effector cell" refers to an immune cell involved in the effector phase of an immune response, as opposed to the recognition and activation phases of the immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells such as natural killer cells have the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). For example, monocytes and macrophages that express FcR are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system, or the binding to antigen-presenting cells. In some embodiments, effector cells may phagocytose target antigens or target cells.

[0078] A "human effector cell" is a leukocyte that expresses receptors such as a T cell receptor or FcR and performs an effector function. The cell preferably expresses at least FcγRIII and performs an ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. The effector cells can be isolated from their natural source, e.g., blood or PBMCs as described herein.

[0079] The term "immune cell" is used herein in the broadest sense and includes cells of bone marrow or lymphoid origin, such as lymphocytes (including B cells and T cells such as cytotoxic T cells (CTLs)); killer cells; natural killer (NK) cells; macrophages; monocytes; eosinophils; polymorphonuclear cells such as neutrophils, granulocytes, mast cells, and basophils, but is not limited thereto.

[0080] The "effector function" of an antibody refers to the biological activity resulting from the Fc region of the antibody (natural sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptor; BCR).

[0081] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing an Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and subsequently lyse the target cell. NK cells, which are primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay as described in U.S. Patent No. 5,500,362 or 5,821,337 may be performed. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition to the above, the ADCC activity of a molecule of interest may be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0082] "Complement-dependent cytotoxicity", i.e., "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) that has formed a complex with a cognate antigen. To evaluate complement activation, for example, a CDC assay such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) may be performed.

[0083] A "blocking" antibody or "antagonist" antibody or "antagonistic" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which the antibody binds. Preferred blocking or antagonist antibodies have the ability to substantially or completely inhibit the biological activity of the antigen.

[0084] An antibody that "binds" to a target antigen, e.g., an AS-SPIK or NS-SPIK polypeptide, is an antibody that binds to the antigen with sufficient affinity such that, as a result, it is useful as a therapeutic agent in 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 terms "specific binding" to a particular polypeptide or to an epitope on a particular polypeptide target or "specifically binds" to the foregoing or "specifically" to the foregoing mean a binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by comparing the binding of a molecule to the binding of a control molecule, where the control molecule is generally a molecule of 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 shown to be specific binding. In one embodiment, the term "specific binding" refers to a binding in which a molecule binds to a particular polypeptide or to an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0085] The terms "cancer" and "cancerous" generally refer to or describe a physiological state in mammals characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More detailed examples of such cancers include lung cancers including squamous cell carcinoma (e.g., epithelial squamous cell cancer), skin cancer, melanoma, 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 cholangiocarcinoma (ICC)), bladder cancer (e.g., urothelial bladder cancer), testicular cancer (germ cell tumor), hepatocellular carcinoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer 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. Further examples of cancers include, but are not limited to, retinoblastoma, theca cell tumor, masculinizing cell tumor, hepatocellular carcinoma, non-Hodgkin lymphoma (NHL), multiple myeloma, and hematologic malignancies including 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 sarcoma, melanoma, skin cancer, schwannoma, anaplastic glioma, neuroblastoma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and urinary tract cancer.

[0086] The term "metastatic cancer" means a state of cancer in which cancer cells from the primary tissue move via blood vessels or lymphatic vessels from the primary site to one or more other sites in the body and form one or more secondary tumors in one or more organs other than the primary tissue.

[0087] As used herein, the term "AS-SPIK-related disorder" of "disorders characterized by the expression of AS-SPIK" refers to a disorder associated with the expression or overexpression of the AS-SPIK gene or gene product (AS-SPIK polypeptide), and the disorder may be any disorder characterized by cells that express normal or high levels of AS-SPIK as compared to appropriate control cells. Appropriate control cells may be cells derived from an individual not suffering from cancer that expresses or overexpresses AS-SPIK, or non-cancerous cells from any subject in need thereof, or non-cancerous cells from another individual suffering from cancer that expresses or overexpresses AS-SPIK. One prominent example of an AS-SPIK-related disorder is liver cancer.

[0088] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0089] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0090] As used herein, the terms "predicted" and "foreseen" are also synonymous in the sense that a prediction method or a foresight method enables a person performing the method to select a patient who is more likely to respond to treatment with an anti-cancer agent comprising an anti-AS-SPIK antibody (usually before treatment, but not necessarily so).

[0091] As used herein, the terms "treat," "treatment," or "treating" refer to both therapeutic treatment and prophylactic or preventative measures, the latter of which are intended to prevent or slow down (reduce) a targeted pathologic condition or disorder. A subject in need of treatment includes a subject already suffering from a particular disease or disorder, as well as a subject susceptible to the disorder or in whom prevention of the disorder is to be prevented.

[0092] Detailed Description of the Invention The present invention is based at least in part on the discovery that certain disorders are characterized by the expression of a specific form of Kazal-type serine protease inhibitor (SPIK). Exemplary but non-limiting examples are liver cancers including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). More specifically, the inventors have found that certain cancers such as liver cancer express a form of SPIK that includes an additional 23 amino acids at the N-terminus of the secreted SPIK polypeptide. This 23-amino acid segment (SEQ ID NO: 81) is not present in the SPIK polypeptide secreted from normal cells such as pancreatic cells. This is consistent with the inventors' previous report that the first 9 amino acids of this 23-amino acid segment may be present in the unprocessed SPIK secreted by liver cancer cell lines. Lu et al., Immunology 2011;134(4):398-408. This longer form of SPIK may be referred to as AS-SPIK, i.e., Abnormal Secreted SPIK. Also, the AS-SPIK produced by liver cancer cells may be referred to as LC-SPIK, i.e., Liver Cancer Secreted SPIK. The terms AS-SPIK and LC-SPIK are used synonymously herein. An exemplary AS-SPIK polypeptide may have the amino acid sequence of SEQ ID NO: 82. The inventors may refer to the form of SPIK secreted by normal cells such as pancreatic cells as NS-SPIK, i.e., Normal Secreted SPIK. An exemplary NS-SPIK polypeptide may have the amino acid sequence of SEQ ID NO: 83. The inventors have also found that the conformation (e.g., 3D structure) of AS-SPIK is different from that of NS-SPIK.

[0093] Accordingly, aspects of the invention include compositions such as antibodies that specifically or preferentially bind to AS-SPIK and do not bind to NS-SPIK. AS-SPIK complexes are also provided. An AS-SPIK complex according to an embodiment of the invention includes an antibody that specifically or preferentially binds to AS-SPIK, and an AS-SPIK polypeptide, or a fragment thereof.

[0094] Aspects of the invention include antibody-drug conjugates (ADCs) comprising an antibody (Ab), a linker (L), and a drug moiety (D) described herein. In some embodiments, the ADC has the formula Ab-(L-D)p, where p is an integer in the range of 1 to 8.

[0095] Aspects of the invention also include methods of using the antibodies of the subject matter for the detection of disorders characterized by the expression of AS-SPIK, such as liver cancer, such as liver disorders such as HCC or ICC.

[0096] The inventors believe that they understand certain events that occur upon the expression of AS-SPIK, but the compositions and methods of the invention are not limited to compositions and methods that function by affecting any particular cellular mechanism. Without being bound by theory, the inventors hypothesize that because SPIK is a protease inhibitor, overexpression of SPIK in cancer cells suppresses the activity of signal peptide peptidase, a type of protease, resulting in the secretion of non-attenuated full-length proteins from cancer cells.

[0097] Compositions The compositions provided herein include antibodies that specifically or preferentially bind to AS-SPIK and do not bind to NS-SPIK.

[0098] Kazal-type serine protease inhibitor (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 the inhibition of apoptosis. Lu et al., Immunology 2011;134(4):398-408. Exemplary human SPIK amino acid sequences include GenBank accession number: M11949, GI number: 190687; GenBank accession number: NM003122, GI: 657940887; and GeneBank accession number: BC025790, GI: 19343607.

[0099] Antibody 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 that includes at least one amino acid within this region. The above epitope may be a conformational epitope (conformational specific epitope) or a linear epitope. In some embodiments, the antibody specifically or preferentially binds to an epitope within the AS-SPIK protein sequence shown in SEQ ID NO: 81. In some embodiments, the antibody specifically or preferentially binds to a conformational specific epitope that includes at least one amino acid of SEQ ID NO: 81.

[0100] The antibodies according to the embodiments of the present invention may be polyclonal or monoclonal, particularly monoclonal, and can be produced by cells of humans, mice, rabbits, sheep, or goats, or by hybridomas derived from these cells. In some embodiments, the antibody may be a humanized antibody or a chimeric antibody.

[0101] The antibodies according to the embodiments of the present invention can adopt various conformations and can include proteins composed of one or more polypeptides substantially encoded by immunoglobulin genes. Any one of various antibody structures can be used, including intact antibodies, antibody multimers, or antibody fragments or other variants thereof, including the above antibody fragments or other variants containing the functional antigen-binding regions of the above antibodies. The term "immunoglobulin" can be used synonymously with "antibody". The above antibodies may be of monoclonal or polyclonal origin. Regardless of the origin of the above antibodies, suitable antibodies include intact antibodies, such as IgG tetramers having two heavy (H) chains and two light (L) chains, single-chain antibodies, chimeric antibodies, humanized antibodies, complementarity-determining region (CDR) grafted antibodies, and antibody fragments, such as Fab, Fab’, F(ab’)2, scFv, Fv, and recombinant antibodies derived from such fragments, such as camelbodies, microantibodies, diabodies, and bispecific antibodies.

[0102] An intact antibody is an antibody that includes an antigen-binding variable region (VH and VL) and light-chain constant domains (CL) and heavy-chain constant domains, CH1, CH2, and CH3. The above constant domains may be natural sequence constant domains (e.g., human natural sequence constant domains) or amino acid sequence variants thereof. As is well known in the art, the above VH and VL regions are further subdivided into regions of hypervariability called "complementary determining regions" (CDRs) interspersed with more conserved framework regions (FRs). The above CDRs of an antibody usually include amino acid sequences that together define the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site.

[0103] The anti-AS-SPIK antibody may be derived from any class of immunoglobulin, such as IgA, IgG, IgE, IgD, IgM (and their subtypes (e.g., IgG1, IgG2, IgG3, and IgG4)), and the light chain of the above immunoglobulin may be of the kappa or lambda type. The recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes.

[0104] The term "antigen-binding portion" of an immunoglobulin or antibody generally refers to the portion of the immunoglobulin that specifically or preferentially binds to an epitope containing an amino acid residue (SEQ ID NO: 81) on the target, in this case, AS-SPIK but not on NS-SPIK. Thus, the antigen-binding portion of an immunoglobulin is a molecule in which one or more of the immunoglobulin chains are not full-length, but the molecule that specifically or preferentially binds to a cellular target. Examples of antigen-binding portions or fragments include (i) a monovalent fragment consisting of the Fab fragment, VLC, VHC, CL, and CH1 domains; (ii) an F(ab’)2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fv fragment consisting of the VLC and VHC domains of a single arm of the antibody; and (v) an isolated CDR having a framework sufficient to specifically or preferentially bind, such as the antigen-binding portion of the variable region. The antigen-binding portions of the light chain variable region and the heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined by a synthetic linker that can be used to join VLC and VHC into a single protein chain such that the VLC region and the VHC region pair to form a monovalent molecule (known as a single-chain Fv (scFv)). Such scFv is included within the term "antigen-binding portion" of an antibody.

[0105] The "Fv" fragment is the smallest antibody fragment that contains a complete antigen recognition site and an antigen binding site. This region is composed of a dimer of one tightly covalently linked heavy chain variable domain and one light chain variable domain. In this configuration, the three hypervariable regions of each variable domain interact to delineate an antigen binding site on the surface of the VH-VL dimer. The six hypervariable regions confer antigen binding specificity, but even a single variable domain (i.e., half of the Fv that contains only three hypervariable regions specific for the antigen), although with a lower affinity than the entire binding site, has the ability to recognize and bind the antigen. To improve stability, the VH-VL domains may be connected by a flexible peptide linker such as (Gly4Ser)3 to form a single-chain Fv or scFV antibody fragment, or may be engineered to form a disulfide bond by introducing two cysteine residues into the framework region to generate a disulfide-stabilized Fv (dsFv).

[0106] Antibody fragments are suitable for use in the provided methods as long as the fragments retain the desired specificity of the full-length antibody and / or sufficient specificity to bind to AS-SPIK and not bind to NS-SPIK.

[0107] The antigen binding domains of the antibodies described herein can be utilized in the production of T cell inducing molecules (e.g., bispecific T cell inducing, also known as BiTE molecules) as well as CAR-T constructs. T cell inducing molecules are described, for example, in Huehls et al., Bispecific T cell engagers for cancer immunotherapy, Immunol Cell Biol. 2015 Mar;93(3):290-296. CAR-T constructs containing single domain antibodies as binding (target) domains are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386.

[0108] Methods for preparing antibody fragments include both biochemical methods (e.g., proteolytic digestion of intact antibodies followed by chemical cross-linking) and recombinant DNA-based methods that genetically engineer immunoglobulin sequences to direct the synthesis of the desired fragments. Antibody fragments can be obtained by proteolysis of whole immunoglobulins with papain, a nonspecific thiol protease. Papain digestion yields two identical antigen-binding fragments called "Fab fragments," each having a single antigen-binding site, and a residual "Fc fragment." The various fractions can be separated by protein A-Sepharose or ion-exchange chromatography. The usual procedure for preparing F(ab’)2 fragments from rabbit and human IgG is limited proteolysis with the enzyme pepsin. Pepsin treatment of intact antibodies yields F(ab’)2 fragments that have two antigen-binding sites and still have the ability 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 in that a few residues are added to the carboxyl terminus of the heavy-chain CH1 domain, which contains one or more cysteines (s) from the antibody hinge region. F(ab’)2 antibody fragments were originally generated as pairs of Fab’ fragments having hinge cysteines between them.

[0109] Methods for producing anti-AS-SPIK antibodies are also within the scope of the present invention. For example, the variable regions can be constructed by modifying the DNA sequences encoding the immunoglobulin chains using, for example, the methods used to generate humanized immunoglobulins, such as the PCR mutagenesis method.

[0110] A monoclonal antibody is a homogeneous antibody of the same antigen specificity produced by a single clone of antibody-producing cells, while a polyclonal antibody generally recognizes different epitopes on the same antigen and is produced by multiple clones of antibody-producing cells. Each monoclonal antibody is directed against a single determinant on the antigen. The modifier, monoclonal, indicates that the character of the antibody in question is obtained from a substantially homogeneous population of antibodies and should not be construed to require that the antibody in question be produced by any particular method.

[0111] The monoclonal antibodies herein include chimeric antibodies, i.e., antibodies that usually have a part of the heavy chain and / or light chain that is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remaining part of the chain(s) is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and may also include fragments of such antibodies as long as they exhibit the desired biological activity. Examples of chimeric antibodies of interest include primatized antibodies containing variable domain antigen-binding sequences derived from non-human primates (e.g., anthropoids, Old World monkeys, New World monkeys, prosimians) and human constant region sequences.

[0112] Mouse or rabbit monoclonal antibodies were generated by immunizing mice or rabbits with a specially designed recombinant protein that has an additional 23-amino acid sequence (SEQ ID NO: 81) in addition to the common region (SEQ ID NO: 83), an amino acid sequence that is present in AS-SPIK and in both NS-SPIK and AS-SPIK. In some embodiments, the recombinant protein need not have the entire 23-amino acid sequence (SEQ ID NO: 81) to be effective in generating antibodies that bind to AS-SPIK only and not to NS-SPIK. Methods for screening and selecting antibodies that specifically or preferentially bind to AS-SPIK are described in Example 1.

