Epitope of Anti-serine protease inhibitor kazal (SPIK) antibody

Abstract

To provide a new method for diagnosing and treating liver cancer.SOLUTION: Provided are isolated antibodies that specifically bind to a conformational epitope of AS-SPIK proteins and do not specifically bind to NS-SPIK proteins, wherein the conformational epitope of the AS-SPIK proteins comprises a specific amino acid sequence of one or more amino acids selected from the group consisting of L14, L15, S16, L17, D24, and S25, and one or more amino acids selected from the group consisting of C58, V59, L60,, and. C61 F62. Methods of producing such antibodies, compositions, such as pharmaceutical compositions, comprising such antibodies, and uses thereof for diagnosing and / or treating disorders characterized by expression of AS-SPIK (e.g., liver cancer) are disclosed. Diagnostic methods and kits containing anti-AS-SPIK antibodies are also disclosed.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to the filing date of U.S. Provisional Patent Application No. 62 / 899,024, filed September 11, 2019, the entire disclosure of which is incorporated herein by reference.

[0002] 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] FIELD OF THE INVENTION The present invention relates to two classes of anti-AS-SPIK antibodies that specifically bind to one of two distinct conformational epitopes, as well as methods for producing such antibodies, compositions, such as pharmaceutical compositions, containing such antibodies, and their use for diagnosing and / or treating disorders characterized by expression of AS-SPIK (e.g., liver cancer). Diagnostic methods and kits containing anti-AS-SPIK antibodies are also disclosed. [Background technology]

[0004] The liver is one of the largest organs in the body. It has many functions, including the production of enzymes and bile required for food digestion, regulation of glycogen storage, plasma protein synthesis, hormone production, and detoxification of various metabolic products. Liver disorders, including liver cancers such as hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), viral infections, cirrhosis, and other inflammatory disorders of the liver, affect millions of people worldwide. For example, more than 5 million people in the United States and more than 450 million people worldwide suffer from hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, and more than 30% of these infected individuals are at high risk of developing liver cancer. (1-4) Despite advances in diagnosis and treatment, liver cancer remains a significant cause of both morbidity and mortality. Primary liver cancer, or cancer originating in the liver, has a 5-year survival rate of less than 10%. However, if liver cancer is detected early, at its most treatable stage, survival rates increase to nearly 40%. Patients with early-stage 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, can be unreliable due to low sensitivity and the possibility of operator error. Expensive imaging techniques may also be less accurate for detecting small, early-stage tumors (1, 2). Liver biopsy, which is still considered the most reliable method for distinguishing benign from malignant tumors, is invasive and requires surgery (3). There is a continuing need for new methods to diagnose and treat liver cancer, especially for people affected by cirrhosis, viral infections, and inflammatory disorders of the liver.

[0005] The serine protease inhibitor Kazal (SPIK / SPINK1) is a small secreted protein with 79 amino acids (4). It was first discovered in the pancreas as an inhibitor of trypsinogen autoactivation (5, 6). Recent studies have shown that SPIK expression outside the pancreas is elevated in liver cancers, such as hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), but its activity is limited or absent in normal tissues, including the liver (7-9). The use of SPIK as a cancer biomarker has been hindered by the fact that serum SPIK levels are also elevated in the presence of other diseases, particularly pancreatitis (10-12). We discovered that SPIK secreted by hepatoma cells retains an additional fragment of at least 9 residues at the N-terminus, whereas SPIK secreted from normal cells, such as pancreatic cells, is deleted (13). We refer to this type of SPIK as NS-SPIK (normal secretory SPIK). However, the present inventors discovered that SPIK secreted by hepatoma cells is larger than NS-SPIK, and that an additional fragment of at least 9 residues of the 23 amino acids is retained during secretion (11). The present inventors named SPIK secreted by hepatoma cells AS-SPIK or LC-SPIK (abnormally secreted SPIK or hepatoma-secreted SPIK). AS-SPIK and LC-SPIK have the same meaning herein. Further research by the present inventors revealed that, in addition to the 9 amino acids at the N-terminus of SPIK, an additional 23 amino acids (SEQ ID NO: 6) are retained in AS-SPIK, but are removed from NS-SPIK. The reason why cancer cells secrete uncleaved SPIK is unknown. Our ongoing hypothesis is that because SPIK is a serine protease inhibitor, overexpression of SPIK in cancer cells suppresses the activity of signal peptide peptidase, a type of serine protease, resulting in the secretion of the full-length protein from the cancer cells.While the inventors believe they understand the specific events that occur during the process of AS-SPIK expression, the compositions and methods of the present invention are not limited to those that work by affecting any particular cellular mechanism. Therefore, by using antibodies that can recognize and specifically bind to this domain, it should be possible to distinguish SPIK produced by cancerous liver cells from SPIK produced by other non-cancerous diseases. To this end, the inventors developed a series of monoclonal antibodies that recognize only cancerous SPIK (AS-SPIK, abnormally secreted SPIK) with high sensitivity and specificity, but not normal SPIK (NS-SPIK, normally secreted SPIK). Using these antibodies, the inventors further developed an ELISA test kit (SERAVUE®) to evaluate the performance of AS-SPIK in distinguishing HCC from other liver diseases such as HBV and HCV infection, cirrhosis, and non-liver diseases such as pancreatitis, as well as healthy subjects. Clinical trial results suggest that the subject antibodies and related methods can be used to selectively and reliably detect HCC, even at very early stages, without interference from other liver or non-liver diseases, which would greatly improve the diagnosis and treatment of HCC. Summary of the Invention

[0006] An embodiment of the present invention includes an isolated antibody that specifically binds to a conformational epitope of AS-SPIK protein but not to NS-SPIK protein, wherein the conformational epitope of AS-SPIK protein comprises one or more amino acids selected from the group consisting of L14, L15, S16, L17, D24 and S25 of SEQ ID NO: 2, and one or more amino acids selected from the group consisting of C58, V59, L60, C61 and F62 of SEQ ID NO: 2.

[0007] In some embodiments, the conformational epitope comprises amino acids L14, L15, S16, and L17 of SEQ ID NO:2. In some embodiments, the conformational epitope comprises amino acids L60 and C61 of SEQ ID NO:2. In some embodiments, the conformational epitope comprises amino acids L14, L15, S16, L17, L60, and C61 of SEQ ID NO:2. In some embodiments, the conformational epitope further comprises amino acids D24 and S25 of SEQ ID NO:2. In some embodiments, the conformational epitope further comprises amino acids C58, V59, and F62 of SEQ ID NO:2. In some embodiments, the conformational epitope comprises amino acids L14, L15, S16, L17, D24, S25, C58, V59, L60, C61, and F62 of SEQ ID NO:2.

[0008] In some embodiments, the isolated antibody comprises a CDRH1 sequence comprising S6; and / or a CDRH2 sequence comprising I2, G5, G6, Y10 and K16; and / or a CDRH3 sequence comprising G4 and Y7; and / or a CDRL1 sequence comprising Q4 and S9; and / or a CDRL2 sequence comprising A2, S3, T4 and S7; and / or a CDRL3 sequence comprising Q1, Q2, Y4 and S5.

[0009] In some embodiments, the isolated antibody comprises a CDRH1 sequence comprising S6, a CDRH2 sequence comprising I2, G5, G6, Y10 and K16, a CDRH3 sequence comprising G4 and Y7, a CDRL1 sequence comprising Q4 and S9, a CDRL2 sequence comprising A2, S3, T4 and S7, and a CDRL3 sequence comprising Q1, Q2, Y4 and S5.