[0113] The test results of the monoclonal antibodies are shown in Figure 1, and these results indicate that the antibodies of the present invention can specifically or preferentially recognize AS-SPIK, but cannot recognize NS-SPIK. These antibodies 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 the description of the test, please refer to Example 1.

[0114] The sequences of the variable regions of the light chain (VL) and heavy chain (VH) of monoclonal antibodies that specifically or preferentially bind to AS-SPIK (and do not bind to NS-SPIK) were determined (SEQ ID NOs: 1-20). All CDRs of these antibodies were also determined (SEQ ID NOs: 21-80). Figures 3-6 show the consensus amino acid sequences of these regions. Tables 1-3 show the SEQ ID NOs of the various VH and VL regions of 10 exemplary antibodies of the present invention.

[0115] For example, the IM-A1 antibody has a VH region shown in SEQ ID NO: 1 and a VL region shown in SEQ ID NO: 11. The above VH region has three CDR regions corresponding to SEQ ID NOs: 21, 31, and 41. The above VL region has three CDR regions corresponding to SEQ ID NOs: 51, 61, and 71.

[0116] The IM-A6 antibody has a VH region shown in SEQ ID NO: 2 and a VL region shown in SEQ ID NO: 12. The above VH region has three CDR regions corresponding to SEQ ID NOs: 22, 32, and 42. The above VL region has three CDR regions corresponding to SEQ ID NOs: 52, 62, and 72.

[0117] The IM-B10 antibody has a VH region shown in SEQ ID NO: 3 and a VL region shown in SEQ ID NO: 13. The above VH region has three CDR regions corresponding to SEQ ID NOs: 23, 33, and 43. The above VL region has three CDR regions corresponding to SEQ ID NOs: 53, 63, and 73.

[0118] The IM-C6 antibody has a VH region shown in SEQ ID NO: 4 and a 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.

[0119] The IM-D3 antibody has a VH region shown in SEQ ID NO: 5 and 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.

[0120] The IM-D5 antibody has a VH region shown in SEQ ID NO: 6 and a 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.

[0121] The IM-E2 antibody has a VH region shown in SEQ ID NO: 7 and a 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.

[0122] The IM-F5 antibody has a VH region shown in SEQ ID NO: 8 and a 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.

[0123] The IM-G6 antibody has a VH region shown in SEQ ID NO: 9 and a 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.

[0124] The IM-G7 antibody has a VH region shown in SEQ ID NO: 10 and 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.

[0125] 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.

[0126] In some embodiments, the antibody comprises a VH region having a sequence shown in any one of SEQ ID NOs: 1 to 10, or a VH region having a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of any one of SEQ ID NOs: 1 to 10, provided that the resulting antibody or antigen-binding fragment specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK.

[0127] In some embodiments, the antibody comprises a VL region having a sequence shown in any one of SEQ ID NOs: 11 to 20, or a VL having a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of any one of SEQ ID NOs: 11 to 20, provided that the resulting antibody or antigen-binding fragment specifically or preferentially binds to AS-SPIK and does not bind to NS-SPIK.

[0128] In some embodiments, the antibody has a sequence set forth in any one of SEQ ID NOs: 21-30, or comprises a VH CDR1 region with no more than two amino acid mutations contained in any one of SEQ ID NOs: 21-30, so long as the resulting antibody specifically or preferentially binds to AS-SPIK and not NS-SPIK. In some embodiments, the VH CDR1 region may comprise the following structural formula, where X represents a variable amino acid, which may be any of the identified amino acids: S X1X2X3X4S (SEQ ID NO: 107), where X1 is S or A, X2 is Y or N, X3 is A, G, P, or V, and X4 is I or V.

[0129] In some embodiments, the antibody has a sequence set forth in any one of SEQ ID NOs: 31-40, or comprises a VH CDR2 region with no more than two amino acid mutations contained in any one of SEQ ID NOs: 31-40, so long as the resulting antibody specifically or preferentially binds to AS-SPIK and not NS-SPIK. In some embodiments, the VH CDR2 region may comprise the following structural formula, where X represents a variable amino acid, which is one of the identified amino acids: X5I X6X7X8G X9X 10 X 11 YASWAKS (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.

[0130] In some embodiments, the antibody has a sequence set forth in any one of SEQ ID NOs: 41-50, or comprises a VH CDR3 region with no more than two amino acid mutations contained in any one of SEQ ID NOs: 41-50, so long as the resulting antibody specifically or preferentially binds to AS-SPIK and not NS-SPIK. In some embodiments, the VH CDR3 region may comprise the following structural formula, where X represents a variable amino acid, which is one of the identified amino acids: RX 12 X 13 X 14 X 15 X16 X 17 X 18 X 19 X 20 X 21 (SEQ ID NO: 109), where 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.

[0131] In some embodiments, the antibody has a sequence shown in any one of SEQ ID NOs: 51 - 60 or contains no more than two amino acid mutations included in any one of SEQ ID NOs: 51 - 60 in the VL CDR1 region, as 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 include the following structural formula, where X represents a variable amino acid, which is the identified amino acid: Q A S X 22 X 23 I X 24 X 25 X 26 L X 27 (SEQ ID NO: 110), where X 22 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, X 27 is any amino acid.

[0132] In some embodiments, the antibody has a sequence shown in any one of SEQ ID NOs: 61 to 70 or contains no more than two amino acid mutations contained in any one of SEQ ID NOs: 61 to 70 in the VL CDR2 region, as 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 include the following structural formula, where X represents a variable amino acid, which is the identified amino acid: X 28 A X 29 X 30 L X 31 S (SEQ ID NO: 111), where X 28 is any amino acid, X 29 is S or T, X 30 is T, Y, or K, X 31 is A, P, or V.

[0133] In some embodiments, the antibody has a sequence shown in any one of SEQ ID NOs: 71 to 80 or contains no more than two amino acid mutations contained in any one of SEQ ID NOs: 71 to 80 in the VL CDR3 region, as 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 include the following structural formula, where X represents a variable amino acid, which is the identified amino acid: X 32 Q X 33 Y X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 (SEQ ID NO: 112), where 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, X39 is D, G, or N, and X 40 is N or V, and X 41 is any amino acid.

[0134] An antibody according to an embodiment of the present invention may include any of the 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, as long as the antibody binds to AS-SPIK and does not bind to NS-SPIK, starting with a sequence having the homology or percent sequence identity described above.

[0135] The structural formulas and consensus sequences described herein were determined using the software program "BioEdit" developed by North Carolina State University.

[0136] The high binding activity of all these antibodies against AS-SPIK was examined. The results are shown in Example 1.

[0137] The method for producing monoclonal antibodies may include a purification step. For example, the present antibody may be further purified generally using various chromatography methods such as filtration, centrifugation, and HPLC or affinity chromatography, all of which are techniques well known to those skilled in the art. Each of these purification techniques includes a fractionation for separating the desired antibody from other components of the mixture. Analytical methods particularly suitable for the preparation of antibodies include, for example, protein A-sepharose and / or protein G-sepharose chromatography.

[0138] The anti-AS-SPIK antibodies of the present invention may contain CDRs derived from human or non-human sources. "Humanized" antibodies are generally chimeric or mutant monoclonal antibodies derived from mice, rats, hamsters, rabbits, or other species that have human constant region domains and / or variable region domains or specific modifications. The framework of the immunoglobulin may be a human, humanized, or non-human framework (e.g., a mouse framework modified to reduce antigenicity in humans), or a synthetic framework (e.g., a consensus sequence). A humanized immunoglobulin is an immunoglobulin in which the framework residues correspond to human germline sequences and the CDRs are derived from V(D)J recombination and somatic hypermutation. However, a humanized immunoglobulin may also contain amino acid residues not encoded by the human germline immunoglobulin nucleic acid sequence (e.g., mutations introduced by random or site-directed mutagenesis ex vivo). Antibody variable domain genes that are based on germline sequences but have framework mutations introduced, for example, by the in vivo somatic hypermutation process are called "human."

[0139] Humanized antibodies may be engineered by various methods known in the art, including, for example, (1) grafting non-human complementarity-determining regions (CDRs) onto human frameworks and constant regions (a process referred to in the art as humanization), or (2) grafting an entire non-human variable domain, provided that surface residues are substituted to give the domain a human-like surface (a process referred to in the art as veneering). Humanized antibodies may include both humanized antibodies and veneered antibodies. Similarly, human antibodies can be produced by introducing the human immunoglobulin locus into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Loading results in the production of human antibodies, which is closely similar to that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.

[0140] In addition to chimeric and humanized antibodies, fully human antibodies can be derived from transgenic mice having human immunoglobulin genes. In some embodiments, the antibodies can be produced and identified by scFv-phage display libraries.

[0141] The anti-AS-SPIK antibody can also be modified to modulate the antigen-binding affinity, effector function, or pharmacokinetics of the antibody. In particular, random mutations can be made in the CDRs, and the products screened to identify antibodies having higher affinity and / or higher specificity. Usually, it is sufficient if one or two amino acids in the CDRs are different.

[0142] CDR shuffling and grafting techniques may be used, for example, with the antibodies provided herein. CDR shuffling inserts CDR sequences into specific framework regions. By CDR grafting techniques, CDR sequences can be randomly combined into a single master framework. Such techniques can be used, for example, to subject the CDR sequences of the anti-AS-SPIK antibody to mutagenesis to generate a plurality of different sequences, incorporate these into a scaffold sequence, and screen the resulting antibody variants for desired properties, such as higher affinity.

[0143] Studies by the inventors on the function of SPIK have revealed that SPIK can bind to granzyme A (GzmA) and inhibit GzmA from inducing apoptosis. Lu et al., Immunology 2011;134(4):398-408. GzmA is a cytotoxic serine protease secreted by activated CTLs and NK cells that kills target cells during immune surveillance. The role of GzmA-induced apoptosis in the elimination of 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 render these cells resistant to apoptosis induced by GzmA during immune clearance. As a result, these cancer cells evade killing by the immune pathway. Lu et al., Immunology 2011;134(4):398-408. Based on this hypothesis, without being bound by theory, it is concluded that suppressing the overexpression of the above AS-SPIK or inhibiting the activity of overexpressed AS-SPIK may restore the immune killing of cancer cells induced by GzmA during the immune clearance of the human body.

[0144] The anti-AS-SPIK antibody according to an embodiment of the present invention can inhibit the activity of AS-SPIK as shown 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 the anti-AS-SPIK antibody to block the binding of AS-SPIK to GzmA, release GzmA, and restore the killing of these cancer cells by apoptosis via immune clearance. For this purpose, the anti-SPIK antibody can be used in the treatment of disorders characterized by the expression of AS-SPIK, including but not limited to cancer, viral infections, and inflammation.

[0145] One therapeutic use of antibodies is through humanization. Treatment with humanized monoclonal antibodies is a rapidly evolving area, and the specificity and efficacy of the above antibodies have been well studied. Rothernberg, ME, Cell 2016;165(3):509. The anti-AS-SPIK monoclonal antibodies of the present subject matter, 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, as well as other antibodies of the present invention that can inhibit the activity of SPIK, are also humanized and can be used for the treatment of diseases.

[0146] For example, recombinant techniques using phagemid technology enable the preparation of antibodies with desired specificities from recombinant genes encoding various antibodies. Certain recombinant techniques involve the isolation of antibody genes by immunological screening of a combinatorial immunoglobulin phage expression library prepared from RNA isolated from the spleen of an immunized animal. In 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.

[0147] In addition to the combinatorial immunoglobulin phage expression library disclosed above, one approach by molecular cloning is to prepare antibodies from transgenic mice containing a human antibody library. Such transgenic animals can be used to produce human antibodies of a single isotype, more particularly, isotypes essential for B cell maturation such as IgM and possibly IgD.

[0148] This anti-AS-SPIK immunoglobulin can be modified to reduce or eliminate glycosylation. The immunoglobulin lacking glycosylation may be an immunoglobulin that is not glycosylated at all, not fully glycosylated, or irregularly glycosylated (i.e., the glycosylation pattern of the mutant differs from that of the corresponding wild-type immunoglobulin). The IgG polypeptide contains one or more (e.g., one, two, or three or more) mutations that attenuate glycosylation, i.e., mutations that result in an IgG CH2 domain that lacks glycosylation, is not fully glycosylated, or is irregularly glycosylated. The oligosaccharide structure may also be modified, for example, by removing the fucose moiety from the N-linked glycan.

[0149] The antibody can also be modified to enhance its stability and / or solubility in vivo by complexing it with a non-protein polymer, such as polyethylene glycol. Any PEGylation method can be used as long as the anti-AS-SPIK antibody retains its ability to selectively bind to AS-SPIK and not to NS-SPIK.

[0150] A wide variety of antibody / immunoglobulin frameworks or scaffolds can be used as long as the resulting polypeptide contains at least one binding region 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 in this specification), and immunoglobulins with a preferably humanized context from other animal species. Single-chain antibodies, such as those identified in camelids, are particularly interesting in this regard.

[0151] The anti-AS-SPIK antibody of the present invention specifically or preferentially binds to the epitope on AS-SPIK and does not bind to the epitope on NS-SPIK. An epitope refers to a paratope, that is, an antigenic determinant on a target that is specifically bound by the binding site of an antibody. An epitopic determinant is usually composed of chemically active surface groups of a molecule, such as amino acids or sugar side chains, and usually has specific three-dimensional structural features as well as specific charge features. An epitope generally has about 4 to about 10, preferably 4 to 8, adjacent amino acids (linear or continuous epitope), or may be a set of non-adjacent amino acids that define a specific structure (for example, a conformational epitope). Therefore, 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 examples include X-ray crystallography, two-dimensional nuclear magnetic resonance, and precise epitope mapping using a CLIPS (Chemically Linked Peptides on Scaffolds) peptide array (Timmerman, Puijk et al., J Mol Recognit, 20(5), 283-299, (2007)).

[0152] Methods for predicting other potential epitopes to which an antibody can bind include 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-Schulz analysis (Kolaskar and Tongaonkar Analysis Kolaskar & Tongaonkar, FEBS Letters, 172-174 (1990)), as well as 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 crystallographic data. Techniques for predicting epitopes also include those that predict both continuous and discontinuous epitopes. Methods for predicting discontinuous epitopes include, but are not limited to, DiscoTope, BEpro, ElliPro, SEPPA, EPITOPIA, EPCES, Bpredictor, and EPMeta (Yao et al., PLOS ONE, (2013)). In some embodiments, potential epitopes are identified by determining the theoretical extracellular domain.Such predictions can be made using analysis algorithms such as TMpred (Hofmann and Stoffel, Biol. Chem. 374:166 (1993)) or TMHMM (see Krogh et al., J. Mol. Biol., 305(3):567-580 (2001)). 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 the locations where those peptides are cleaved from the full-length protein. The portion of the protein that is outside the cell can function as a target for antibody interaction.

[0153] The compositions of the present invention comprise an antibody as described herein that (1) exhibits a threshold level of binding activity; (2) does not significantly cross-react with known related polypeptide molecules; (3) binds to AS-SPIK, and (4) does not bind to NS-SPIK. The binding affinity of the antibody can be readily measured by those skilled in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad, Sci. 51:660-672 (1949)).

[0154] In some embodiments, the anti-AS-SPIK antibodies of the present invention bind to the target AS-SPIK at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, 106-fold, or more than the other proteins, such as NS-SPIK, that are predicted to have some degree of homology to AS-SPIK with respect to their target epitopes or mimetic decoys.

[0155] In some embodiments, the anti-AS-SPIK antibody is 10 -4 M or less, 10 -7 M or less, 10 -9It binds with a high affinity below M or a sub-nanomolar affinity (0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM or less). In some embodiments, the binding affinity of the anti-AS-SPIK antibody for each target of the antibody is at least 1×10 6 Ka. In some embodiments, the binding affinity of the anti-AS-SPIK antibody for AS-SPIK is at least 5×l0 6 Ka, at least 1×10 7 Ka, at least 2×l0 7 Ka, at least 1×10 8 Ka, or more. The antibody can also be described or identified in terms of its binding affinity for AS-SPIK. In some embodiments, the binding affinity is such that the Kd is 5×10 -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -3 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or 10 -15Binding affinities of M or less are exemplified. That is, in the present specification, terms such as "specific binding", "specifically binding", or "specifically bind" with respect to / against AS-SPIK refer to an antibody that binds to AS-SPIK and does not bind to NS-SPIK. Without being bound by theory, the antibody of the present invention that specifically binds to AS-SPIK is considered to bind to an epitope that exists only within the first 1 to 23 amino acids (SEQ ID NO: 82) that are present in AS-SPIK but not in NS-SPIK. The antibody of the present invention may bind to an epitope that spans not only the 1 to 23 amino acids of AS-SPIK but also includes some amino acids in the common region (SEQ ID NO: 83). Alternatively, the present antibody may bind not only to at least one amino acid in the first 1 to 23 amino acids (SEQ ID NO: 82) of AS-SPIK but also to at least one amino acid in the common region (SEQ ID NO: 83). In these cases, the present antibody may also bind to NS-SPIK, but the level of binding is below the background level. This is referred to in the present specification as "preferentially binding" or "preferential binding". Since assays can be developed to subtract the background level of binding as "noise", antibodies that preferentially bind to AS-SPIK are still useful in diagnostic methods. Therefore, the above assay indicates that only a specific level of binding (above a specific threshold level and above background noise) is allowed for diagnosing disorders characterized by the expression of AS-SPIK, such as liver disorders in patients with liver cancer and the like.

[0156] The antibody of the present invention is 10 -4 M or less, 10 -7 M or less, 10 -9It can bind with an affinity of less than 1 μM or an affinity of less than nanomolar (0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM, or less). In some embodiments, the binding affinity of each of the antibodies of the anti-AS-SPIK antibody to its respective target is at least 1×10 6 Ka. In some embodiments, the binding affinity of the anti-AS-SPIK antibody to AS-SPIK is at least 5×10 6 Ka, at least 1×10 7 Ka, at least 2×10 7 Ka, at least 1×10 8 Ka, or more. In some embodiments, the above binding affinity is such that the Kd is 5×10 -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -3 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or an affinity of less than 10 -15 M or less. In contrast, as used herein, the term "binds non-specifically" with respect to, for example, NS-SPIK means 1 / 1.5, 1 / 2, 5, 1 / 10, 1 / 100, 1 / 10 3 、1 / 10 4, 1 / 10 5 , 1 / 10 6 Refers to a small binding affinity that is the following or less. Affinities such as Kd can be measured by a radiolabeled antigen binding assay (radioimmunoassay, RIA) performed using the Fab version of the antibody of interest and its antigen. According to another embodiment, Kd can be measured using a surface plasmon resonance assay with 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, and the affinity of the present antibody for AS-SPIK is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, 10 3 -fold, 10 4 -fold, 10 5 -fold, or 10 6 -fold greater than that for NS-SPIK.

[0157] In some embodiments, the present antibody does not bind to known related polypeptide molecules. For example, the present antibody binds to AS-SPIK but does not bind to known related polypeptides such as NS-SPIK. Antibodies can be screened against known related polypeptides to isolate a population of antibodies that specifically or preferentially bind to AS-SPIK. For example, an antibody specific for AS-SPIK will pass through a column containing NS-SPIK attached to an insoluble matrix under appropriate buffer conditions. Such screening enables the isolation of polyclonal and monoclonal antibodies that are non-cross-reactive with closely related polypeptides. Other methods for screening and isolating specific antibodies include, but are not limited to, simultaneous immunoelectrophoresis, radioimmunoassay (RIA), radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assays, inhibition or competition assays, and sandwich assays.

[0158] The antibody according to an embodiment of the present invention may contain a detectable label, which may sometimes be referred to as a reporter (for example, a detectable reporter). In some embodiments, the detectable label may be any molecule covalently bound to an antibody (for example, an anti-AS-SPIK antibody) or a biologically active fragment thereof that enables qualitative and / or quantitative evaluation of the expression or activity of a tagged peptide. Examples of the above activity include biological activity, physicochemical activity, or a combination thereof. As long as the labeled antibody retains its biological activity, both the form and position of the detectable label may change. Many different labels can be used, and the selection of a particular label will depend on the desired application. The labeled anti-AS-SPIK antibody can be used, for example, to evaluate the level of AS-SPIK in a biological sample, such as urine, saliva, cerebrospinal fluid, blood, or a biopsy sample.

[0159] Detectable labels can include enzymes, photoaffinity ligands, radioisotopes, and fluorescent or chemiluminescent compounds. Exemplary enzyme labels can include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and urease. Covalent binding of the anti-AS-SPIK antibody to the enzyme can be carried out by various methods, for example, by binding to glutaraldehyde via free amino groups. Alternatively, the anti-AS-SPIK antibody can be bound to the enzyme via sugar residues. Other enzymes containing carbohydrates can also be bound to the antibody in this way. Binding of the enzyme can also be carried out by using a heterobifunctional linker such as N-succinimidyl 6-(N-maleimidyl)hexanoate to link the amino group of the antibody to the free thiol group of an enzyme such as β-galactosidase. The horseradish peroxidase detection system can be used, for example, with the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB), which produces a soluble product detectable at 450 nm in the presence of hydrogen peroxide. The alkaline phosphatase detection system can be used, for example, with the chromogenic substrate p-nitrophenyl phosphate, which produces a soluble product easily detectable at 405 nm. Similarly, the β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which produces a soluble product detectable at 410 nm. The urease detection system can be used with substrates such as urea-bromocresol purple.

[0160] Detectable labels may include, but are not limited to, fluorescent labels such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine; chemiluminescent compounds selected from the group consisting of luminol, isoluminol, aromatic acridinium esters, imidazole, acridinium salts, and oxalate esters; liposomes or dextrans; or bioluminescent compounds such as luciferin, luciferase, and aequorin. Alternatively, or in addition to these, detectable labels may include radiopaque agents or contrast agents such as barium, diatrizoates, ethyl iodized oil, gallium citrate, iocarmic acid, iotetric acid, iodamide, iopamidol, iopanoic acid, iopromide, iosefamic acid, ioselonic acid, iosramide meglumine, iosemetic acid, iotasul, iotetronic acid, iotalamic acid, iotroxate, ioxaglic acid, ioxotrizoic acid, iopodate, meglumine, metrizamide, metrizoates, propyliodone, and thallous chloride, but are not limited thereto.

[0161] Labels can be added during or after synthesis. Recombinant anti-AS-SPIK antibodies or their biologically active variants can also be labeled by adding a label precursor (e.g., a radiolabeled amino acid) to the culture medium in which the transformed cells are growing. In some embodiments, peptidomimetics or variants may be used to facilitate the incorporation of detectable markers. For example, the N-terminal phenylalanine residue may be replaced with a closely related aromatic amino acid such as tyrosine, which can be readily labeled with 125 I. In some embodiments, additional functional groups may be added to fragments of the anti-AS-SPIK antibody or its biologically active variants to assist in effective labeling. For example, a 3-tributylstannylbenzoyl group may be added to the N-terminus of the native structure, and then the tributylstannyl group may be 125 substituted with I to generate a radiolabeled iodobenzoyl group.

[0162] Antibody-drug conjugate (ADC) Aspects of the invention include immunocomplexes, or antibody-drug conjugates (ADCs), comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin from bacteria, fungi, plants, or animals, or fragments thereof), or a radioisotope (i.e., a radioactive conjugate). In another aspect, the invention further provides methods of using the immunocomplexes. In one aspect, the immunocomplex comprises any of the anti-AS-SPIK antibodies covalently bound to a cytotoxic agent or a detectable agent. ADCs are described, for example, in U.S. Patent No. 8,362,213, the disclosure of which is incorporated herein by reference in its entirety.

[0163] Use of an ADC for the local delivery of a cytotoxic agent or cytostatic agent, i.e., a drug that kills or inhibits tumor cells in cancer treatment (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. Patent No. 4,975,278) enables targeted delivery of the drug moiety to the tumor and intracellular accumulation in the tumor, while systemic administration of these unbound drug agents can result in unacceptable levels of toxicity not only to the tumor cells to be eliminated but also to normal cells (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 (ed.s), pp. 475-506). Efforts to improve the therapeutic index (i.e., the maximum efficacy and minimum toxicity of the ADC) 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 the properties of drug conjugation and drug release (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549).Drug moieties used in ADCs include bacterial protein toxins such as diphtheria toxin, plant protein toxins such as ricin, auristatin, small molecules such as 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), calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342), daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al (1986) supra). The above drug moieties may affect cytotoxic and cytostatic mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic agents tend to become inactive or less active when complexed with large antibodies or protein receptor ligands.

[0164] The auristatin peptides, auristatin E (AE), and monomethyl auristatin (MMAE), which are synthetic analogs of dolastatin, (WO02 / 088172) are conjugated, as the drug moiety, to (i) the chimeric monoclonal antibody cBR96, specific for Lewis Y on carcinomas; (ii) cAC10, specific for CD30 on hematologic 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 rituxan for the treatment of CD20-expressing cancers and immune disorders (WO04 / 032828); (iv) the anti-EphB2R antibody 2H9 for the treatment of colorectal cancer (Mao et al (2004) Cancer Research 64(3):781-788); (v) the E-selectin antibody (Bhaskar et al (2003) Cancer Res. 63:6387-6394); (vi) trastuzumab (HERCEPTIN®, US2005 / 0238649); and (vii) an anti-CD30 antibody (WO03 / 043583). Variants of auristatin E are disclosed in U.S. Patent No. 5,767,237 and U.S. Patent 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, Volume 45, Abstract Number 623, presented Mar. 28, 2004. Auristatin analogs MMAE and MMAF are conjugated to various antibodies (US2005 / 0238649).

[0165] Conventional means of attaching a drug moiety to an antibody, i.e., attaching via a covalent bond, generally result in a heterogeneous mixture of molecules in which the drug moiety is attached at several sites on the antibody. For example, a cytotoxic agent typically binds to the antibody through the lysine residues of many, often multiple, antibodies, producing a heterogeneous mixture of antibody-drug conjugates. Depending on the reaction conditions, the heterogeneous mixture usually contains antibodies with a distribution having from 0 to about 8 or more attached drug moieties. Further, within each subpopulation of conjugates having a specific integer ratio of drug moiety to antibody, there is a potentially heterogeneous mixture in which the drug moiety is attached at various sites on the antibody. Analytical and preparative methods may be insufficient to separate and characterize the antibody-drug conjugate species molecules within the heterogeneous mixture resulting from the conjugation reaction. Antibodies are large, complex, and structurally diverse biomolecules that often have many reactive functional groups. The reactivity of these functional groups with linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration, and co-solvent. Further, the multi-step conjugation process may lack reproducibility because of the difficulty in controlling reaction conditions and characterizing the reagents and intermediates.

[0166] Cysteine thiols are reactive at neutral pH, unlike most amines which are protonated and have low nucleophilicity near pH 7. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often exist in an oxidized form as disulfide-linked oligomers or have internally cross-linked disulfide groups. Extracellular proteins generally do not have free thiols (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London, at page 55). The cysteine thiol groups of antibodies are generally more reactive, i.e., more nucleophilic, towards electrophilic conjugation reagents than the amine or hydroxyl groups of the antibody. Cysteine residues have been introduced into proteins by genetic engineering techniques to form covalent bonds 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. Patent No. 6,248,564). However, manipulation of cysteine thiol groups by mutating various amino acid residues of a protein to cysteine amino acids can be potentially problematic, especially in the case of unpaired (free Cys) residues or residues that are prone to reaction or oxidation.In concentrated protein solutions, unpaired Cys residues on the surface of the protein pair and oxidize to form intermolecular disulfides, thus protein dimers or multimers, whether in the periplasm of E. coli, the culture supernatant, or partially or fully purified protein. Newly introduced Cys due to the formation of disulfide dimers becomes non-reactive towards complexation with drugs, ligands, or other labels. Furthermore, when the protein oxidatively forms an intramolecular disulfide bond between a newly engineered Cys and an existing Cys residue, both Cys thiol groups become unavailable for active site involvement and interaction. Additionally, the protein can become inactive or non-specific due to misfolding or loss of three-dimensional structure (Zhang et al (2002) Anal. Biochem. 311:1-9).

[0167] Antibodies engineered with cysteine are designed as FAB antibody fragments (thioFab) and expressed as full-length IgG monoclonal (thioMab) antibodies (Junutula, J. R. et al. (2008) J Immunol Methods 332:41-52; US2007 / 0092940, the contents of which are incorporated by reference). ThioFab and thioMab antibodies were conjugated via a linker at the position of the newly introduced cysteine thiol with a thiol-reactive linker reagent and a drug-linker reagent to prepare antibody-drug conjugates (thioADC).

[0168] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0169] polypeptide Aspects of the invention include compositions comprising a SPIK polypeptide, e.g., an AS-SPIK polypeptide encoded by any one of the nucleic acid sequences of SEQ ID NOs: 104 and 105. As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, although generally these terms refer to peptide sequences of various sizes. The inventors refer to the amino acid-based compositions of the invention as “polypeptides,” to convey that the compositions are linear polymers of amino acid residues, and this may be useful to distinguish the compositions from full-length proteins. The polypeptides according to embodiments of the invention may “consist of” or “comprise” fragments of an AS-SPIK polypeptide or an NS-SPIK polypeptide, and the invention encompasses polypeptides that consist of or comprise biologically active variants of an AS-SPIK polypeptide or an NS-SPIK polypeptide. Thus, it will be understood that the polypeptides may contain only fragments of an AS-SPIK polypeptide or an NS-SPIK polypeptide (or biologically active variants thereof), but may also contain additional residues thereon. Biologically active variants retain sufficient activity to inhibit proteases.

[0170] The bond between the above amino acid residues may be a conventional peptide bond or another covalent bond (such as an ester or ether bond), and the above polypeptide may be modified by amidation, phosphorylation, or glycosylation. The modification may affect the polypeptide backbone and / or one or more side chains. Chemical modification may be a naturally occurring modification (e.g., glycosylation in a bacterial host) that occurs in vivo following translation of the mRNA encoding the polypeptide, or a synthetic modification that occurs in vitro. A biologically active variant of the AS-SPIK polypeptide or NS-SPIK polypeptide may contain one or more structural modifications derived from any combination of naturally occurring (i.e., naturally occurring in vivo) and synthetic modifications (i.e., naturally occurring or non-naturally occurring modifications that occur in vitro). Examples of modifications include, but are not limited to, amidation (e.g., substitution of the free carboxyl group at the C-terminus with an amino group); biotinylation (e.g., acylation of a lysine or other reactive amino acid residue with a biotin molecule); glycosylation (e.g., production of a glycoprotein or glycopeptide by addition of a glycosyl group to any of an asparagine, hydroxylysine, serine, or threonine residue); acetylation (e.g., addition of an acetyl group, usually at the N-terminus of the polypeptide); alkylation (e.g., addition of an alkyl group); isoprenylation (e.g., addition of an isoprenoid group); lipoylation (e.g., attachment of a lipoic acid moiety); and phosphorylation (e.g., addition of a phosphate group to serine, tyrosine, threonine, or histidine).