[0010] An embodiment of the present invention includes an isolated antibody that specifically binds to a conformational epitope of AS-SPIK protein but not to NS-SPIK protein, wherein the conformational epitope of AS-SPIK protein comprises one or more amino acids selected from the group consisting of L36, N37, I42 and Y43 of SEQ ID NO: 2, and one or more amino acids selected from the group consisting of R67, Q68, I71 and L72 of SEQ ID NO: 2.

[0011] In some embodiments, the conformational epitope comprises amino acids L36 and N37 of SEQ ID NO: 2. In some embodiments, the conformational epitope comprises amino acids 142 and Y43 of SEQ ID NO: 2. In some embodiments, the conformational epitope comprises amino acids L36, N37, 142, and Y43 of SEQ ID NO: 2. In some embodiments, the conformational epitope comprises amino acids R67, Q68, 171, and L72 of SEQ ID NO: 2. In some embodiments, the conformational epitope comprises amino acids L36, N37, 142, Y43, R67, Q68, 171, and L72 of SEQ ID NO: 2.

[0012] In some embodiments, the antibody comprises a CDRH1 sequence comprising Y3, S7, and W9; and / or a CDRH2 sequence comprising A1, I2, G4, G6, and Y10; and / or a CDRH3 sequence comprising R1 and D7; and / or a CDRL1 sequence comprising A2, S3, Q4, I6, Y9, L10, and S11; and / or a CDRL2 sequence comprising A2, S3, L5, and S7; and / or a CDRL3 sequence comprising Q1, Q2, and T5.

[0013] In some embodiments, the antibody comprises a CDRH1 sequence comprising Y3, S7, and W9; a CDRH2 sequence comprising A1, I2, G4, G6, and Y10; a CDRH3 sequence comprising R1 and D7; a CDRL1 sequence comprising A2, S3, Q4, I6, Y9, L10, and S11; a CDRL2 sequence comprising A2, S3, L5, and S7; and a CDRL3 sequence comprising Q1, Q2, and T5.

[0014] In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody has binding affinity for an effector cell. In some embodiments, the antibody has binding affinity for a T cell antigen. In some embodiments, the T cell antigen comprises a CD3 protein. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a CAR-T type.

[0015] Aspects of the invention include immunoconjugates comprising an antibody described herein covalently linked to a cytotoxic agent, in some embodiments, the cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, a radioisotope, and a nucleolytic enzyme.

[0016] In some embodiments, the immunoconjugate has the formula Ab-(LD)p, where Ab is an antibody described herein, L is a linker, D is a drug moiety, and p is an integer ranging from 1 to 8. In some embodiments, D is selected from the group consisting of maytansinoids, auristatins, and dolostatins. In some embodiments, L comprises one or more linkers selected from the group consisting of 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4-(2-pyridyldithio)butyric acid-N-hydroxysuccinimide ester (SPDB), and N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB).

[0017] Aspects of the invention include pharmaceutical compositions comprising the antibodies or immunoconjugates described herein.

[0018] An embodiment of the present invention includes a method for the treatment of a disorder characterized by expression of AS-SPIK, comprising administering to a subject having the disorder an antibody or immunoconjugate described herein, or a pharmaceutical composition described herein.

[0019] An embodiment of the invention includes the use of an antibody or immunoconjugate described herein in the preparation of a medicament for the treatment of a disorder characterized by expression of AS-SPIK.

[0020] An embodiment of the present invention includes an antibody or immunoconjugate described herein for use in treating a disorder characterized by expression of AS-SPIK.

[0021] 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.

[0022] Aspects of the invention include polynucleotides encoding the antibodies described herein. Aspects of the invention include vectors comprising the polynucleotides described herein. Aspects of the invention include host cells comprising the vectors described herein.

[0023] Aspects of the invention include methods of producing an antibody or immunoconjugate described herein, comprising growing a host cell described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell.

[0024] Embodiments of the present invention include diagnostic methods for determining whether a subject has or is at risk of developing a disorder characterized by expression of AS-SPIK, the method comprising contacting a biological test sample from the subject with an AS-SPIK antibody described herein to form 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 to a reference value to determine whether the subject has or is at risk of developing the disorder.

[0025] Embodiments of the present invention include diagnostic methods for determining whether a subject has or is at risk of developing a disorder characterized by expression of AS-SPIK, the method comprising contacting a biological test sample from the subject with a first antibody or antigen-binding fragment that specifically binds to SPIK to form a SPIK-antibody complex, contacting the SPIK-antibody complex with an AS-SPIK antibody or antigen-binding fragment described herein to form 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 to a reference value to determine whether the subject has or is at risk of developing the disorder.

[0026] 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 infection of the liver, inflammatory disorders of the liver, and cirrhosis.

[0027] Aspects of the invention include kits comprising an antibody or immunoconjugate described herein. In some embodiments, the kit further comprises an antibody that specifically binds to SPIK. [Brief explanation of the drawings]

[0028] [Figure 1]Gel electrophoresis was used to demonstrate the sizes of AS-SPIK and NS-SPIK. [Figure 2] The N-terminal sequence of AS-SPIK by Edman N-terminal analysis is shown. [Figure 3] Comparison of the amino acid sequences of AS-SPIK and NS-SPIK [Figure 4] A comparison of the three-dimensional structure of AS-SPIK with that of NS-SPIK is shown. [Figure 5] 1 shows the results of testing the binding activity of antibodies that bind to AS-SPIK and NS-SPIK. [Figure 6] 1 shows the results of a binding test of anti-AS-SPIK antibodies to synthetic peptides. [Figure 7] The three-dimensional structure (crystal model) of AS-SPIK and its epitope is shown. [Figure 8] 1 shows the results of an inhibition study to identify the binding site of class I anti-AS-SPIK antibodies. [Figure 9] The consensus amino acids of the CDRs in class I anti-AS-SPIK antibodies are shown. [Figure 10] The consensus amino acids of the CDRs in class II anti-AS-SPIK antibodies are shown. [Figure 11] A mechanism explanation for the AS-SPIK detection kit is provided. [Figure 12] Panel A shows that IM-CA22 specifically binds to AS-SPIK but not to NS-SPIK, while antibody IM-BA2 binds to both AS-SPIK and NS-SPIK. Panel B shows that AS-SPIK is elevated in HCC but not in pancreatitis or healthy patients. Panel C shows that NS-SPIK is elevated in pancreatitis patients but does not interfere with the performance of AS-SPIK-based detection kits. [Figure 13] Table 1 shows the binding characteristics of class I and class II anti-AS-SPIK antibodies. [Figure 14]Table 2 shows the regions related to anti-AS-SPIK binding predicted by CLIPS analysis and the amino acid residues constituting the epitopes therein. [Figure 15] 1 is a table (Table 3) showing the amino acid sequences of four example Class I antibodies that bind to epitope I as further set forth herein. [Figure 16] 1 is a table (Table 4) showing the amino acid sequences of four example Class II antibodies that bind to epitope II as further set forth herein. [Figure 17] 5 is a table (Table 5) showing serum AS-SPIK levels in a clinical trial with 512 samples. [Figure 18] Table 6 shows a summary of the performance of AS-SPIK versus AFP in detecting HCC. [Figure 19] 7 is a table (Table 7) showing AS-SPIK levels in early versus late stage HCC. [Figure 20] 8 is a table (Table 8) showing AS-SPIK levels in HCC by BCLC stage. DETAILED DESCRIPTION OF THE INVENTION

[0029] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in "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 regularly updated); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and These are fully described in such publications as Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0030] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed herein, subject to any specific excluded limits in the stated range. Where a stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the invention.

[0031] Unless otherwise specified, antibody residues herein 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)).

[0032] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent 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 known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0033] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0034] definition For the purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any definition conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0035] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Typically, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes. The term includes any molecular determinant capable of specific binding to an antibody. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, 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 to which a binding molecule binds.