[0171] One or more of the above amino acid residues in the biologically active variant may be non-naturally occurring amino acid residues. Naturally occurring amino acid residues include amino acid residues naturally encoded by the genetic code, as well as non-standard amino acids (e.g., amino acids having a D-configuration instead of an L-configuration). The peptides of the present invention may also include amino acid residues that are modified versions of standard residues (e.g., pyrrolysine may be used instead of lysine, and selenocysteine may be used instead of cysteine). Non-naturally occurring amino acid residues are amino acid residues that are not found in nature but conform to the basic formula of an amino acid and can be incorporated into a peptide. Examples of non-naturally occurring amino acid residues include D-alloisoleucine (2R,3S)-2-amino-3-methylpentanoic acid and L-cyclopentylglycine (S)-2-amino-2-cyclopentylacetic acid. For other examples, reference can be made to textbooks or the World Wide Web (the site is currently maintained by the California Institute of Technology and displays the structures of non-natural amino acids that have been successfully incorporated into functional proteins).

[0172] Alternatively, or in addition to the above, one or more of the above amino acid residues in the biologically active variant may be a naturally occurring residue that is different from the naturally occurring residue present at 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 inventors may sometimes refer to substitutions, additions, or deletions of amino acid residues as mutations of the wild-type sequence. As described above, substitutions may involve replacing a naturally occurring amino acid residue with a non-naturally occurring residue or simply a different naturally occurring residue. Further, substitutions may constitute conservative substitutions or non-conservative substitutions. Conservative amino acid substitutions typically include 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.

[0173] Polypeptides that are biologically active variants of AS-SPIK can be characterized in terms of the degree to which their sequences are similar or homologous to the corresponding wild-type polypeptides. For example, the sequence of a biologically active variant may be at least 80% 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 an NS-SPIK polypeptide may have an amino acid sequence having at least 80% or about 80% sequence homology (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% or about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence homology) (or the percent identity shown by way of example) to an AS-SPIK or NS-SPIK polypeptide (SEQ ID NOs: 81, 82, 83) or a homolog or ortholog thereof.

[0174] Biologically active variants of AS-SPIK polypeptides or NS-SPIK polypeptides will retain sufficient biological activity to be useful in the methods of the invention. The biologically active variants described above will retain sufficient activity to function as inhibitors of protease activity. The biological activity can be evaluated by methods known to those of skill in the art, including, but not limited to, in vitro cleavage assays or functional assays.

[0175] Polypeptides can be produced by a variety of methods including, for example, recombinant techniques or chemical synthesis. Once the polypeptides are produced, they can be isolated and purified to any desired extent. For example, reverse phase (preferably) or normal phase HPLC, or size exclusion or partition chromatography on a polysaccharide gel medium such as Sephadex G-25, followed by lyophilization may be used. The composition of the final polypeptide can be confirmed by amino acid sequencing or amino acid analysis by FAB-MS techniques after degradation of the peptide by standard means. Salts, esters, amides, and N-acyl derivatives of the amino groups of the polypeptide containing acidic salts can be prepared using methods known in the art, and such peptides are useful in the context of the present invention.

[0176] An AS-SPIK complex is also provided. The AS-SPIK complex according to an embodiment of the present invention includes AS-SPIK and an antibody of the present invention described herein that specifically or preferentially binds to the AS-SPIK polypeptide or a fragment thereof. The above fragment particularly has a length of at least 23 amino acids (SEQ ID NO: 81), preferably at least 10 amino acids, more preferably has at least the 7th to 23rd amino acids (including both ends) of the amino acids of SEQ ID NO: 81, and even more preferably has at least the 8th to 17th amino acids (including both ends) of SEQ ID NO: 81. The above antibody may be any of the aforementioned anti-AS-SPIK antibodies. The above AS-SPIK polypeptide or a fragment thereof may be the aforementioned AS-SPIK polypeptide or a fragment thereof. In some embodiments, the above antibody is an anti-AS-SPIK monoclonal antibody, 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 above AS-SPIK polypeptide is a polypeptide having an amino acid sequence with at least 98% homology (or identity) to the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 81. In some embodiments, the above AS-SPIK polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 82.

[0177] 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 may form immune complexes with AS-SPIK or AS-SPIK peptides under certain conditions. The above complex may be precipitated from the solution for further analysis, for example, in a sandwich ELISA assay. The above immune complex can be captured by a 96-well plate immobilized with a second anti-SPIK antibody as a carrier. When the antibody in the above complex is labeled with a reporter such as horseradish peroxidase (HPR), the amount of the formed AS-SPIK immune complex can be measured. The above AS-SPIK immune complex can also be captured by agarose beads conjugated with protein A or G for Western blot analysis.

[0178] Nucleic acid The terms "nucleic acid" and "polynucleotide" are used synonymously herein and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) including nucleic acid analogs, any of which may encode a polypeptide of the present invention and all of which are encompassed by the present invention. The polynucleotide may have essentially any three-dimensional structure. The nucleic acid may be double-stranded or single-stranded (i.e., sense or antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA) and portions 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, as well as nucleic acid analogs. In the context of the present invention, the nucleic acid may encode a fragment of a naturally occurring AS-SPIK polypeptide or NS-SPIK polypeptide or a biologically active variant thereof. Non-limiting examples of nucleic acid sequences according to embodiments of the present invention include SEQ ID NOs: 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 one preferred embodiment, the nucleic acid sequence comprises nucleotide residues 28 to 105 of SEQ ID NO: 105. In one preferred embodiment, the nucleic acid sequence comprises nucleotide residues 49 to 105 of SEQ ID NO: 105. Lu et al., Immunology 2011;134(4):398-408.

[0179] An "isolated" nucleic acid may be, for example, a DNA molecule or fragment thereof that occurs in nature, provided that at least one of the nucleic acid sequences that are normally adjacent to the DNA molecule in the naturally occurring genome has been removed or is absent. Thus, isolated nucleic acids can include, but are not limited to, DNA molecules that exist as separate molecules independent of other sequences (e.g., chemically synthesized nucleic acids, or cDNA or genomic DNA fragments generated by polymerase chain reaction (PCR) or restriction endonuclease treatment). Isolated nucleic acids also refer to DNA molecules incorporated into a vector, an autonomously replicating plasmid, a virus, or the genomic DNA of a prokaryotic or eukaryotic organism. Additionally, isolated nucleic acids may include engineered nucleic acids such as DNA molecules that are part of a hybrid or fusion nucleic acid. For example, a cDNA library or genomic library, or a nucleic acid present among many (e.g., dozens, or hundreds to millions) of other nucleic acids in a gel slice containing a genomic DNA restriction digest is not an isolated nucleic acid.

[0180] Isolated nucleic acid molecules can be generated, for example, by polymerase chain reaction (PCR) techniques that can be used to obtain isolated nucleic acids containing the nucleotide sequences described herein, including nucleotide sequences encoding the polypeptides described herein. PCR can be used to amplify specific sequences derived from DNA as well as RNA, including sequences derived from total genomic DNA or total cellular RNA. Generally, oligonucleotide primers are designed that have the same or similar sequence to the opposite strand of the template to be amplified, using sequence information from the region of interest or subsequent termini. A variety of PCR strategies are also available that can introduce site-specific nucleotide sequence modifications into the template nucleic acid.

[0181] The isolated nucleic acid can be chemically synthesized either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite techniques) or as a series of oligonucleotides. For example, pairs of longer oligonucleotides (e.g., >50 - 100 nucleotides) can be synthesized that contain the desired sequence, each pair containing short segments of complementarity (e.g., about 15 nucleotides) such that a double-strand is formed when the oligonucleotide pairs are annealed. The oligonucleotides can be extended using DNA polymerase to generate a single double-stranded nucleic acid molecule for each oligonucleotide pair, which can then be ligated into a vector. The isolated nucleic acid of the present invention can also be obtained, for example, by mutagenesis of a naturally occurring portion of DNA encoding AS-SPIK or NS-SPIK (e.g., by the aforementioned production methods).

[0182] The two nucleic acids or the polypeptides encoded by the nucleic acids described above can be described as having a certain degree of homology or identity to each other. For example, the AS-SPIK polypeptide or NS-SPIK polypeptide and their biologically active variants can be described as showing a certain degree of homology or identity. Alignments can be collected by placing short AS-SPIK polypeptide or NS-SPIK polypeptide sequences on the Protein Information Research (PIR) site (http: / / pir.georgetown.edu) and then analyzing them using the Basic Local Alignment Search Tool (BLAST) algorithm for "short nearly identical sequences" on the NCBI website (http: / / www.ncbi.nlm.nih.gov / blast).

[0183] To determine the homology or identity of sequences, for example, BioEdit (version 4.8.5, North Carolina State University) (in BioEdit, the alignment of nucleic acid sequences or protein sequences can be performed over the entire length of those sequences (global alignment)), or a computer program such as ALIGN-2 described above can be used to align a query nucleic acid sequence or a query amino acid sequence against one or more subject nucleic acid sequences or subject amino acid sequences, respectively.

[0184] BioEdit can calculate the optimal match between a query sequence and one or more subject sequences, perform the alignment of the above sequences, and as a result, determine identity, similarity, and differences. Gaps of one or more residues can be inserted into the query sequence, the subject sequence, or both to maximize the sequence alignment. To speed up the pairwise alignment of nucleic acid sequences, the following default parameters are used. That is, word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used. That is, gap start penalty: 10.0; gap extension penalty: 5.0; and weight transition: yes. To speed up the pairwise alignment of protein sequences, the following parameters are used. That is, word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; and gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used. That is, weighting matrix: blosum; gap start penalty: 10.0; gap extension penalty: 0.05; hydrophilic gap: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalty: on. The output is a sequence alignment reflecting the relationship between the sequences.

[0185] To determine the percent sequence identity between a query sequence and a subject sequence, in BioEdit, divide the number of identities in the optimal alignment by the number of residues being compared (excluding gap positions), and multiply the result by 100. This output is the percent sequence identity of the subject sequence relative to the query sequence. Note that this percent sequence identity value may be rounded to the nearest tenth of a decimal. For example, 78.11, 78.12, 78.13, and 78.14 are truncated to 78.1, and 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0186] The nucleic acids and polypeptides described herein may be referred to as "exogenous." The term "exogenous" indicates 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 to another, i.e., a heterologous nucleic acid. Typically, such exogenous nucleic acids are introduced into other species via recombinant nucleic acid constructs. An exogenous nucleic acid may be one that is native to an organism and is reintroduced into the cells of that organism. Exogenous nucleic acids containing native sequences can often be distinguished from the native sequences by the presence of non-native sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences adjacent to the native sequences in a recombinant nucleic acid construct. Further, exogenous nucleic acids that have been stably transformed are typically integrated at positions other than where the native sequences are found.

[0187] Also provided herein are recombinant constructs that 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 operably linked to regulatory regions suitable for expressing AS-SPIK or NS-SPIK in a particular cell. It will be appreciated that multiple nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known in the art. For many amino acids, there are multiple nucleotide triplets that function as codons for that amino acid. For example, the codons in the coding sequence of AS-SPIK or NS-SPIK can be changed using the appropriate codon bias table for that organism to obtain optimal expression in a particular organism.

[0188] Also provided are vectors comprising nucleic acids such as the nucleic acids described herein. A "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted and replicated. Generally, a vector has the ability to replicate when associated with appropriate control elements. Suitable vector backbones include, for example, those commonly used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An "expression vector" is a vector that contains regulatory regions. A variety of host / expression vector combinations can be used to express the nucleic acid sequences described herein. Suitable expression vectors include, for example, but are not limited to, plasmids and viral vectors derived from bacteriophage, baculovirus, and retrovirus.

[0189] The vectors provided in this specification may include, for example, an origin of replication, a scaffold attachment region (SAR), and / or a marker. The marker gene can confer a selectable 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 the 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™ tag (Kodak, New Haven, CT) are typically expressed as a fusion with the encoded polypeptide and such tags can be inserted at any position within the polypeptide, including either the carboxyl or amino terminus.

[0190] Additional expression vectors may include, for example, segments of chromosomal, episomal, and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, such as the E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9, and their derivatives, plasmids such as RP4; phage DNA, such as derivatives of phage λ, e.g., NM989, and other phage DNA, such as M13 and filamentous single-stranded phage DNA; yeast plasmids such as the 2μ plasmid or its derivatives, vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to use phage DNA or other expression control sequences.

[0191] The above vector may also include a regulatory region. The term "regulatory region" refers to a nucleotide sequence that affects the initiation and rate of transcription or translation, as well as the stability and / or mobility of the transcription or translation product. Examples of 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, stop sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0192] As used herein, the term "operably linked" refers to the arrangement of a regulatory region and a sequence to be transcribed in a nucleic acid such that transcription or translation of such a sequence is affected. For example, to place a coding sequence under the control of a promoter, the translation start site of the translation reading frame of the polypeptide is typically located between about 1 and about 50 nucleotides downstream of the promoter. However, the promoter may be located up to about 5,000 nucleotides upstream of the translation start site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically includes at least a core (basic) promoter. A promoter may also include at least one control element such as an enhancer sequence, upstream element, or upstream activation region (UAR). The choice of 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. It is routine for those skilled in the art to regulate the expression of a coding sequence by appropriately selecting and arranging a promoter and other regulatory regions for the coding sequence.

[0193] Vectors containing AS-SPIK or NS-SPIK nucleic acid sequences can be constructed in a manner that promotes uptake by cells, i.e., prokaryotic or eukaryotic cells, such as mammalian cells. Useful vector systems and constructs are as described above. In some embodiments, the vectors may deliver the above compositions to specific cell types. However, the invention is not so limited, and other DNA delivery methods are contemplated, such as chemical transfection using, for example, calcium phosphate, DEAE dextran, liposomes, lipoplexes, surfactants, and perfluorinated chemical liquids, as well as physical delivery methods such as electroporation, microinjection, ballistic particles, and "gene gun" systems. In some embodiments, the polynucleotides of the invention may also be used in conjunction with microdelivery vehicles such as cationic liposomes, other lipid-containing complexes, and other polymeric complexes having the ability to mediate delivery of polynucleotides to host cells. Another delivery method is to use single-stranded DNA production vectors that can produce the expressed product intracellularly.

[0194] Method of Use The compositions disclosed herein are generally and diversely useful for the diagnosis and / or treatment of disorders characterized by the expression of AS-SPIK. Such disorders include, but are not limited to, cancer, viral infections, and inflammatory disorders. One notable example is liver cancer. Other non-limiting examples include the cancers described herein in relation to the definition of the term "cancer". Accordingly, aspects of the invention include methods of diagnosing and / or treating cancer (e.g., liver cancer) in a subject having the above cancer or at risk of developing the above cancer. The terms "subject", "patient", and "individual" are used interchangeably herein.

[0195] In some embodiments, the method comprises contacting a biological test sample from a subject with an AS-SPIK antibody or antigen-binding fragment thereof to generate 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 has developed the disorder or is at risk of developing the disorder. In certain embodiments, the method comprises contacting a biological test sample with a first antibody or antigen-binding fragment that binds to SPIK to generate a SPIK-antibody complex; contacting the SPIK-antibody complex with an AS-SPIK antibody or antigen-binding fragment to generate 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 has developed the disorder or is at risk of developing the disorder. Some non-limiting examples of antibodies that can be used in such methods are described herein.

[0196] In some embodiments, the method comprises administering to a patient suffering from a disease or disorder characterized by AS-SPIK expression a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, or antibody-drug conjugate described herein.