[0036] "Epitope mapping" is the process of identifying the binding site or epitope of an antibody on a target antigen. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0037] "Epitope binning," as defined herein, is the process of classifying antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for distinguishing the epitope recognition properties of different antibodies and identifying antibodies with different binding specificities, in combination with a computational process for clustering antibodies based on their epitope recognition properties.

[0038] If two antibodies recognize the same or sterically overlapping epitopes, the antibodies bind to "essentially the same epitope" as the reference antibody. The most widely used and rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay, which can be configured in any number of different formats using either labeled antigen or labeled antibody. Typically, the antigen is immobilized on a 96-well plate, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive or enzyme label.

[0039] As used herein, a "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, resulting from a sequence alteration involving that amino acid residue / position. For example, common modifications include substitution of a residue (or at that position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more (generally fewer than five or three) amino acids adjacent to that residue / position, and deletion of that residue / position. An "amino acid substitution" or a variation thereof refers to the replacement of an existing amino acid residue in a given (starting) amino acid sequence with a different amino acid residue. Generally, and preferably, the modification results in an alteration in at least one physical or biochemical activity of the mutant polypeptide compared to a polypeptide comprising the starting (or "wild-type") amino acid sequence. For example, in the case of an antibody, the altered physical or biochemical activity may be binding affinity, binding ability, and / or binding efficacy for a target molecule.

[0040] The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), single-chain molecules, and antibody fragments (e.g., Fab, F(ab'), and Fv). The term "immunoglobulin" (Ig) is used interchangeably herein 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 the broadest sense and specifically includes all isotypes, subclasses, and forms of antibodies, including IgG, IgM, IgA, IgD, and IgE antibodies and fragments thereof, preferably antigen-binding fragments.

[0041] Unless otherwise specified, the term "antibody" specifically includes naturally occurring human and non-human IgG1, IgG2 (IgG2a, IgG2b), IgG3, IgG4, IgE, IgA, IgD and IgM antibodies, including naturally occurring variants.

[0042] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597.

[0043] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0044] "Humanized" forms of non-human (e.g., murine) antibodies are antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are also replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that 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.

[0045] "Amino acid sequence identity percentage (%)" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity.Alignment for determining the percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the entire length of the sequences being compared.However, for the purposes herein, amino acid sequence identity percentage values ​​are generated using the sequence comparison computer program ALIGN-2.

[0046] As used herein, the term "percent sequence identity" refers to the degree of homology between any given query sequence and a subject sequence. For example, a naturally occurring AS-SPIK or NS-SPIK polypeptide may be the query sequence, and a fragment of the AS-SPIK or NS-SPIK polypeptide may be the subject sequence. Similarly, a fragment of an AS-SPIK or NS-SPIK polypeptide may be the query sequence, and a biologically active variant thereof may be the subject sequence.

[0047] The term "consensus sequence," as used herein, means a sequence of amino acid or nucleotide residues that represents the most common residue found at each position in a sequence alignment after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence correspondence, without considering any conservative substitutions as part of the sequence identity.

[0048] An "isolated" antibody, as used herein, is one that has been identified and separated and / or recovered from components of its natural environment in recombinant host cells. Contaminant components of its natural environment are materials 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 unwanted by-products of production. In preferred embodiments, an isolated antibody, as used herein, will be purified (1) to greater than 95%, or greater than 98%, or greater than 99% by weight, as determined by SDS-PAGE or SEC-HPLC methods, (2) sufficiently to obtain at least 15 residues of 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 or, preferably, silver stain. Ordinarily, an isolated antibody will be prepared by at least one purification step.

[0049] In the case of IgG, a four-chain unit generally has a size of approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. The pairing of the VH and VL together forms a single antigen-binding site.

[0050] The term "polypeptide" is used herein in the broadest sense and includes 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 joined by peptide bonds.

[0051] The terms "specific binding" or "specifically binds to" or "specific for" refer to the binding of an antibody to a target antigen, e.g., an epitope on a particular polypeptide, peptide, or other target (e.g., a glycoprotein target), and mean binding that is measurably different from a nonspecific interaction (e.g., a nonspecific interaction could be binding with bovine serum albumin or casein). Specific binding can be measured, for example, by determining the binding of the antibody to the target molecule compared to binding to a control molecule. For example, specific binding can be determined by competition between the target, e.g., excess unlabeled target, and a similar control molecule. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target, as used herein, can be exhibited by a molecule having a Kd for the target of, for example, at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or more. In certain instances, the term "specific binding" refers to binding in which a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0052] "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 specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). For example, 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 higher. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind antigens slowly and dissociate easily, while high-affinity antibodies generally tend to bind antigens more quickly and remain bound longer. Various methods for measuring binding affinity are known in the art.

[0053] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured by a technique appropriate for the antibody and target pair, e.g., using a surface plasmon resonance assay using, e.g., a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip at approximately 10 response units (RU) at 25°C.

[0054] The term "valent," as used herein, refers to the presence of a specific number of binding sites in an antibody. Thus, the term "bivalent" refers to the presence of two binding sites.

[0055] "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Monospecificity" refers to the ability to bind to only one epitope. In some embodiments, the antibody binds to each epitope with an affinity of at least 10 M, or 10 M or greater.

[0056] The terms "target" or "binding target" are used in the broadest sense and specifically include, but are not limited to, polypeptides, nucleic acids, carbohydrates, lipids, cells, and other molecules that occur in nature, with or without biological function.

[0057] The term "antigen" refers to an entity or fragment thereof that can bind to an antibody or elicit a cellular immune response. 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, as defined above, includes regions known as antigenic determinants or epitopes.

[0058] As used herein, the term "immunogenic" refers to a substance that induces the production of antibodies and / or activates T cells and / or other reactive immune cells directed against the immunogenic antigen.

[0059] The "antigen-binding site" or "antigen-binding region" of an antibody of the present invention typically contains six hypervariable regions (HVRs), which contribute to varying degrees to the affinity of the binding site for the antigen. The term "complementarity-determining region" or "CDR" is used interchangeably herein 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 extent of the HVRs and framework regions (FRs) is determined by comparison with a compiled database of amino acid sequences in which such regions are defined by intersequence variability and / or structural information from antibody / antigen complexes. Functional antigen-binding sites composed of fewer HVRs (i.e., binding specificity determined by three, four, or five HVRs) are also within the scope of the present invention. Fewer than the complete set of six HVRs may be sufficient to bind to some binding targets. Thus, in some cases, the HVRs of the VH or VL domain alone may be sufficient. Furthermore, a particular antibody may have non-HVR-associated binding sites for the antigen, and such binding sites are specifically included within this definition.

[0060] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0061] "Antibody drug conjugate" (ADC) or immunoconjugate means an antibody, or antigen-binding fragment thereof, conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0062] The term "host cell," as used herein, refers to any type of cell line that can be engineered to produce an antibody according to the invention. In one embodiment, Chinese hamster ovary (CHO) cells are used as host cells.

[0063] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0064] A nucleic acid is "operably linked" when it is 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 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 contiguous with the DNA sequences being linked, and, in the case of a secretory leader, contiguous and in reading frame, although enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0065] 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 so that the antibody is useful as a diagnostic and / or therapeutic agent when targeting AS-SPIK.

[0066] The term "variable" refers to the fact that certain segments of variable domains differ significantly in sequence among antibodies. The "variable" or "V" domain is involved in antigen binding and defines the specificity of a particular antibody for a particular antigen. However, variability is not evenly distributed across the 110-amino acid span of the variable domain. Instead, V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions," each 9-12 amino acids long. Native heavy and light chain variable domains each contain four FRs, largely in a β-sheet configuration, connected by three hypervariable regions that form, in some cases, loops connecting the β-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0067] A "complete" antibody is one that contains an antigen-binding site and a light chain constant domain (CL) and at least a heavy chain constant domain of a particular antibody class. For example, a complete IgG antibody contains an antigen-binding site, a light chain constant domain CL, and at least heavy chain constant domains CH1 (Cγ1), CH2 (Cγ2), and CH3 (Cγ3). A complete IgM antibody contains an antigen-binding site, a light chain constant domain CL, and at least heavy chain constant domains CM1 (Cμ1), CM2 (Cμ2), CM3 (Cμ3), and CM4 (Cμ4). A complete IgA antibody contains an antigen-binding site, a light chain constant domain CL, and at least heavy chain constant domains CA1 (Cα1), CA2 (Cα2), and CA3 (Cα3). A complete IgD antibody contains an antigen-binding site, a light chain constant domain CL, and at least heavy chain constant domains CD1 (Cδ1), CD2 (Cδ2), and CD3 (Cδ3). An intact IgE antibody comprises 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 native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0068] An "antibody fragment" or "antigen-binding fragment" of an antibody comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Non-limiting examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises the antigen-binding site of an intact antibody, thereby retaining the ability to bind to antigen. Those skilled in the art will understand that antibody fragments can be formed from any intact antibody, e.g., IgG, IgM, IgA, IgD, or IgE antibody, by separating at least the antigen-binding portion of the antibody from the remainder of the light and heavy chains to produce an antigen-binding fragment. In certain embodiments, an antibody fragment may comprise the antigen-binding region of an antibody and one or more additional domains of the antibody's light and / or heavy chains. For example, in some embodiments, an antibody fragment may comprise an antigen-binding region comprising the VH and VL domains, the light chain constant domain CL, and one or more heavy chain constant domains, such as 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 IgG antibody fragments, papain digestion produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting the ability to readily crystallize. Fab fragments consist of the entire L chain together with the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of IgG antibodies produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments in that they contain several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0070] The Fc fragment of an IgG antibody contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of the antibody are determined by the sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain cell types.

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

[0072] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to the native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in 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 with the native-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0073] The human IgG1 amino acid sequence is provided by UniProtKB number P01857, which is incorporated herein by reference in its entirety. The human IgG2 amino acid sequence is provided by UniProtKB number P01859, which is incorporated herein by reference in its entirety. The human IgG3 amino acid sequence is provided by UniProtKB number P01860, which is incorporated herein by reference in its entirety. The human IgG4 amino acid sequence is provided by UniProtKB number P01861, which is incorporated herein by reference in its entirety.

[0074] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. In single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain may be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in two-chain Fv species. The folding of these two domains generates six hypervariable loops (three loops each from the H and L chains) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0075] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains connected in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995.

[0076] The term "chimeric antigen receptor" or "CAR" is used herein in the broadest sense to refer to an engineered receptor that grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) onto a transmembrane domain and an intracellular signaling domain. Typically, receptors are used to graft the specificity of a monoclonal antibody onto T cells to create chimeric antigen receptors (CARs) (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 that is involved in the effector phase of an immune response, as opposed to the recognition and activation phases of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, are capable of inducing antibody-dependent cellular cytotoxicity (ADCC). For example, FcR-expressing monocytes and macrophages are involved in the specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, effector cells are capable of phagocytosing target antigens or target cells.

[0078] A "human effector cell" is a leukocyte that expresses a receptor, such as a T cell receptor or FcR, and performs effector function. Preferably, the cell expresses at least FcγRIII and performs 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. Effector cells may be isolated from their native source, e.g., from blood or PBMCs, as described herein.

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

[0080] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an 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, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like.

[0081] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0082] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0083] A "blocking" antibody or "antagonist" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies are capable of substantially or completely inhibiting the biological activity of the antigen.

[0084] An antibody that "binds" to an antigen of interest, e.g., an AS-SPIK or NS-SPIK polypeptide, is one that binds to the antigen with sufficient affinity so that it is useful as a therapeutic agent for targeting cells or tissues expressing the antigen and does not significantly cross-react with other proteins. With respect to antibody binding to a target molecule, the terms "specific binding" or "specifically binding to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which is generally a molecule of similar structure that lacks binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., excess unlabeled target. In this case, specific binding is indicated when binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. In one embodiment, the term "specific binding" refers to binding of a molecule to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0085] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer, including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, gastric 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 (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer. Cancers include, but are not limited to, retinoblastoma, theca cell carcinoma, virilizing tumor, hepatoma, non-Hodgkin's lymphoma (NHL), hematologic malignancies including multiple myeloma and acute hematologic malignancies, endometrial or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, hepatic cancer, anal cancer, penile cancer, nasopharyngeal cancer, laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, Schwannoma, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and urinary tract cancer.

[0086] The term "metastatic cancer" refers to a cancerous condition in which cancer cells from a primary tissue are carried by blood or lymphatic vessels from the primary site to one or more other parts of the body, forming one or more secondary tumors in one or more organs in addition to the primary tissue.

[0087] As used herein, an "AS-SPIK-associated disorder" in a "disorder characterized by AS-SPIK expression" refers to a disorder associated with the expression or overexpression of the AS-SPIK gene or gene product (AS-SPIK polypeptide), and may be any disorder characterized by cells expressing normal or elevated levels of AS-SPIK compared to suitable control cells. Suitable control cells may be cells from an individual not affected by an AS-SPIK-expressing or overexpressing cancer, or may be non-cancerous cells from a subject in need thereof, or may be non-cancerous cells from another individual affected by an AS-SPIK-expressing or overexpressing cancer. One well-known example of an AS-SPIK-associated 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] "Tumor," as used herein, 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 "predictive" and "forecasting" are interchangeable in the sense that a method for prediction or forecasting enables the person practicing the method to select (usually, but not necessarily, before treatment) patients who are believed to be more likely to respond to treatment with anti-cancer drugs, including anti-AS-SPIK antibodies.

[0091] The terms "treat," "treatment," or "treating," as used herein, refer to both therapeutic treatment and prophylactic measures, in which the objective is to prevent or slow (alleviate) the targeted pathological condition or disorder. Those in need of treatment include those already with the particular condition or disorder, as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[0092] Detailed Description of the Invention As shown herein, AS-SPIK has the complete amino acid sequence of the SPIK gene, whereas NS-SPIK is shorter than AS-SPIK due to the removal of 23 amino acids at the N-terminus during the secretion process. This size difference was confirmed by gel electrophoresis and Edman degradation protein sequencing.

[0093] Furthermore, the data herein demonstrate that the three-dimensional conformations of AS-SPIK and NS-SPIK differ. Retaining an additional 23 amino acids at the N-terminus significantly alters the conformation of AS-SPIK compared to NS-SPIK. Three structural / conformational changes are visualized by comparing the three-dimensional structures (crystals) of AS-SPIK and NS-SPIK. The first change is at the N-terminus of the protein. An extra 23-residue fragment in AS-SPIK protrudes outward and extends beyond the body of the protein, while the N-terminus of NS-SPIK does not have an additional fragment protruding from the body of the protein. Second, because the N-terminus of AS-SPIK is longer, the first loop of AS-SPIK is flatter and bends differently compared to the corresponding loop in NS-SPIK. This difference results in a larger space between the first loop and the alpha helix in AS-SPIK, exposing amino acids that are internal and inaccessible in NS-SPIK. Finally, the longer N-terminus of AS-SPIK also changes the relative position and distance between the N-terminus of the protein and the alpha helix, increasing the space in this local region (Figure 4).