[0197] Liver cancer One prominent example of a disorder characterized by AS-SPIK expression is liver cancer. Liver cancer encompasses a wide range of diseases that cause damage to the liver and liver dysfunction. Liver cancer can be caused, for example, by infectious agents, diseases, trauma, or genetic diseases, or combinations of infectious agents, diseases, trauma, and genetic diseases.

[0198] Liver cancers include primary liver cancers, such as diseases associated with abnormal cell proliferation, for example, hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma, and hepatoblastoma. Such cancers can include cancers at any stage of disease progression, such as very early HCC (stage 0 of the Barcelona Clinic Liver Cancer staging system (BCLC) and tumor size <2 cm), early stage (BCLC stage A, tumor size 2 cm to 5 cm), intermediate stage (BCLC stage B, medium tumor size >5 cm), late stage (BCLC stages C and D, advanced), or metastatic stage (Pons et al., HPB 2005;7(1):35-41), and also early stage ICC of ICC (stages I, II, and IIIa, tumor size <2 cm), intermediate stage (stages IIIb and IIIc, tumor size ≧2 cm), and late stage (stage IV) (Farges et al., Cancer 2011;117(10):2170-2177).

[0199] Liver cancer may 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 infectious diseases can be either acute or chronic.

[0200] Liver cancer may also occur from liver damage, for example, cirrhosis. Cirrhosis is late-stage scarring or fibrosis of the liver and may be caused by various forms of liver disease and illness. Cirrhosis may occur as a result of genetic diseases, such as hemochromatosis, cystic fibrosis, Wilson's disease, and autoimmune diseases. Cirrhosis may also be due to hepatitis virus infection and alcohol consumption.

[0201] Liver cancer may also occur from other diseases, including, but not limited to, alcoholic liver disease; disorders associated with abnormal fat content of the liver, such as fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatosis, and hepatic fibrosis.

[0202] Biological sample "Biological sample", "test sample", or "sample" refers to a sample obtained from or derived from a patient. The sample may 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 may be a tissue sample. Exemplary tissue samples include biopsy specimens such as liver biopsy specimens, or primary cell culture specimens prepared from the patient's cells, or the supernatant of a primary culture.

[0203] Immunoassay Aspects of the present invention include diagnostic assay methods, such as diagnostic immunoassays, which can be used to detect the presence or absence of AS-SPIK in a test sample. The immunoassay format used for the detection of AS-SPIK can be configured in various forms. Examples of such immunoassays include homologous and heterologous assays, competitive and non-competitive assays, both direct and indirect assays, and "sandwich" assays. Useful formats include enzyme immunoassays, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), electrochemiluminescent assay, radioimmunoassay, immunofluorescence, fluorescence polarization assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, immunoblotting, agglutination, luminescent proximity assay, and nephelometry, but are not limited thereto.

[0204] Regardless of the method, the biological sample is contacted with the anti-AS-SPIK antibody of the present invention. In some embodiments, the biological sample may be immobilized on a solid support. In some embodiments, the biological sample is contacted with the anti-SPIK antibody of the present invention immobilized on a solid support. The solid support may be, for example, a plastic surface, a glass surface, a paper or fiber surface, or a particle surface. More specifically, examples of the support include microplates, beads, polyvinylidene difluoride (PVDF) membranes, nitrocellulose membranes, nylon membranes, porous membranes, and non-porous membranes. The composition of the substrate can be changed. 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 made of particulate matter (e.g., glass or various thermoplastic polymers), or a cast membrane film made of nitrocellulose, nylon, or polysulfone. In a general embodiment, the substrate is any surface or support on which an antibody or polypeptide can be immobilized, including one or more solid supports (e.g., glass such as a slide glass or a coated plate, silica, plastic or derivatized plastic, paramagnetic or non-magnetic metal), semi-solid supports (e.g., polymeric materials, gels, agarose, or other matrices), and / or porous supports (e.g., filters, nylon or nitrocellulose membranes or other membranes). In some embodiments, for example, synthetic polymers including polystyrene, polypropylene, glycidyl methacrylate, aminated or carboxylated polystyrene, polyacrylamide, polyamide, and polyvinyl chloride may be used as the substrate.

[0205] In some embodiments, the immunoassay method may be a two-antibody "sandwich" assay. The biological sample is contacted with an anti-SPIK antibody of the invention immobilized on a solid support, such as a microtiter plate. The sample and the first antibody are incubated under conditions favorable for specific binding and formation of the SPIK-antibody complex. Following the contacting step, unbound components of the biological sample are removed. The complex is then contacted with a second anti-SPIK antibody. The second antibody binds to a SPIK epitope different from the epitope to which the first antibody is bound. Thus, the first and second antibodies do not competitively inhibit each other with respect to binding to SPIK. In some embodiments, the first antibody can recognize an epitope, i.e., an antigenic determinant, present on both AS-SPIK and NS-SPIK. The inventors may sometimes refer to such an antibody as a "pan-SPIK" antibody. Alternatively, the first antibody can recognize an epitope 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 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 binds specifically or preferentially to AS-SPIK and does not bind specifically to NS-SPIK. Alternatively, the sandwich assay can be configured such that both the first antibody and the second antibody bind specifically or preferentially to AS-SPIK and do not bind specifically to NS-SPIK.

[0206] Antibody binding can be measured in various ways. For example, the signal generated by a detectable label may be analyzed, and in the case where applicable, quantified using an optical scanner or other image acquisition device and software that enables measurement of signals related to complex formation, such as fluorescence signals, luminescence signals, or phosphorescence signals, or radioactive signals. Exemplary devices for measuring detectable signals include, but are not limited to, microplate readers, fluorometers, spectrophotometers, and gamma counters.

[0207] Reference sample The level of AS-SPIK in a biological sample may be compared to the level of a reference sample. Standard reference levels typically correspond to the average AS-SPIK levels derived from a population of individuals. The reference population may include individuals of similar age, body size, ethnic background, or general health status as the individual of interest. Thus, the AS-SPIK level in a patient's sample may be compared to values from 1) individuals with known liver cancer who express AS-SPIK and have AS-SPIK in their body fluids; and 2) individuals without liver cancer who have low levels of AS-SPIK in their body fluids.

[0208] Generally, a high level of AS-SPIK is greater than either the level of AS-SPIK present in a control sample or the average level (reference value) of AS-SPIK present in samples from a population of normal and healthy individuals without liver cancer, preferably at least 1, 2, 3, 4, or 5% greater, more preferably at least 5% greater, and may be any level of AS-SPIK. A low level of AS-SPIK may be any level of AS-SPIK that is less than either the level of AS-SPIK present in a control sample or the average level of AS-SPIK present in samples from a population of individuals with liver cancer. The average level of AS-SPIK present in samples from a population of normal and healthy individuals can be measured using any population size. For example, a population of 2 to 250 individuals, such as 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250 or more individuals can be used to measure the average level of AS-SPIK in samples from a population of normal and healthy individuals, and the larger the sample population, the higher the accuracy of the measurement.

[0209] In some embodiments, a reference chart can be used to determine whether a particular level of AS-SPIK in a sample is elevated compared to a control sample or a larger population. For example, the reference chart may include the normal range of AS-SPIK present in healthy individuals of similar age, ethnic background, or general health status as the individual being tested. This reference chart can be used to classify any level of AS-SPIK measured in a sample as low, normal, or high compared to a control sample or compared to the average value from a larger population. The term "high level" is defined as a level that is higher than the reference level, preferably at least 2% higher, more preferably at least 5% higher.

[0210] Alternatively, or in addition to the above, the level of AS-SPIK in a biological sample may be "normalized" against the level of one or more additional biological markers, for example, another marker whose expression is independent of the expression of AS-SPIK. That is, the levels of the additional markers can be evaluated in parallel with the level of AS-SPIK, either simultaneously or on separate occasions. The additional markers can serve as internal controls for sample preparation, handling, storage, and day-to-day assay variability. The values of the levels of AS-SPIK and the additional markers may be expressed as a ratio, which can be compared to similar ratios obtained for a reference sample or population. A useful second marker is alpha-fetoprotein.

[0211] 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 SPIK polypeptide or fragments thereof. When multiple samples are used, these samples may be samples of different concentrations. In one embodiment, the reference set may include six samples of recombinant AS-SPIK at concentrations of AS-SPIK of 50 ng / ml, 30 ng / ml, 8 ng / ml, 3 ng / ml, 1 ng / ml, and 0 ng / ml. The recombinant AS-SPIK may be purified by affinity chromatography (HPLC) using either an anti-AS-SPIK antibody such as IM-CA22 or an anti-tag antibody. Reference values in blood or other body fluids can vary. However, those skilled in the art are in a position to determine the average level of AS-SPIK in different body fluids for each population and to determine each reference value, such that the level of AS-SPIK in patients determined to have liver cancer is well above the reference value, while the level in patients detected not to have liver cancer or healthy individuals is well below each 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 present in samples from a population of normal and healthy individuals. Note that the levels of AS-SPIK in the biological sample and the control sample are measured by the same method and are thus comparable. For example, the absolute value of the AS-SPIK level can be measured via a calibration curve using the above recombinant AS-SPIK.

[0212] control sample 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 medium containing 100 ng / ml of AS-SPIK from the stable cell line S2-3. This was created 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.

[0213] The methods disclosed herein are useful for the detection of liver cancer in patients suspected of having or at risk of liver cancer. This method can also be used for the analysis of samples from patients who have received treatment for liver cancer, such as hepatocellular carcinoma, to determine whether the patient's hepatocellular carcinoma is at risk of remission. This method can also be used to monitor the course of treatment, such as treatment with a therapeutic agent such as a small molecule drug or a therapeutic antibody, chemotherapy, radiation therapy, or surgery, to determine the effectiveness of the treatment and to enable the treating clinician to change the treatment regimen as needed. This method can also be used for the detection, monitoring, or analysis of patients suffering from or at risk of any disorder associated with an increase in the level of AS-SPIK in a biological sample, such as a blood or serum sample obtained from a patient.

[0214] The methods disclosed herein may be used in combination 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.

[0215] Product The compositions described herein may be packaged in a suitable container, which is indicated to be, for example, for the detection, identification, and quantification of AS-SPIK in biological samples. The above product, also referred to as a "kit", may comprise an antibody, an antigen-binding fragment of an antibody, and / or an antibody-drug conjugate of the present invention, a medium, a purified sample of an antigen for use as a positive control, or any combination thereof. The container included in the kit may contain a composition comprising an antibody of the present invention that specifically or preferentially binds to AS-SPIK but does not bind to NS-SPIK. The kit may comprise an antibody that binds to both AS-SPIK and NS-SPIK. A suitable buffer for diluting or reconstituting the test sample and the antibody may be provided. Some components may be provided in a dry form and may need to be reconstituted. The above anti-SPIK antibody may be pre-bound to an assay device, such as a microplate. Thus, in one embodiment, a kit for the detection, identification, and quantification of AS-SPIK comprises an anti-AS-SPIK antibody and a pan-SPIK antibody. The kit may optionally comprise a detectable label.

[0216] Accordingly, a packaged product (e.g., a sterile container containing one or more of the compositions described herein, packaged at a concentrated or ready-to-use concentration for storage, transportation, or sale), and a kit comprising at least one composition of the present invention, such as an anti-AS-SPIK antibody, are also within the scope of the present invention. The product may comprise a container (e.g., a vial, jar, bottle, bag, etc.) containing one or more compositions of the present invention. The product may also further comprise, for example, a packaging material, instructions for use, a syringe, a delivery device, a buffer, or other control reagents for treating or monitoring a disease for which diagnosis or treatment is required.

[0217] The kit of the present invention may provide reagents for a specific type of assay. Thus, the kit may comprise a group of beads (e.g., suitable for an agglutination assay or a lateral flow assay), or a plate (e.g., a plate suitable for an ELISA assay). In other embodiments, the kit comprises a lateral flow immunoassay device, an analytical rotor, or a device such as an electrochemical, optical, or optoelectronic sensor. The above-mentioned group of beads, the above-mentioned plate, and the above-mentioned device are useful for performing an immunoassay. For example, they may be useful for detecting the formation of a first agent - analyte - second agent complex.

[0218] Furthermore, the kit may comprise various diluents and buffers, a labeling complex or other agents for detecting specifically bound antigens or antibodies, and other signal generating reagents such as enzyme substrates, cofactors, chromogens. The kit may comprise one or more reference samples at various concentrations, for example, purified recombinant AS-SPIK. The kit may also comprise a positive control, for example, a cell supernatant derived from a cell line overexpressing AS-SPIK. Other components of the kit include coating reagents, polyclonal or monoclonal capture antibodies specific for the test antigen or analyte, or a cocktail 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 having an indicator molecule conjugated to the antibody, an indication color chart for comparison by colorimetric analysis, disposable gloves, decontamination procedure instructions, application rods or containers, sample preparation cups, etc. In one embodiment, the kit comprises a buffer or other reagent suitable for constituting a reaction medium that enables the formation of a peptide - antibody complex.

[0219] Such a kit provides a convenient and efficient method for a clinician to determine whether a subject has liver cancer or is at risk thereof. Thus, in certain embodiments, the kit further comprises instructions for use. The product may include instructions (e.g., a printed label or accompanying document, or other medium that describes how to use the product (e.g., an audio tape or video tape)). The instructions may be attached to the container (e.g., affixed to the container) and may describe the method to follow when performing the assay, the indications of the kit, and other uses.

[0220] The following examples are presented for illustrative purposes only and are not intended to limit the scope of the invention. Although several embodiments are presented in this disclosure, it should be understood that the disclosed compositions and methods can be implemented in many other specific forms without departing from the spirit or scope of this disclosure. The examples are to be considered illustrative and not restrictive, and their intent is not to be limited to the details presented herein. Various examples of changes, substitutions, and modifications will be apparent to those skilled in the art and can be made without departing from the spirit and scope disclosed herein.

Example

[0221] Example 1: Production of a Rabbit Monoclonal Antibody Specific for AS-SPIK The 10 monoclonal antibodies described herein were generated from rabbits. Briefly, rabbits were immunized with recombinant AS-SPIK containing different subsets of 23 additional amino acids (SEQ ID NO: 81). After 3 or 4 immunizations, the blood was tested by ELISA. Briefly, blood from the immunized rabbits was reacted with plates coated with recombinant AS-SPIK. After washing, the plates were developed by incubation with an anti-rabbit antibody labeled with HRP (horseradish peroxidase), and the optical density was measured after reaction with the substrate TMB. Rabbits that were positive in the test (antibodies produced) were sacrificed. Then, monoclonal antibodies were established and further screened by ELISA using plates immobilized with partially purified AS-SPIK from S2-3 cells (a cell line that expresses large amounts of AS-SPIK by the integrated full-length SPIK gene, Lu et al. Tumor-associated protein SPIK / TATI suppresses serine protease dependent cell apoptosis, Apoptosis, 2008 13(4), 483-494) and NS-SPIK from pancreatic cells. Finally, 10 monoclonal antibodies that strongly bind to AS-SPIK but not to NS-SPIK were selected. Figure 1 shows 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 show high binding activity to AS-SPIK, while the binding activity to NS-SPIK is only at the background level as a negative control (negative control in Figure 1). This data suggests that the rabbit monoclonal antibodies identified herein recognize only AS-SPIK. Furthermore, all 10 antibodies can strongly bind to AS-SPIK and have similar binding affinities for AS-SPIK.