[0094] Because of these size and conformational differences, many different antibodies can be generated that specifically bind to AS-SPIK but not to NS-SPIK. This was confirmed by developing approximately 20 different monoclonal antibodies that specifically bind to AS-SPIK but not to NS-SPIK in mice and rabbits. Figure 5 shows that eight of the developed monoclonal antibodies (IM-A1, IM-B10, IM-C6, IM-E2, IM-CA22, IM-CA18, IM-CA46, and IMCA77) strongly bind to AS-SPIK, regardless of whether they are mouse or rabbit, but have little to no binding activity to NS-SPIK (similar to the negative control). Poly S, a polyclonal antibody developed in sheep, contains multiple antibodies that bind to various epitopes and therefore strongly binds to both AS-SPIK and NS-SPIK.

[0095] Further analysis of the eight antibodies above confirmed that all of them bind to conformational epitopes. This was supported by testing each antibody with various synthetic peptides to evaluate their potential for binding to various combinations of linear epitope regions of AS-SPIK and NS-SPIK. Four peptides, each containing sequences from a different region of AS-SPIK, were synthesized by BioMatik (Wilmington, DE), including peptide A, which contains the entire sequence of AS-SPIK but does not contain any disulfide bonds due to inactivation of cysteine ​​side chains. Peptide B, which contains AS-SPIK fragments M1 to G, was synthesized by BioMatik (Wilmington, DE). 50 Peptide C contains AS-SPIK fragment D 23 ~G 50 and peptide D comprises AS-SPIK fragment N 51 ~C 79 The test results showed that none of these eight antibodies bound to any of the synthetic peptides, supporting the hypothesis that the anti-AS-SPIK antibodies developed by the inventors bind to a conformational epitope (Figure 6).

[0096] The data presented herein demonstrate that the anti-AS-SPIK antibodies described herein can be divided into two classes: class I and class II. Class I antibodies function similarly to IM-CA22 and include IM-A1, IM-B10, IM-CA18, IM-D2, IM-D3, IM-D5, and IM-G2. Class II antibodies function similarly to IM-E2 and include IM-C6, IM-CA46, IM-CA77, IM-A6, IM-B3, IM-F5, and IM-G6. Most importantly, we demonstrate that any class I antibody can pair with any class II antibody in a sandwich ELISA test, and vice versa. This is due to the fact that class I antibodies bind to conformational epitopes that are different from those bound by class II antibodies. Furthermore, when used as pairs in a sandwich ELISA, class I antibodies completely inhibit binding to any other class I antibody, and class II antibodies completely inhibit binding to any other class II antibody (Figure 13; Table 1).

[0097] In addition to the competitive study results described herein, the inventors performed Precision Epitope Mapping using CLIPS (Chemically Linked Peptides on a Scaffold) peptide arrays to further conclusively demonstrate that the binding epitopes of class I antibodies are distinct from those of class II antibodies. CLIPS epitope mapping suggests that class I antibodies bind to epitope I, a discontinuous conformational epitope composed of at least two separate regions within AS-SPIK. Similarly, class II antibodies bind to epitope II, a discontinuous conformational epitope composed of another two separate regions within AS-SPIK (Figure 14; Table 2).

[0098] CLIPS studies have identified 7FLLSALALLSLSGNTGADSLGREA as an essential binding site for all class I antibodies. 29 (SEQ ID NO: 7) and 58 CVLCFENRKRQ 68(SEQ ID NO: 8) were identified. Within these binding sites of Epitope I, the key residues are: 14 LLSL 17 (SEQ ID NO: 12), 24 DS 25 (SEQ ID NO: 13), and 58 CVLCF 62 (SEQ ID NO: 14) (Figure 14; Table 2).

[0099] CLIPS studies have identified the essential binding site for all class II antibodies. 36 LNGCTKIYD 44 (SEQ ID NO: 9) and 64 NRKRQTSILIQ 74 (SEQ ID NO: 10) were also identified. Within these binding sites of Epitope II, the key residues are: 36 LN 37 (SEQ ID NO: 15), 42 IY 43 (SEQ ID NO: 16), 67 Race Queen 68 (SEQ ID NO: 17), and 71 IL 72 (SEQ ID NO: 18) (Figure 14; Table 2).

[0100] In addition to defining epitopes for both classes of antibodies, structural similarities within each class of antibodies that define the characteristics of each antibody type are also provided herein. Class I antibodies have notable similarities in their CDRs, with the following residues being highly conserved: a) S6 in CDRH1, b) I2, G5, G6, Y10, and K16 in CDRH2, c) G4 and Y7 in CDRH3, d) Q4 and S9 in CDRL1, e) A2, S3, T4, and S7 in CDRL2, and f) Q1, Q2, Y4, and S5 in CDRL3 (Figure 9). Class II antibodies also have notable similarities in their CDRs, with the following residues being highly conserved: a) Y3, S7, and W9 in CDRH1, b) A1, I2, G4, G6, and Y10 in CDRH2, c) R1 and D7 in CDRH3, d) A2, S3, Q4, I6, Y9, L10, and S11 in CDRL1, e) A2, S3, L5, and S7 in CDRL2, and f) Q1, Q2, and T5 in CDRL3 (Figure 10). In some embodiments, anti-AS-SPIK antibodies may comprise any suitable combination of CDR sequences containing the conserved amino acid residues listed above. For example, in some embodiments, a Class I anti-AS-SPIK antibody may comprise one, two, three, four, five, or all six of the following CDR sequences, or any combination thereof: a) S6 in CDRH1, b) I2, G5, G6, Y10, and K16 in CDRH2, c) G4 and Y7 in CDRH3, d) Q4 and S9 in CDRL1, e) A2, S3, T4, and S7 in CDRL2, and f) Q1, Q2, Y4, and S5 in CDRL3 (Figure 9). In some embodiments, a Class II anti-AS-SPIK antibody may comprise one, two, three, four, five, or all six of the following CDR sequences, or any combination thereof: a) Y3, S7, and W9 in CDRH1, b) A1, I2, G4, G6, and Y10 in CDRH2, c) R1 and D7 in CDRH3, d) A2, S3, Q4, I6, Y9, L10, and S11 in CDRL1, e) A2, S3, L5, and S7 in CDRL2, and f) Q1, Q2, and T5 in CDRL3 (Figure 10).

[0101] The amino acid sequences of various examples of Class I antibodies that bind to epitope I are shown in Table 3 (Figure 15). The amino acid sequences of various examples of Class II antibodies that bind to epitope II are shown in Table 4 (Figure 16).

[0102] Aspects of the present invention are based, at least in part, on the discovery that certain disorders are characterized by the expression of unique forms or variants of the serine protease inhibitor Kazal (SPIK). One prominent example is liver cancer, including, but not limited to, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). More specifically, the inventors have discovered that certain cancers, such as liver cancer, express a form of SPIK that contains an additional 23 amino acids at the N-terminus of the secreted SPIK polypeptide. This 23-amino acid segment (SEQ ID NO: 6) is not found in SPIK polypeptides secreted from normal cells, such as pancreatic cells. This is consistent with our previous report that the first 9 amino acids of this 23-amino acid segment may be present in unprocessed SPIK secreted by liver cancer cell lines. (Lu et al., Immunology 2011;134(4):398-408). We sometimes refer to the longer form of SPIK as AS-SPIK or aberrant secreted SPIK. We may also refer to AS-SPIK produced by hepatoma cells as LC-SPIK or hepatoma-secreted SPIK. The terms AS-SPIK and LC-SPIK are used interchangeably herein. An exemplary AS-SPIK polypeptide may have the amino acid sequence of SEQ ID NO:2. We may also refer to the form of SPIK secreted by normal cells, such as pancreatic cells, as NS-SPIK or normal-secreted SPIK. An exemplary NS-SPIK polypeptide may have the amino acid sequence of SEQ ID NO:4.

[0103] The present inventors have demonstrated herein that AS-SPIK differs from NS-SPIK in both size and conformation (three-dimensional structure). Antibodies that selectively bind to AS-SPIK but not NS-SPIK can be divided into two classes (Class I and Class II) based on the epitope they bind to. Class I anti-AS-SPIK antibodies bind to epitope I, and Class II anti-AS-SPIK antibodies bind to epitope II, both of which are described herein. Epitope mapping and analysis of the three-dimensional structure of AS-SPIK have shown that each epitope is discontinuous in the conformation, consisting of at least two separate regions, and specific epitope sequences have also been identified. Furthermore, further analysis of antibodies from each class has identified consensus sequences for Class I and Class II antibodies that are important for functionality. Methods of using such antibodies to diagnose and / or treat disorders characterized by AS-SPIK expression (e.g., liver cancer) are also described herein.

[0104] Thus, 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. AS-SPIK complexes according to embodiments of the invention comprise AS-SPIK and an antibody or fragment thereof that specifically or preferentially binds to an AS-SPIK polypeptide.

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

[0106] Embodiments of the present invention also include methods of using the subject antibodies for the detection of disorders characterized by expression of AS-SPIK, eg, liver disorders such as liver cancer, eg, HCC or ICC.

[0107] While the inventors believe they understand the specific events that occur during expression of AS-SPIK, the compositions and methods of the present invention are not limited to working 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 peptidases, a type of protease, resulting in the secretion of the unreleased full-length protein from the cancer cells.

[0108] composition The compositions provided herein include antibodies that specifically or preferentially bind to AS-SPIK and do not bind to NS-SPIK.

[0109] Serine protease inhibitor Kazal (SPIK), also known as SPINK1, PSTI, and TATI, is a small protein that has been shown to broadly regulate the activity of many cellular proteases, including trypsin-like 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 be involved in the inhibition of apoptosis. Lu et al., Immunology 2011;134(4):398-408. Exemplary human SPIKE amino acid sequences include GenBank Accession No. M11949, GI No. 190687; GenBank Accession No. NM003122, GI: 657940887; and GeneBank Accession No. BC025790, GI: 19343607.

[0110] antibody The antibodies provided herein may include antibodies that specifically or preferentially bind to conformational epitopes on the AS-SPIK protein. In some embodiments, the antibodies specifically or preferentially bind to discontinuous conformational epitopes, as described above. Antibodies according to embodiments of the present invention may be polyclonal or monoclonal, particularly monoclonal, and may be produced by human, mouse, rabbit, sheep, or goat cells, or by hybridomas derived from these cells. In some embodiments, the antibodies may be humanized or chimeric.

[0111] Antibodies according to embodiments of the present invention can take various configurations and can include proteins consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Any one of a variety of antibody structures may be used, including intact antibodies, antibody multimers, or antibody fragments or other variants thereof that contain functional antigen-binding regions of an antibody. The term "immunoglobulin" may be used synonymously with "antibody." Antibodies may be monoclonal or polyclonal in origin. Regardless of the source of the antibody, suitable antibodies include intact antibodies, e.g., 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, e.g., Fab, Fab', F(ab')2, scFv, Fv, and recombinant antibodies derived from such fragments, e.g., camelbodies, microantibodies, diabodies, and bispecific antibodies.

[0112] An intact antibody comprises antigen-binding variable regions (VH and VL) as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. The VH and VL regions are further subdivided into regions of hypervariability called "complementarity-determining regions" (CDRs) interspersed with more conserved framework regions (FR). The CDRs of an antibody generally contain amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.

[0113] Anti-AS-SPIK antibodies may be of any class of immunoglobulin, e.g., IgA, IgG, IgE, IgD, IgM (and their subtypes, e.g., IgG1, IgG2, IgG3, and IgG4), and the immunoglobulin light chains may be of the kappa or lambda type. Recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.

[0114] The term "antigen-binding portion" of an immunoglobulin or antibody generally refers to a portion of an immunoglobulin that specifically or preferentially binds to a target, in this case, a conformational epitope of an AS-SPIK protein. Thus, an antigen-binding portion of an immunoglobulin is a molecule in which one or more immunoglobulin chains are not full-length but that specifically or preferentially binds to a target. Examples of antigen-binding portions or fragments include: (i) a Fab fragment, which is a monovalent fragment consisting of the VLC, VHC, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fv fragment consisting of the VLC and VHC domains of a single antibody arm; and (v) an isolated CDR, e.g., an antigen-binding portion of a variable region, having sufficient framework for specific or preferential binding. The antigen-binding portion of a light chain variable region and the antigen-binding portion of a heavy chain variable region, e.g., the two domains of an Fv fragment, VLC and VHC, can be joined using recombinant methods by a synthetic linker that allows them to be produced as a single protein chain in which the VLC and VHC domains pair to form a monovalent molecule (known as a single-chain Fv (scFv)). Such scFvs are encompassed by the term "antigen-binding portion" of an antibody.

[0115] An "Fv" fragment is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH·VL dimer. Although the six hypervariable regions confer antigen-binding specificity, a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) can recognize and bind antigen, albeit with lower affinity than the entire binding site. For improved stability, the VH·VL domains can be connected by a flexible peptide linker, such as (Gly4Ser)3, to form single-chain Fv or scFv antibody fragments, or can be engineered to form disulfide bonds by introducing two cysteine ​​residues into the framework regions to form disulfide-stabilized Fvs (dsFvs).

[0116] Fragments of antibodies are suitable for use in the provided methods, so long as they retain the desired epitope specificity of the full-length antibody and / or sufficient specificity to bind to AS-SPIK and not to NS-SPIK.

[0117] The antigen-binding domains of the antibodies described herein can be used to generate T cell binding molecules (e.g., bispecific T cell engagement (also known as BiTE) molecules) as well as CAR-T structures. T cell binding 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 structures comprising single-domain antibodies as binding (targeting) 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.

[0118] Methods for producing antibody fragments include both biochemical methods (e.g., proteolytic digestion of intact antibodies, optionally followed by chemical cross-linking) and recombinant DNA-based methods in which immunoglobulin sequences are genetically engineered to direct the synthesis of desired fragments. Antibody fragments can be obtained by proteolysis of whole immunoglobulins with the nonspecific thiol protease, papain. Papain digestion results in two identical antigen-binding fragments, called "Fab fragments," each with a single antigen-binding site, and a residual "Fc fragment." The various fractions can be separated by protein A-Sepharose or ion-exchange chromatography. The usual procedure for producing F(ab')2 fragments from IgG derived from rabbits and humans is limited hydrolysis with the enzyme pepsin. Pepsin treatment of intact antibodies yields F(ab')2 fragments that have two antigen-binding sites and are still capable of cross-linking antigen. Fab fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteine(s) of the antibody hinge region. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them.

[0119] Methods for producing anti-AS-SPIK antibodies are also within the scope of the present invention. For example, variable regions can be constructed using PCR mutagenesis to alter the DNA sequences encoding immunoglobulin chains (e.g., using methods utilized to form humanized immunoglobulins).

[0120] Monoclonal antibodies are homogeneous antibodies of identical antigen specificity produced by a single clone of antibody-producing cells, whereas polyclonal antibodies generally recognize different epitopes on the same antigen and are produced by two or more clones of antibody-producing cells. Each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method.

[0121] The monoclonal antibodies herein may also include chimeric antibodies, i.e., antibodies having portions of the heavy and / or light chains identical or homologous to corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical or homologous to corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest include primatized antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., ape, Old World monkey, New World monkey, prosimian) and human constant region sequences.