[0222] Example 2: Detection of antibody-antigen complexes in samples containing IM-E2 In the media of S2-3, PanC1, the sera of HCC patients, and the sera of healthy individuals, 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 and AS-SPIK were measured by sandwich ELISA. Briefly, the immune complexes of antibodies and AS-SPIK in different samples were formed by incubating the antibodies with the above samples at 37 °C for 0.5 h. Then, this mixture was reacted with a 96-well plate immobilized with a polyclonal anti-SPIK antibody at 37 °C for 1 h. Next, this plate was incubated with an anti-rabbit antibody labeled with HRP at 37 °C for 45 min. The amount of the antibody-antigen complex was measured by the optical density after the reaction with the substrate TMB. Figure 2 shows the test results of IM-E2. Similar results were obtained from other antibodies. These results clearly show that IM-E2 can form an antibody-antigen complex with AS-SPIK (Figure 2; S2-3: OD (1.389)), but does not form a complex with NS-SPIK (Figure 2, PanC1: OD (0.061)). The high OD (1.477) is also seen in the sera of HCC patients, but not in healthy individuals (Health: OD (0.106)), suggesting that antibodies such as IM-E2 described herein can be used for the detection of serum AS-SPIK.

[0223] Example 3: Sequences of the variable heavy and light chains of the antibodies and their CDRs The sequences of the variable heavy chains (VH) and variable light chains (VL) of all 10 antibodies were determined. Next, the CDRs of the above antibodies were identified. Table 1 shows the sequence numbers of VH and VL of 10 antibodies, namely 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 sequence numbers of the VH CDRs of these 10 antibodies, and Table 3 shows the sequence numbers of the VL CDRs of these 10 antibodies.

Table 1

Table 2

Table 3

[0224] Example 4: Consensus of VH and VL sequences Using the program named BioEdit Sequence Alignment Editor, University of North Carolina, the consensus of the sequences was determined by aligning the full sequences of VH and VL of 10 antibodies. The bottom rows of FIGS. 3 and 4 show the sequence identities of these 10 antibodies in either VH or VL. Obviously, there is a high similarity in the sequences in VH and VL for these 10 antibodies. The sequence identities of the above 10 antibodies are 85-95%.