[0122] Mouse and rabbit monoclonal antibodies were generated by immunizing mice or rabbits with a specifically designed recombinant protein that contains a common region (SEQ ID NO: 4) of amino acid sequences found in both NS-SPIK and AS-SPIK, plus an additional 23 amino acid sequence (SEQ ID NO: 6) found in AS-SPIK but not NS-SPIK. In some embodiments, the recombinant protein may not need to contain the entire 23 amino acid sequence (SEQ ID NO: 6) to generate antibodies that are effective in binding only to AS-SPIK but not to NS-SPIK.

[0123] Methods for producing monoclonal antibodies may include a purification step. For example, antibodies may commonly be further purified using, for example, filtration, centrifugation, and various chromatographic methods, such as HPLC or affinity chromatography, all of which are well known to those of skill in the art. Each of these purification techniques involves fractionation to separate the desired antibody from other components of the mixture. Analytical methods particularly suited to antibody production include, for example, protein A-Sepharose and / or protein G-Sepharose chromatography.

[0124] The anti-AS-SPIK antibodies of the present invention may contain CDRs of human or non-human origin. "Humanized" antibodies are generally chimeric or mutant monoclonal antibodies derived from mouse, rat, hamster, rabbit, or other species, with human constant and / or variable region domains or specific alterations. The immunoglobulin framework may be human, humanized, or non-human (e.g., a mouse framework modified to reduce antigenicity in humans), or a synthetic framework (e.g., a consensus sequence). A humanized immunoglobulin is one in which framework residues correspond to human germline sequences and CDRs result from V(D)J recombination and somatic mutation. However, humanized immunoglobulins may also contain amino acid residues not encoded by human germline immunoglobulin nucleic acid sequences (e.g., mutations introduced by ex vivo random or site-specific mutagenesis). Antibody variable domain genes based on germline sequences but with framework mutations introduced, for example, by in vivo somatic mutation processes, are referred to as "human."

[0125] Humanized antibodies may be engineered by a variety of methods known in the art, including, for example: (1) grafting nonhuman complementarity-determining regions (CDRs) onto human framework and constant regions (a process referred to in the art as humanization); or (2) grafting entire nonhuman variable domains but conferring a human-like surface by substitution of surface residues (a process referred to in the art as veneering). Humanized antibodies may include both humanized and veneered antibodies. Similarly, human antibodies may be generated by introducing human immunoglobulin loci into transgenic animals, e.g., mice, in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.

[0126] In addition to chimeric and humanized antibodies, fully human antibodies can be derived from transgenic mice carrying human immunoglobulin genes. In some embodiments, antibodies may be generated and identified by scFv-phage display libraries.

[0127] Anti-AS-SPIK antibodies may be modified to adjust antigen-binding affinity, effector function, or pharmacokinetics. In particular, random mutations may be made in the CDRs and the products screened to identify antibodies with higher affinity and / or higher specificity. Generally, the CDRs may differ by one or two amino acids.

[0128] CDR shuffling and insertion techniques may be used, for example, with the antibodies provided herein. CDR shuffling inserts CDR sequences into specific framework regions. CDR insertion techniques allow for the random combination of CDR sequences into a single basic framework. Using such techniques, for example, the CDR sequences of an anti-AS-SPIK antibody may be mutagenized to generate multiple different sequences that can be incorporated into the scaffold sequence, and the resulting antibody variants may be screened for desired properties, such as higher affinity.

[0129] Our research on the function of SPIK has shown that SPIK can bind to granzyme A (GzmA) and inhibit its apoptosis-inducing activity. (Lu et al., Immunology 2011;134(4):398-408) GzmA is a cytotoxic serine protease secreted by activated CTLs and NK cells to kill target cells during immune surveillance. The role of GzmA-induced apoptosis in the elimination of malignant cells, such as tumor precursor / tumor germ cells, has been confirmed. (Pardo et al., Eur J Immunol 2002;32(10):2881-2887) Therefore, overexpression of AS-SPIK in hepatocarcinoma cells may render them resistant to GzmA-induced apoptosis during immune clearance, thereby evading these cancer cells from immune-mediated killing. (Lu et al., Immunology 2011;134(4):398-408) Based on this hypothesis, and without being bound by theory, the inventors conclude that suppressing the overexpression of AS-SPIK or inhibiting the activity of overexpressed AS-SPIK can restore the immune killing of cancer cells induced by GzmA during immune clearance in the human body.

[0130] Anti-AS-SPIK antibodies according to embodiments of the present invention can inhibit the activity of AS-SPIK, as demonstrated by the disclosure of PCT Application No. PCT / US19 / 20999, the entire disclosure of which is incorporated herein by reference. Thus, anti-AS-SPIK antibodies can be used to block the binding of AS-SPIK to GzmA, free GzmA, thereby reversing the apoptotic death of such cancer cells due to immune clearance. To this end, anti-SPIK antibodies may be used to treat disorders characterized by AS-SPIK expression, including, but not limited to, cancer, viral infection, and inflammation.

[0131] One therapeutic use of antibodies is through humanization. Therapy with humanized monoclonal antibodies is a rapidly developing field, and their specificity and efficacy have been thoroughly studied. Rothernberg, ME, Cell 2016;165(3):509. The subject anti-AS-SPIK monoclonal antibodies, including but not limited to IM-CA22, IM-A1, IM-B10, IM-CA18, IM-D2, IM-D3, IM-D5, IM-G2, IM-E2, IM-C6, IM-CA46, IM-CA77, IM-A6, IM-B3, IM-F5, and IM-G6, as well as other antibodies of the present invention, such as antibodies that bind to epitope I or epitope II and can inhibit SPIK activity as described herein, can also be humanized and used to treat diseases.

[0132] For example, recombinant techniques using phagemid technology allow the production of antibodies with desired specificity from recombinant genes encoding a range of antibodies. A particular recombinant technique involves the isolation of antibody genes by immunological screening of a combinatorial immunoglobulin phage expression library prepared from RNA isolated from the spleens of immunized animals. For such methods, a combinatorial immunoglobulin phagemid library may be prepared from RNA isolated from the spleens of immunized animals, and phagemids expressing the appropriate antibody may be selected by panning using cells expressing the antigen and control cells.

[0133] In addition to the combinatorial immunoglobulin phage expression libraries disclosed above, one molecular cloning approach is to generate antibodies from transgenic mice containing human antibody libraries. Such transgenic animals can be used to generate human antibodies of a single isotype, more specifically, an isotype essential for B cell maturation, such as IgM and, optionally, IgD.

[0134] Anti-AS-SPIK immunoglobulins may be modified to reduce or eliminate glycosylation. A glycosylated immunoglobulin may be an immunoglobulin that is not glycosylated at all, is not fully glycosylated, or is atypically glycosylated (i.e., the glycosylation pattern of the variant 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 reduce glycosylation, i.e., mutations that result in an IgG CH2 domain that is not glycosylated, is not fully glycosylated, or is atypically glycosylated. The oligosaccharide structure may also be modified, for example, by removing the fucose moiety of N-linked glycans.

[0135] Antibodies may also be modified to enhance in vivo stability and / or solubility by conjugation with a nonprotein polymer, such as polyethylene glycol. Any PEGylation method may be used as long as the anti-AS-SPIK antibody retains the ability to selectively bind to AS-SPIK and not to NS-SPIK.

[0136] A wide variety of antibody / immunoglobulin frameworks or scaffolds may be utilized, so long as the resulting polypeptide contains at least one binding region specific for the target, i.e., AS-SPIK. Such frameworks or scaffolds include human immunoglobulins of the five major idiotypes, or fragments thereof (such as those disclosed elsewhere herein), and preferably immunoglobulins of other animal species with humanized versions. Single heavy chain antibodies, such as those identified in camelids, are of particular interest in this regard.