[0225] Example 5: Consensus of CDR sequences The consensus of the sequences of the CDRs of the variable heavy chain was determined using the above software. FIG. 5 shows that all 10 antibodies have a high similarity in VH CDR1, and their identities are 66-100%. There is a 66% similarity in VHCRD2 of the 10 antibodies, and the identities are 50-66%. In contrast, the similarity of VH CDR3 of these 10 antibodies is very small. Only the amino acids in VH CDR3 are identical in all 10 antibodies. The consensus sequence shows that the similarity of the CDRs of VL is higher than that of VH in these 10 antibodies. There is an 82% similarity in VL CDR1 of the 10 antibodies, and their identities are 73-82% (FIG. 6). There is an 86% similarity in VL CDR2 of the 10 antibodies, and their identities are 72-86%. Regarding VL CDR3, there is a 75% similarity among 10 antibodies, but the identities are 45-75% (FIG. 6). SEQUENCE LISTING <110> IMCARE BIOTECH, LLC. <120> ANTI-SERINE PROTEASE INHIBITOR KAZAL (SPIK) ANTIBODIES, IMMUNOCONJUGATES, AND METHODS OF USE <130> IMC-0002-WO <140> PCT / US2020 / 041228 <141> 2020-07-08 <150> 62 / 871,565 <151> 2019-07-08 <160> 113 <170> PatentIn version 3.5 <210> 1 <211> 151 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 1 Leu Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser 1 5 10 15 Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr 20 25 30 Val Ser Gly Phe Ser Leu Ser Ser Asn Ala Ile Ser Trp Val Arg Gln 35 40 45 Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly Ala Ile Gly Ser Ser Gly 50 55 60 Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Val Thr Arg 65 70 75 80 Asn Thr Asn Leu Asn Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala 85 90 95 Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Trp Glu Asn Ile Gly Tyr 100 105 110 Thr Asn Val Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 125 Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys 130 135 140 Cys Gly Asp Thr Pro Ser Ser 145 150 <210> 2 <211> 148 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser Glu 1 5 10 15 Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr Val 20 25 30 Ser Gly Phe Ser Leu Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala 35 40 45 Pro Gly Asn Gly Leu Glu Trp Ile Gly Arg Ile Asn Ser Gly Gly Ala 50 55 60 Thr Asp Tyr Ala Ser Trp Ala Arg Ser Arg Ser Thr Ile Thr Arg Asp 65 70 75 80 Thr Asn Leu Asn Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala 85 90 95 Asp Thr Ala Thr Tyr Phe Cys Ala Lys Glu Glu Tyr Ser Tyr Gly Gly 100 105 110 Ala Tyr Gly Met Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp 130 135 140 Thr Pro Ser Ser 145 <210> 3 <211> 135 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 3 Leu Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser 1 5 10 15 Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr 20 25 30 Val Ser Gly Phe Ser Leu Ser Ser Tyr Gly Val Ser Trp Val Arg Gln 35 40 45 Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Trp Ser Gly Gly 50 55 60 Thr Thr Asp Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Ile Thr Arg 65 70 75 80 Asn Thr Asn Glu Asn Thr Val Thr Leu Lys Val Thr Ser Leu Thr Ala 85 90 95 Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Tyr Gly 100 105 110 Tyr Ala Ser Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 125 Gly Gln Pro Lys Ala Pro Ser 130 135 <210> 4 <211> 153 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 4 Leu Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser 1 5 10 15 Glu Gly Gly Leu Phe Lys Pro Thr Asp Ala Leu Thr Leu Thr Cys Thr 20 25 30 Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln 35 40 45 Ala Pro Gly Ser Gly Leu Glu Trp Ile Gly Ala Ile Asn Thr Tyr Gly 50 55 60 Gly Thr Tyr Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Ile Thr Arg 65 70 75 80 Asn Thr Asn Glu Asn Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala 85 90 95 Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Phe Asp Ser Asp Ala 100 105 110 Tyr Thr Ser Ala Ser Gly Gly Met Asp Pro Trp Gly Pro Gly Thr Leu 115 120 125 Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Phe Phe Pro Leu 130 135 140 Ala Pro Cys Cys Gly Asp Thr Pro Arg 145 150 <210> 5 <211> 149 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 5 Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser Glu 1 5 10 15 Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr Val 20 25 30 Ser Gly Phe Ser Leu Ser Ser Tyr Ala Ile Gly Trp Val Arg Gln Ala 35 40 45 Pro Gly Asn Gly Leu Glu Trp Ile Gly Thr Ile Val Thr Ser Gly Ile 50 55 60 Pro Tyr Tyr Ala Asn Trp Ala Lys Ser Arg Ser Thr Ile Thr Arg Asn 65 70 75 80 Thr Asn Leu Asn Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala Ala 85 90 95 Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Leu Asp Pro Ala Tyr Ser 100 105 110 Thr Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys 130 135 140 Gly Asp Thr Pro Ser 145 <210> 6 <211> 149 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 6 Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser Glu 1 5 10 15 Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr Val 20 25 30 Ser Gly Phe Ser Leu Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala 35 40 45 Pro Gly Asn Gly Leu Glu Trp Ile Gly Ala Ile Gly Lys Ser Gly Ser 50 55 60 Ala Tyr Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Ile Thr Arg Asn 65 70 75 80 Thr Asn Leu Asn Thr Val Ser Leu Lys Met Thr Ser Leu Thr Ala Ala 85 90 95 Asp Thr Ala Thr Tyr Phe Cys Ala Arg Trp Asp Ser Val Gly Trp Thr 100 105 110 Asp Ala Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys 130 135 140 Gly Asp Thr Pro Ser 145 <210> 7 <211> 136 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 7 Leu Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser 1 5 10 15 Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr 20 25 30 Val Ser Gly Phe Ser Leu Ser Ala Tyr Ala Ile Ser Trp Val Arg Gln 35 40 45 Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly Ala Ile Asn Ser Gly Gly 50 55 60 Ser Ala Tyr Tyr Ala Asn Trp Ala Lys Ser Arg Ser Thr Ile Thr Arg 65 70 75 80 Asn Thr Asn Leu Asn Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala 85 90 95 Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Asp Ile Tyr Asp Tyr 100 105 110 Gly Gly Ala Phe Asp Pro Trp Gly Pro Gly Thr Leu Val Thr Val Ser 115 120 125 Thr Gly Gln Pro Lys Leu His His 130 135 <210> 8 <211> 147 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 8 Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser Glu 1 5 10 15 Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr Val 20 25 30 Ser Gly Phe Ser Leu Ser Ile Tyr Gly Val Ser Trp Val Arg Gln Ala 35 40 45 Pro Gly Asn Gly Leu Glu Trp Ile Gly Ile Ile Tyr Ala Ser Gly Ser 50 55 60 Ala Asp Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Ile Thr Arg Asn 65 70 75 80 Thr Asn Leu Asn Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala Ala 85 90 95 Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Asp Asp Thr Tyr Gly Tyr 100 105 110 Thr Ser Ser Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp 130 135 140 Thr Pro Ser 145 <210> 9 <211> 146 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 9 Leu Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser 1 5 10 15 Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr 20 25 30 Val Ser Gly Phe Ser Leu Ser Ser Tyr Pro Ile Ser Trp Val Arg Gln 35 40 45 Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly Asp Ile Tyr Ala Ser Gly 50 55 60 Ser Ile Leu Tyr Ala Ser Trp Ala Thr Gly Arg Ser Thr Ile Thr Arg 65 70 75 80 Asn Thr Asn Leu Asn Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala 85 90 95 Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Val Ser Tyr Ser Gly Gly 100 105 110 Thr Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln 115 120 125 Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr 130 135 140 Pro Ser 145 <210> 10 <211> 148 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 10 Leu Val Ala Val Leu Lys Gly Val Gln Cys Gln Ser Val Lys Glu Ser 1 5 10 15 Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr Leu Thr Leu Thr Cys Thr 20 25 30 Val Ser Gly Phe Ser Leu Ser Ser Asn Val Ile Ser Trp Val Arg Gln 35 40 45 Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Asp Ile Tyr Val Ser Gly 50 55 60 Asn Thr Asp Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Ile Thr Arg 65 70 75 80 Asn Ala Asn Leu Asn Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala 85 90 95 Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Tyr Asp Met Ser Ser Asp 100 105 110 Ala Phe Asp Pro Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp 130 135 140 Thr Pro Ser Ser 145 <210> 11 <211> 114 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 11 Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala 1 5 10 15 Ser Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 20 25 30 Ser Gln Ser Ile Ser Thr Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly 50 55 60 Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 65 70 75 80 Thr Ile Ser Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 85 90 95 Gln Gly Tyr Ser Thr Ser Asp Val Asp Asn Ala Phe Gly Gly Gly Thr 100 105 110 Glu Gly <210> 12 <211> 134 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 12 Leu Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro 1 5 10 15 Ala Ser Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln 20 25 30 Ala Ser Glu Ser Ile Ser Thr Tyr Leu Ser Trp Leu Gln Gln Lys Pro 35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser 50 55 60 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr 65 70 75 80 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 85 90 95 Gln Gln Asp Tyr Thr Ile Ser Asn Val Gly Asn Val Phe Gly Gly Gly 100 105 110 Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile 115 120 125 Phe Pro Pro Ser Ala Asp 130 <210> 13 <211> 134 <212> PRT <213> Artificial Sequence <220> <22l> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 13 Leu Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro 1 5 10 15 Ala Ser Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln 20 25 30 Ala Ser Glu Ser Ile Ser Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro 35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser 50 55 60 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 65 70 75 80 Leu Thr Ile Ser Asp Gly Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 85 90 95 Gln Gln Gly Tyr Ser Val Ser Asn Val Asp Asn Ile Phe Gly Gly Gly 100 105 110 Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile 115 120 125 Phe Pro Pro Ser Ala Asp 130 <210> 14 <211> 132 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 14 Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala 1 5 10 15 Ser Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 20 25 30 Ser Gln Ser Ile Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Ile Pro Gly 35 40 45 Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly 50 55 60 Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 65 70 75 80 Thr Ile Ser Gly Val Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 85 90 95 Gln Gly Tyr Thr Ser Asn Val Asp Asn Val Phe Gly Gly Gly Thr Glu 100 105 110 Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro 115 120 125 Pro Ser Ala Asp 130 <210> 15 <211> 130 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 15 Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala 1 5 10 15 Ser Val Ser Glu Pro Val Arg Gly Thr Val Thr Ile Lys Cys Gln Ala 20 25 30 Ser Gln Ser Ile Ser Thr Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Ala Ser Gly 50 55 60 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu 65 70 75 80 Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys His 85 90 95 Gln Gly Tyr Ser Ala Ser Asn Val Asp Asn Thr Phe Gly Gly Gly Thr 100 105 110 Glu Gly Gly Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe 115 120 125 Pro Pro 130 <210> 16 <211> 133 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 16 Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala 1 5 10 15 Ser Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 20 25 30 Ser Gln Ser Ile Ser Thr Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Gln Arg Pro Lys Leu Leu Ile Tyr Gly Ala Ser Lys Leu Ala Ser Gly 50 55 60 Val Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu 65 70 75 80 Thr Ile Ser Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 85 90 95 Gln Gly Tyr Glu Thr Ser Asn Val Asp Asn Ala Phe Gly Gly Gly Thr 100 105 110 Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe 115 120 125 Pro Pro Ser Ala Asp 130 <210> 17 <211> 133 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 17 Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala 1 5 10 15 Ser Val Glu Val Thr Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 20 25 30 Ser Gln Gly Ile Ser Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly ​35 40 45 Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Thr Thr Leu Val Ser Gly 50 55 60 Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 65 70 75 80 Thr Ile Ser Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 85 90 95 Gln Asp Tyr Thr Thr Ser Asn Val Asp Asn Thr Phe Gly Gly Gly Thr 100 105 110 Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe 115 120 125 Pro Pro Ser Ala Asp 130 <210> 18 <211> 130 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 18 Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala 1 5 10 15 Ser Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 20 25 30 Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Gln Pro Pro Lys Arg Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly 50 55 60 Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 65 70 75 80 Thr Ile Ser Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 85 90 95 Gln Asp Tyr Ser Ser Asn Asn Ile Asp Asn Thr Phe Gly Gly Gly Thr 100 105 110 Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe 115 120 125 Pro Pro 130 <210> 19 <211> 130 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 19 Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala 1 5 10 15 [[ID=3)] Ser Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 20 25 30 Ser Glu Asp Ile Glu Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Pro Ser Gly 50 55 60 Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu 65 70 75 80 Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 85 90 95 Gln Asp Tyr Ser Ser Ser Asn Val Asp Asn Thr Phe Gly Gly Gly Thr 100 105 110 Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe 115 120 125 Pro Pro 130 <210> 20 <211> 133 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 20 Leu Trp Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro 1 5 10 15 Ala Ser Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln 20 25 30 Ala Ser Gln Asn Ile Gly Ser Tyr Leu Ser Trp Tyr Gln His Lys Pro 35 40 45 Gly Gln Arg Pro Arg Leu Leu Met Tyr Arg Ala Ser Thr Leu Ala Ser 50 55 60 Gly Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr 65 70 75 80 Leu Thr Ile Ser Gly Val Gln Cys Asp Asn Ala Ala Thr Tyr Tyr Cys 85 90 95 Gln Gln Gly Tyr Thr Asn Ser Gly Val Asp Asn Thr Phe Gly Gly Gly 100 105 110 Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile 115 120 125 Phe Pro Pro Ser Ala 130 <210> 21 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 21 Ser Ser Asn Ala Ile Ser 1 5 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 22 Ser Ser Tyr Ala Ile Ser 1 5 <210> 23 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 23 Ser Ser Tyr Gly Val Ser 1 5 <210> 24 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 24 Ser Ser Tyr Ala Ile Ser 1 5 <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 25 Ser Ser Tyr Ala Ile Gly 1 5 <210> 26 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 26 Ser Ser Tyr Ala Ile Ser 1 5 <210> 27 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 27 Ser Ala Tyr Ala Ile Ser 1 5 <210> 28 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 28 Ser Ile Tyr Gly Val Ser 1 5 <210> 29 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 29 Ser Ser Tyr Pro Ile Ser 1 5 <210> 30 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 30 Ser Ser Asn Val Ile Ser 1 5 <210> 31 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 31 Ala Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Ser 1 5 10 15 <210> 32 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 32 Arg Ile Asn Ser Gly Gly Ala Thr Asp Tyr Ala Ser Trp Ala Arg Ser 1 5 10 15 <210> 33 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 33 Ser Ile Trp Ser Gly Gly Thr Thr Asp Tyr Ala Ser Trp Ala Lys Ser 1 5 10 15 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <![CDATA[ ]]<400> 34 Ala Ile Asn Thr Tyr Gly Gly Thr Tyr Tyr Ala Ser Trp Ala Lys Ser 1 5 10 15 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 35 Thr Ile Val Thr Ser Gly Ile Pro Tyr Tyr Ala Asn Trp Ala Lys Ser 1 5 10 15 <210> 36 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 36 Ala Ile Gly Lys Ser Gly Ser Ala Tyr Tyr Ala Ser Trp Ala Lys Ser 1 5 10 15 <210> 37 <211> 16 [[ID=]62]<212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 37 Ala Ile Asn Ser Gly Gly Ser Ala Tyr Tyr Ala Asn Trp Ala Lys Ser 1 5 10 15 <210> 38 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 38 Ile Ile Tyr Ala Ser Gly Ser Ala Asp Tyr Ala Ser Trp Ala Lys Ser 1 5 10 15 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Asp Ile Tyr Ala Ser Gly Ser Ile Leu Tyr Ala Ser Trp Ala Thr Gly 1 5 10 15 <210> 40 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 40 Asp Ile Tyr Val Ser Gly Asn Thr Asp Tyr Ala Ser Trp Ala Lys Ser 1 5 10 15 <210> 41 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Arg Trp Glu Asn Ile Gly Tyr Thr Asn Val Arg Leu Asp Leu 1 5 10 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 42 Lys Glu Glu Tyr Ser Tyr Gly Gly Ala Tyr Gly Met 1 5 10 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 43 Arg Gly Gly Tyr Asp Tyr Gly Tyr Ala Ser Asn Ile 1 5 10 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 44 Arg Asp Phe Asp Ser Asp Ala Tyr Thr Ser Ala Ser Gly Gly Met Asp 1 5 10 15 Pro <210> 45 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 45 Arg Asn Leu Asp Pro Ala Tyr Ser Thr Thr Arg Leu Asp Leu 1 5 10 <210> 46 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 46 Arg Trp Asp Ser Val Gly Trp Thr Asp Ala Arg Leu Asp Leu 1 5 10 <210> 47 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 47 Arg Glu Asp Ile Tyr Asp Tyr Gly Gly Ala Phe Asp Pro 1 5 10 <210> 48 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 48 Arg Glu Asp Asp Thr Tyr Gly Tyr Thr Ser Ser Ile 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 49 Arg Val Ser Tyr Ser Gly Gly Thr Asp Ile 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 50 Arg Tyr Asp Met Ser Ser Asp Ala Phe Asp Pro 1 5 10 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 51 Gln Ala Ser Gln Ser Ile Ser Thr Ala Leu Ala 1 5 10 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 52 Gln Ala Ser Glu Ser Ile Ser Thr Tyr Leu Ser 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 53 Gln Ala Ser Glu Ser Ile Ser Ser Tyr Leu Ser 1 5 10 <210> 54 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 54 Gln Ala Ser Gln Ser Ile Asn Asn Tyr Leu Ser 1 5 10 <210> 55 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 55 Gln Ala Ser Gln Ser Ile Ser Thr Ala Leu Ala 1 5 10 <210> 56 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 56 Gln Ala Ser Gln Ser Ile Ser Thr Ala Leu Ala 1 5 10 <210> 57 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 57 Gln Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ser 1 5 10 <210> 58 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 58 Gln Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 59 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 59 Gln Ala Ser Glu Asp Ile Glu Ser Tyr Leu Ala 1 5 10 <210> 60 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 60 Gln Ala Ser Gln Asn Ile Gly Ser Tyr Leu Ser 1 5 10 <210> 61 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 61 Gly Ala Ser Thr Leu Ala Ser 1 5 <210> 62 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 62 Lys Ala Ser Thr Leu Ala Ser 1 5 <210> 63 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 63 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 64 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 64 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 65 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 65 Ala Ala Ser Tyr Leu Ala Ser 1 5 <210> 66 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 66 Gly Ala Ser Lys Leu Ala Ser 1 5 <210> 67 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 67 Ala Ala Thr Thr Leu Val Ser 1 5 <210> 68 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 68 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 69 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 69 Arg Ala Ser Thr Leu Pro Ser 1 5 <210> 70 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 70 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 71 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 71 Gln Gln Gly Tyr Ser Thr Ser Asp Val Asp Asn Ala 1 5 10 <210> 72 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 72 Gln Gln Asp Tyr Thr Ile Ser Asn Val Gly Asn Val 1 5 10 <210> 73 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 73 Gln Gln Gly Tyr Ser Val Ser Asn Val Asp Asn Ile 1 5 10 <210> 74 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 74 Gln Gln Gly Tyr Thr Ser Asn Val Asp Asn Val Phe 1 5 10 <210> 75 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 75 His Gln Gly Tyr Ser Ala Ser Asn Val Asp Asn Thr 1 5 10 <210> 76 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 76 Gln Gln Gly Tyr Glu Thr Ser Asn Val Asp Asn Ala 1 5 10 <210> 77 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 77 Gln Gln Asp Tyr Thr Thr Ser Asn Val Asp Asn Thr 1 5 10 <210> 78 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 78 Gln Gln Asp Tyr Ser Ser Asn Asn Ile Asp Asn Thr 1 5 10 <210> 79 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 79 Gln Gln Asp Tyr Ser Ser Ser Asn Val Asp Asn Thr 1 5 10 <210> 80 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 80 Gln Gln Gly Tyr Thr Asn Ser Gly Val Asp Asn Thr 1 5 10 <210> 81 <211> 23 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 81 Met Lys Val Thr Gly Ile Phe Leu Leu Ser Ala Leu Ala Leu Leu Ser 1 5 10 15 Leu Ser Gly Asn Thr Gly Ala 20 <210> 82 <211> 79 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 82 Met Lys Val Thr Gly Ile Phe Leu Leu Ser Ala Leu Ala Leu Leu Ser 1 5 10 15 Leu Ser Gly Asn Thr Gly Ala Asp Ser Leu Gly Arg Glu Ala Lys Cys 20 25 30 Tyr Asn Glu Leu Asn Gly Cys Thr Lys Ile Tyr Asp Pro Val Cys Gly 35 40 45 Thr Asp Gly Asn Thr Tyr Pro Asn Glu Cys Val Leu Cys Phe Glu Asn 50 55 60 Arg Lys Arg Gln Thr Ser Ile Leu Ile Gln Lys Ser Gly Pro Cys 65 70 75 <210> 83 <211> 56 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 83 Asp Ser Leu Gly Arg Glu Ala Lys Cys Tyr Asn Glu Leu Asn Gly Cys 1 5 10 15 Thr Lys Ile Tyr Asp Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Pro 20 25 30 Asn Glu Cys Val Leu Cys Phe Glu Asn Arg Lys Arg Gln Thr Ser Ile 35 40 45 Leu Ile Gln Lys Ser Gly Pro Cys 50 55 <210> 84 <211> 453 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 84 ctggtcgctg tgctcaaagg tgtccagtgt cagtcggtga aggagtccga gggaggtctc 60 ttcaagccaa cggataccct gacactcacc tgcacagtct ctggattctc cctcagtagc 120 aatgcaataa gctgggtccg ccaggctcca gggaacgggc tggaatggat cggagccatt 180 ggtagtagtg gtagcacata ctacgcgagc tgggcgaaaa gccgatccac cgtcaccaga 240 aacaccaacc tgaacacggt gactctaaag atgaccagtc tgacagccgc ggacacggcc 300 acctatttct gtgcgagatg ggagaatatt ggttatacta atgttcggtt ggatctctgg 360 ggccagggca ccctggtcac cgtctcctca gggcaaccta aggctccatc agtcttccca 420 ctggccccct gctgcgggga cacacccagc tcc 453 <210> 85 <211> 343 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 85 tggctcccag gtgccagatg tgcctatgat atgacccaga ctccagcctc tgtggaggta 60 gctgtgggag gcacagtcac catcaagtgc caggccagtc agagcattag cactgcatta 120 gcctggtatc agcagaaacc agggcagcct cccaagctcc tgatctatgg tgcatccact 180 ctggcatctg gggtctcatc gcggttcaaa ggcagtggat ctgggacaca gttcactctc 240 accatcagcg gcgtggagtg tgccgatgct gccacttact actgtcaaca gggttatagt 300 actagtgatg ttgataatgc tttcggcgga gggaccgagg ggg 343 <210> 86 <211> 445 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 86 ggtcgctgtg ctcaaaggtg tccagtgtca gtcggtgaag gagtccgagg gaggtctctt 60 caagccaacg gataccctga cactcacctg cacagtctct ggattctccc tcagtagcta 120 tgcaataagt tgggtccgcc aggctccagg gaacgggctg gaatggatcg ggcgcattaa 180 tagtggtggt gccacagact acgcgagctg ggcgagaagc cgatccacca tcaccagaga 240 caccaacctg aacacggtga ctctgcaaat gaccagtctg accgccgcgg acacggccac 300 ctatttctgt gcgaaagaag agtatagtta tggtggtgct tatggtatgt ggggcccagg 360 cactctggtc accgtctcct cagggcaacc taaggctcca tcagtcttcc cactggcccc 420 ctgctgcggg gacacaccca gctcc 445 <210> 87 <211> 404 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 87 ctctggctcc caggtgccag atgtgcctat gatatgaccc agactccagc ctccgtgtct 60 gcagctgtgg gaggcacagt caccatcaag tgccaggcca gtgagagcat tagtacctac 120 ttatcctggt tgcagcagaa accagggcag cctcccaagc tcctgatcta caaggcttcc 180 actctggcat ctggggtccc atcgcggttc aaaggcagtg gatctgggac agagttcact 240 ctcaccatca gcggtgtgca gtgtgacgat gctgccactt actactgtca acaggattat 300 actattagta atgttggtaa tgttttcggc ggagggaccg aggtggtggt caaaggtgat 360 ccagttgcac ctactgtcct catcttccca ccatcagcgg acca 404 <210> 88 <211> 406 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 88 cctggtcgct gtgctcaaag gtgtccagtg tcagtcggtg aaggagtccg agggaggtct 60 cttcaagcca acggataccc tgacactcac ctgcacagtc tctggattct ccctcagtag 120 ttatggagtg agctgggtcc gccaggctcc agggaagggg ctggagtgga tcgggtccat 180 ttggagtggt ggtaccacag actacgcgag ctgggcgaaa agccgatcca ccataaccag 240 aaacaccaac gagaacacgg tgactctgaa agtgaccagt ctgacagccg cggacacggc 300 cacctatttc tgtgcgaggg ggggttatga ttatggttat gcctcgaaca tctggggccc 360 aggcaccctg gtcaccgtct cctcagggca acctaaggct ccatca 406 <210> 89 <211> 404 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 89 ctctggctcc caggtgccag atgtgcctat gatatgaccc agactccagc ctctgtggag 60 gtagctgtgg gaggcacagt caccatcaag tgccaggcca gtgaaagcat tagcagctac 120 ttatcctggt atcagcagaa accagggcag cctcccaagc tcctgatcta cagggcttcc 180 actctggcat ctggggtccc atcgcggttc agtggcagtg gatctgggac agagttcact 240 ctcaccatca gcgacgggca gtgtgacgat gctgccactt actactgtca acagggttat 300 agtgttagta atgttgataa tattttcggc ggagggaccg aggtggtggt caaaggtgat 360 ccagttgcac ctactgtcct catcttccca ccatcagcgg acca 404 <210> 90 <211> 459 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 90 cctggtcgct gtgctcaaag gtgtccagtg tcagtcggtg aaggagtccg agggaggtct 60 cttcaagcca acggatgccc tgacactcac ctgcacagtc tctggattct ccctcagtag 120 ctatgcaata agctgggtcc gccaggctcc agggagcggg ctggaatgga tcggagccat 180 taatacttat ggtggcacat actacgcgag ctgggcgaaa agccgatcca ccatcaccag 240 aaacaccaac gagaacacgg tgactctgaa aatgaccagt ctgacagccg cggacacggc 300 cacctatttc tgtgcgagag acttcgatag tgatgcttat acttctgcta gtgggggcat 360 ggacccctgg ggcccaggga ccctcgtcac cgtctcttca gggcaaccta aggctccatc 420 attcttccca ctggccccct gctgcgggga cacacccag 459 <210> 91 <211> 400 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 91 tctggctccc aggtgccaga tgtgcctatg atatgaccca gactccagcc tctgtggagg 60 tagctgtggg aggcacagtc accatcaagt gccaggccag tcagagcatt aacaactact 120 tatcctggta tcagcaaata ccagggcagc ctcccaagct cctgatctac agggcatcca 180 ctctggcatc tggggtctca tcgcggttca aaggcagtgg atctgggaca cagttcactc 240 tcaccatcag cggcgtgcag tgtgccgatg ctgccactta ctactgtcaa cagggttata 300 ctagtaatgt tgataatgtt ttcggcggag ggaccgaggt ggtggtcaaa ggtgatccag 360 ttgcacctac tgtcctcatc ttcccaccat cagcggacca 400 <210> 92 <211> 450 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 92 ggtcgctgtg ctcaaaggtg tccagtgtca gtcggtgaag gagtccgagg gaggtctctt 60 caagccaacg gataccctga cactcacctg cacagtctct ggattctccc tcagtagcta 120 tgcaataggc tgggtccgcc aggctccagg gaacgggctg gaatggatcg gaaccattgt 180 tactagtggt atcccatact acgcgaactg ggcgaaaagc cgatccacca tcaccagaaa 240 caccaacctg aacacggtga ctctgaaaat gaccagtctg acagccgcgg acacggccac 300 ctatttctgt gcgagaaatt tagatcctgc ttatagtacc actcggttgg atctctgggg 360 ccagggcacc ctggtcaccg tctcctcagg gcaacctaag gctccatcag tcttcccact 420 ggccccctgc tgcggggaca cacccagctc 450 <210> 93 <211> 394 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 93 tctggctccc aggtgccaga tgtgcctatg atatgaccca gactccagcc tccgtgtctg 60 aacctgtgag aggcacagtc accatcaagt gccaggccag tcagagcatt agcactgcat 120 tagcctggta tcagcagaaa ccagggcagc ctcccaagct cctgatctat gctgcatcct 180 atctggcctc tggggtccca tcgcggttca gcggcagtgg atctgggaca gagttcactc 240 tcaccatcag cgacctggag tgtgccgatg ctgccactta ctactgtcat cagggttata 300 gtgctagtaa tgttgataat actttcggcg gagggaccga ggggggggtc aaaggtgatc 360 cagttgcacc tactgtcctc atcttcccac catc 394 <210> 94 <211> 450 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 94 ggtcgctgtg ctcaaaggtg tccagtgtca gtcagtgaag gagtccgagg gaggtctctt 60 caagccaacg gataccctga cactcacctg cacagtctct ggattctccc tcagtagcta 120 tgcaataagt tgggtccgcc aggctccagg gaacgggctg gaatggatcg gagccattgg 180 taaaagtggt agcgcatact acgcgagctg ggcgaaaagc cgatccacca tcaccagaaa 240 caccaacctg aacacggtgt cgctgaaaat gaccagtctg acagccgcgg acacggccac 300 ctatttctgt gcgagatggg atagtgttgg ttggactgat gctcggttgg atctctgggg 360 ccagggcacc ctggtcaccg tctcctcagg gcaacctaag gctccatcag tcttcccact 420 ggccccctgc tgcggggaca cacccagctc 450 <210> 95 <211> 402 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 95 ctggctccca ggtgccagat gtgcctatga tatgacccag actccagcct ctgtggaggt 60 agctgtggga ggcacagtca ccatcaagtg ccaggccagt cagagcatta gcactgcatt 120 agcctggtat cagcagaaac cagggcagcg tcccaagctc ctgatctatg gtgcatcgaa 180 actggcatct ggggtctcat cgcggttcag tggcagtgga tctgggacag agttcactct 240 caccatcagc ggcgtggagt gtgccgatgc tgccacttac tactgtcaac agggttatga 300 aactagtaat gttgataatg ctttcggcgg agggaccgag gtggtggtca aaggtgatcc 360 agttgcacct actgtcctca tcttcccacc atcagcggac ca 402 <210> 96 <211> 408 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 96 ctggtcgctg tgctcaaagg tgtccagtgt cagtcggtga aggagtccga gggaggtctc 60 ttcaagccaa cggataccct gacactcacc tgcacagtct ctggattctc cctcagtgcc 120 tatgcaataa gctgggtccg ccaggctcca gggaacgggc tggaatggat cggagccatt 180 aatagtggtg gtagcgcata ctacgcgaac tgggcgaaaa gccgatccac catcaccaga 240 aacaccaacc tgaacacggt gactctgaaa atgaccagtc tgacagccgc ggacacggcc 300 acctatttct gtgcgaggga agatatttat gattatggtg gtgcattcga tccctggggc 360 ccaggcaccc tggtcaccgt ctccacaggg caacctaagc tccatcat 408 <210> 97 <211> 401 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 97 ctggctccca ggtgccagat gtgcctatga tatgacccag actccagcct ctgtggaggt 60 aactgtggga ggcacagtca ccatcaagtg ccaggccagt caaggcatta gtagttactt 120 atcctggtat cagcagaaac cagggcagcc tcccaagctc ctgatctatg ctgcgaccac 180 tctggtatct ggggtctcat cgcggttcaa aggcagtgga tctgggacac agttcactct 240 caccatcagc ggcgtggagt gtgccgatgc tgccacttac tactgtcagc aggattatac 300 tactagtaat gttgataata ctttcggcgg agggaccgag gtggtggtca aaggtgatcc 360 agttgcacct actgtcctca tcttcccacc atcagcggac c 401 <210> 98 <211> 443 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 98 gtcgctgtgc tcaaaggtgt ccagtgtcag tcggtgaagg agtccgaggg aggtctcttc 60 aagccaacgg ataccctgac actcacctgc acagtctctg gattctccct cagcatctat 120 ggagtgagct gggtccgcca ggctccaggg aacgggctgg aatggatcgg aatcatttat 180 gctagtggta gcgcagacta cgcgagctgg gcgaaaagcc gatccaccat caccagaaac 240 accaacctga acacggtgac tctgaagatg accagtctga cagccgcgga cacggccacc 300 tatttctgtg cgagagagga cgatacttat ggttatacta gtagtatatg gggcccaggc 360 accctggtca ccgtctcctc agggcaacct aaggctccat cagtcttccc actggccccc 420 tgctgcgggg acacacccag ctc 443 <210> 99 <211> 391 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 99 tggctcccag gtgccagatg tgcctatgat atgacccaga ctccagcctc cgtgtctgca 60 gctgtgggag gcacagtcac catcaagtgc caggccagtc agagcattag tagctactta 120 aactggtatc agcagaaacc agggcagcct cccaagcgcc tgatctacag ggcatccact 180 ctggcatctg gggtctcatc gcggttcaaa ggcagtggat ctgggacaca gttcactctc 240 accatcagcg gcgtggagtg tgccgatgct gccacttact actgtcaaca ggattatagt 300 agtaataata ttgataatac tttcggcgga gggaccgagg tggtggtcaa aggtgatcca 360 gttgcaccta ctgtcctcat cttcccacca t 391 <210> 100 <211> 440 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 100 ctggtcgctg tgctcaaagg tgtccagtgt cagtcggtga aggagtccga gggaggtctc 60 ttcaagccaa cggataccct gacactcacc tgcacagtct ctggattctc cctcagtagc 120 tatccaataa gctgggtccg ccaggctcca gggaacgggc tggaatggat cggagacatt 180 tatgctagtg gtagtatatt gtacgcgagc tgggcgacag gccgatctac catcaccaga 240 aataccaacc tgaacacggt gactctgaaa atgaccagtc tgacagccgc ggacacggcc 300 acctatttct gtgcgagagt aagttatagt ggtggtaccg acatctgggg cccaggcacc 360 ctggtcaccg tctcctcagg gcaacctaag gctccatcag tcttcccact ggccccctgc 420 tgcggggaca cacccagctc 440 <210> 101 <211> 394 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 101 tctggctccc aggtgccaga tgtgcctatg atatgaccca gactccagcc tctgtggagg 60 tagctgtggg aggcacagtc accatcaagt gccaggccag tgaggacatt gaaagctatt 120 tagcctggta tcagcagaaa ccagggcagc ctcccaagct cctgatctac agggcatcca 180 ctctgccatc tggggtccca tcgcggttca aaggcagtgg atctgggaca gagttcactc 240 tcaccatcag cgacctggag tgtgccgatg ctgccactta ctactgtcaa caggattata 300 gtagtagtaa tgttgataat actttcggcg gagggaccga ggtggtggtc aaaggtgatc 360 cagttgcacc tactgtcctc atcttcccac catc 394 <210> 102 <211> 444 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 102 ctggtcgctg tgctcaaagg tgtccagtgt cagtcggtga aggagtccga gggaggtctc 60 ttcaagccaa cggataccct gacactcacc tgcacagtct ctggattctc cctcagtagc 120 aatgtaataa gctgggtccg ccaggctcca gggaaggggc tggagtggat cggagacatt 180 tatgttagtg gtaacacaga ctacgcgagc tgggcgaaaa gccgatccac catcaccaga 240 aacgccaacc tgaacacggt gactctgaaa atgaccagtc tgacagccgc ggacacggcc 300 acctatttct gtgcgagata tgatatgagt agtgatgctt tcgatccctg gggcccaggc 360 accctggtca ccgtctcctc agggcaacct aaggctccat cagtcttccc actggccccc 420 tgctgcgggg acacacccag ctcc 444 <210> 103 <211> 401 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 103 tctctggctc ccaggtgcca gatgtgccta tgatatgacc cagactccag cctctgtgga 60 ggtagctgtg ggaggcacag tcaccatcaa gtgccaggcc agtcagaaca ttggtagcta 120 cttatcctgg tatcagcaca aaccagggca gcgtcccaga ctcctgatgt acagggcatc 180 cactctggca tctggggtct catcgcggtt caaaggcagt ggatctggga cagagttcac 240 tctcaccatc agcggtgtgc agtgtgacaa tgctgccact tactactgtc aacagggtta 300 tactaatagt ggtgttgata atactttcgg cggagggacc gaggtggtgg tcaaaggtga 360 tccagttgca cctactgtcc tcatcttccc accatcagcg g 401 <210> 104 <211> 69 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 104 atgaaggtaa caggcatctt tcttctcagt gccttggccc tgttgagtct atctggtaac 60 actggagct 69 <210> 105 <211> 237 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 105 atgaaggtaa caggcatctt tcttctcagt gccttggccc tgttgagtct atctggtaac 60 actggagctg actccctggg aagagaggcc aaatgttaca atgaacttaa tggatgcacc 120 aagatatatg accctgtctg tgggactgat ggaaatactt atcccaatga atgcgtgtta 180 tgttttgaaa atcggaaacg ccagacttct atcctcattc aaaaatctgg gccttgc 237 <210> 106 <211> 168 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 106 gactccctgg gaagagaggc caaatgttac aatgaactta atggatgcac caagatatat 60 gaccctgtct gtgggactga tggaaatact tatcccaatg aatgcgtgtt atgttttgaa 120 aatcggaaac gccagacttc tatcctcatt caaaaatctg ggccttgc 168 <210> 107 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (2)..(2) <223> / replace="A" <220> <221> VARIANT <222> (3)..(3) <223> / replace="N" <220> <221> VARIANT <222> (4)..(4) <223> / replace="G" or "P" or "V" <220> <221> VARIANT <222> (5)..(5) <223> / replace="V" <220> <221> SITE <222> (1)..(6) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 107 Ser Ser Tyr Ala Ile Ser 1 5 <210> 108 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> MOD_RES <222> (1)..(1) <223> Any amino acid <220> <221> MOD_RES <222> (3)..(4) <223> Any amino acid <220> <221> VARIANT <222> (5)..(5) <223> / replace="G" or "Y" <220> <221> MOD_RES <222> (7)..(9) <223> Any amino acid <220> <221> SITE <222> (1)..(16) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 108 Xaa Ile Xaa Xaa Ser Gly Xaa Xaa Xaa Tyr Ala Ser Trp Ala Lys Ser 1 5 10 15 <210> 109 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> MOD_RES <222> (2)..(11) <223> Any amino acid <400> 109 Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 <210> 110 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (4)..(4) <223> / replace="E" <220> <221> VARIANT <222> (5)..(5) <223> / replace="G" or "D" or "N" <220> <221> VARIANT <222> (7)..(7) <223> / replace="N" or "E" or "G" <220> <221> MOD_RES <222> (8)..(8) <223> Any amino acid <220> <221> VARIANT <222> (9)..(9) <223> / replace="A" <220> <221> MOD_RES <222> (11)..(11) <223> Any amino acid <220> <221> SITE <222> (1)..(11) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 110 Gln Ala Ser Gln Ser Ile Ser Xaa Tyr Leu Xaa 1 5 10 <210> 111 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> MOD_RES <222> (1)..(1) <223> Any amino acid <220> <221> VARIANT <222> (3)..(3) <223> / replace="T" <220> <221> VARIANT <222> (4)..(4) <223> / replace="Y" or "K" <220> <221> VARIANT <222> (6)..(6) <223> / replace="P" or "V" <220> <221> SITE <222> (1)..(7) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 111 Xaa Ala Ser Thr Leu Ala Ser 1 5 <210> 112 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (1)..(1) <223> / replace="H" <220> <221> VARIANT <222> (3)..(3) <223> / replace="D" <220> <221> MOD_RES <222> (5)..(6) <223> Any amino acid <220> <221> VARIANT <222> (7)..(7) <223> / replace="N" <220> <221> VARIANT <222> (8)..(8) <223> / replace="D" or "V" or "G" <220> <221> VARIANT <222> (9)..(9) <223> / replace="D" or "I" <220> <221> VARIANT <222> (10)..(10) <223> / replace="G" or "N" <220> <221> VARIANT <222> (11)..(11) <223> / replace="V" <220> <221> MOD_RES <222> (12)..(12) <223> Any amino acid <220> <221> SITE <222> (1)..(12) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> 112 Gln Gln Gly Tyr Xaa Xaa Ser Asn Val Asp Asn Xaa 1 5 10 <210> 113 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 113 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15

Claims

1. A method for determining whether a subject has or is at risk of developing a disease characterized by expression of AS-SPIK, comprising: (i) obtaining a biological test sample from the subject and contacting it with an isolated AS-SPIK antibody or antigen-binding fragment thereof; wherein the isolated AS-SPIK antibody or antigen-binding fragment thereof specifically binds to an epitope on AS-SPIK (SEQ ID NO: 82) and does not bind to NS-SPIK (SEQ ID NO: 83), and the isolated AS-SPIK antibody or antigen-binding fragment thereof does not exclude binding of other classes of antibodies that specifically bind to an epitope on AS-SPIK and do not bind to NS-SPIK; and (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: 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 (c) a CDRH1 sequence of SEQ ID NO: 24, a CDRH2 sequence of SEQ ID NO: 34, a CDRH3 sequence of SEQ ID NO: 44, a CDRL1 sequence of SEQ ID NO: 54, a CDRL2 sequence of SEQ ID NO: 64, and a CDRL3 sequence of SEQ ID NO: 74; or (d) a CDRH1 sequence of SEQ ID NO: 27, a CDRH2 sequence of SEQ ID NO: 37, a CDRH3 sequence of SEQ ID NO: 47, a CDRL1 sequence of SEQ ID NO: 57, a CDRL2 sequence of SEQ ID NO: 67, and a CDRL3 sequence of SEQ ID NO: 77; and (ii) detecting the concentration of AS-SPIK-antibody complexes in the biological test sample; and (iii) comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject has or is at risk of developing the disease; A method comprising:

2. A method for determining whether a subject has or is at risk of developing a disease characterized by expression of AS-SPIK, comprising: (i) obtaining a biological test sample from the subject and contacting it with a first antibody or antigen-binding fragment thereof that specifically binds to SPIK to form a SPIK-antibody complex; (ii) contacting the SPIK-antibody complex with an isolated AS-SPIK antibody or antigen-binding fragment thereof; wherein the isolated AS-SPIK antibody or antigen-binding fragment thereof specifically binds to an epitope on AS-SPIK (SEQ ID NO: 82) and does not bind to NS-SPIK (SEQ ID NO: 83), and the isolated AS-SPIK antibody or antigen-binding fragment thereof does not exclude binding of other classes of antibodies that specifically bind to an epitope on AS-SPIK and do not bind to NS-SPIK; and (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: 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 (c) a CDRH1 sequence of SEQ ID NO: 24, a CDRH2 sequence of SEQ ID NO: 34, a CDRH3 sequence of SEQ ID NO: 44, a CDRL1 sequence of SEQ ID NO: 54, a CDRL2 sequence of SEQ ID NO: 64, and a CDRL3 sequence of SEQ ID NO: 74; or (d) a CDRH1 sequence of SEQ ID NO: 27, a CDRH2 sequence of SEQ ID NO: 37, a CDRH3 sequence of SEQ ID NO: 47, a CDRL1 sequence of SEQ ID NO: 57, a CDRL2 sequence of SEQ ID NO: 67, and a CDRL3 sequence of SEQ ID NO: 77; and (iii) detecting the concentration of AS-SPIK-antibody complexes in the biological test sample; and (iv) comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject has or is at risk of developing the disease; A method comprising:

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

4. The antibody or antigen-binding fragment thereof, (a) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 1 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 11, or (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 3 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 13, or (c) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO:4 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO:14; or (d) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 7 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 17; The method according to any one of claims 1 to 3, comprising:

5. The antibody or antigen-binding fragment thereof, (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: 3 and a light chain variable region sequence of SEQ ID NO: 13, or (c) a heavy chain variable region sequence of SEQ ID NO: 4 and a light chain variable region sequence of SEQ ID NO: 14, or (d) a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 17; The method of claim 4, comprising:

6. A method described in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises a conformational epitope of AS-SPIK.

7. A method described in any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is multispecific.

8. A method according to any one of claims 1 to 7, wherein the disease is hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), or cirrhosis.

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