[0137] The anti-AS-SPIK antibodies of the present invention specifically or preferentially bind to an epitope on AS-SPIK and not to an epitope on NS-SPIK. The term "epitope" refers to the paratope, i.e., the antigenic determinant on the target to which the antibody binding site specifically binds. Epitopes typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes generally contain between about 4 and about 10, preferably 4 to 8, contiguous amino acids (linear or contiguous epitopes), or they can be a series of noncontiguous amino acids that define a specific structure (e.g., conformational epitopes). Thus, epitopes may consist of at least 4, at least 6, at least 8, at least 10, and at least 12 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography, two-dimensional nuclear magnetic resonance, and Precision Epitope Mapping using CLIPS (Chemically Linked Peptides on Scaffolds) peptide arrays (Timmerman, Puijk et al., J Mol Recognit, 20(5), 283-299, (2007)).

[0138] Other methods for predicting potential epitopes to which an antibody may 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. al., Virology 140:13-20 (1985)), Chou and Fasman analysis (Ponomarenko & Regenmortel, Structural Bioinformatics, 2009), Karplus and Schulz analysis (Kolaskar and Tongaonkar Analysis Kolaskar & Tongaonkar, FEBS Letters, 172-174 (1990)), and Parker analysis. In some embodiments, potential epitopes are determined by correlation with known antigenic sites from other studies, and these prediction techniques may be combined with structural data such as X-ray crystallography data. Epitope prediction may also include techniques 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 theoretical extracellular domains.Analytical algorithms such as TMpred (see Hofmann and Stoffel, Biol. Chem. 374:166 (1993)) or TMHMM (Krogh et al., J. Mol. Biol., 305(3):567-580 (2001)) may be used to make such predictions. Other algorithms, such as SignalP3.0 (Bednsten et al., J. Mol. Biol. 340(4):783-795 (2004)), can be used to predict the presence of signal peptides and where those peptides will be cleaved from the full-length protein. Portions of proteins that are outside the cell can serve as targets for antibody interaction.

[0139] The compositions of the present invention include antibodies described herein that (1) exhibit a threshold level of binding activity, (2) do not significantly cross-react with known related polypeptide molecules, (3) bind to AS-SPIK, and (4) do not bind to NS-SPIK. The binding affinity of an antibody can be readily determined by one skilled in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660-672 (1949)).

[0140] In some embodiments, the anti-AS-SPIK antibody can bind to its target epitope or mimic decoy at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, 106-fold or more to the target AS-SPIK than to another protein predicted to have some homology to AS-SPIK, e.g., NS-SPIK.

[0141] In some embodiments, the anti-AS-SPIK antibody has a titer of 10 M or less, 10 -7 M or less, 10 -9 In some embodiments, the binding affinity of the anti-AS-SPIK antibody for its respective target is at least 1×10 6In 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 higher. Antibodies may also be designated or specified in terms of their binding affinity to AS-SPIK. In some embodiments, the binding affinity is 5×10 -2 Under M, 10 -2 M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -3 M, 10 -4 M, 5 x 10 -5 M, 10 -5 M, 5 x 10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M or 10 -15 M or a binding affinity of Kd less than or equal to M.

[0142] The antibody of the present invention is -4 M or less, 10 -7 M or less, 10 -9 In some embodiments, the binding affinity of the anti-AS-SPIK antibody for its respective target is at least 1×106 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 In some embodiments, the binding affinity is 5×10 -2 M, 10 -2 M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -3 M, 10 -4 M, 5 x 10 -5 M, 10 -5 M, 5 x 10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M or 10 -15 In contrast, the term "non-specific binding," e.g., to NS-SPIK, as used herein, includes binding affinities of at least 1.5, 2, 5, 10, 100, 10 M or less than the binding affinity determined for "specific binding" to AS-SPIK. 3 , 10 4 , 10 5 , 10 6Affinity, such as Kd, refers to a binding affinity that is at least 1.5-fold, 2-fold, 5-fold, 10 ... 3 double, 10 4 double, 10 5 double, or 10 6 Twice as big.

[0143] In some embodiments, the antibody does not bind to a known related polypeptide molecule. For example, it binds to AS-SPIK but not to a known related polypeptide, such as NS-SPIK. Antibodies may be screened against known related polypeptides to isolate a population of antibodies that specifically or preferentially bind to AS-SPIK. For example, antibodies specific for AS-SPIK are passed through a column containing NS-SPIK attached to an insoluble matrix under appropriate buffer conditions. Such screening allows for the isolation of polyclonal and monoclonal antibodies that do not cross-react 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.

[0144] Antibodies according to embodiments of the present invention may include a detectable label, which may also be referred to as a reporter (e.g., a detectable reporter). In some embodiments, the detectable label can be any molecule or biologically active fragment thereof covalently attached to an antibody (e.g., an anti-AS-SPIK antibody) that allows for qualitative and / or quantitative assessment of the expression or activity of the tagged peptide. The activity can include biological activity, physicochemical activity, or a combination thereof. Both the form and location of the detectable label can be varied as long as the labeled antibody retains its biological activity. Many different labels can be used, and the choice of a particular label will depend on the desired application. A labeled anti-AS-SPIK antibody can be used, for example, to assess the level of AS-SPIK in a biological sample, such as urine, saliva, cerebrospinal fluid, blood, or a biopsy sample.

[0145] Detectable labels include enzymes, photoaffinity ligands, radioisotopes, and fluorescent or chemiluminescent compounds. Exemplary enzyme labels include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and urease. Covalent binding of anti-AS-SPIK antibodies to enzymes can be achieved by various methods, such as linking with glutaraldehyde via free amino groups. Alternatively, anti-AS-SPIK antibodies can be conjugated to enzymes via sugar residues. Other enzymes containing carbohydrates can also be conjugated to antibodies using this technique. Enzyme coupling can also be achieved by linking the amino group of the antibody to the free thiol group of an enzyme, such as β-galactosidase, using a heterobifunctional linker, such as succinimidyl 6-(N-maleimido)hexanoate. Horseradish peroxidase detection systems can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which produces a soluble product detectable at 450 nm in the presence of hydrogen peroxide. For example, alkaline phosphatase detection systems may be used with the chromogenic substrate p-nitrophenyl phosphate, which yields a soluble product readily detectable at 405 nm. Similarly, β-galactosidase detection systems may be used with the chromogenic substrate o-nitrophenyl-PD-galactopyranooxide (ONPG), which yields a soluble product detectable at 410 nm. Urease detection systems may be used with substrates such as urea-bromocresol purple.

[0146] The detectable label can be a fluorescent label, including, but not limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine; a chemiluminescent compound selected from the group consisting of luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, and oxalate ester; a liposome or dextran; or a bioluminescent compound such as luciferin, luciferase, and aequorin. Alternatively or additionally, detectable labels include radiopaque or contrast agents such as, but not limited to, barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iosetamic acid, iodamide, iodipamide, iodoxamic acid, ioglamide, iohexol, iopamidol, iopanoic acid, ioprosemic acid, iosephamic acid, ioselic acid, iosuramide meglumine, iosemetic acid, iotasul, iotetolic acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propriodone, and thallium chloride.

[0147] Labels can be added during or after synthesis. Recombinant anti-AS-SPIK antibodies or biologically active variants thereof can also be labeled by adding a label precursor (e.g., a radiolabeled amino acid) to the culture medium in which transformed cells are grown. In some embodiments, peptide analogs or variants may be used to facilitate the incorporation of detectable markers. For example, any N-terminal phenylalanine residue may be replaced with a closely related aromatic amino acid, such as tyrosine, which can be easily labeled with 125I. In some embodiments, additional functional groups that facilitate efficient labeling may be added to fragments of anti-AS-SPIK antibodies or biologically active variants thereof. For example, a 3-tributyltin benzoyl group may be added to the N-terminus of the native structure, followed by replacement of the tributyltin group with 125I to generate a radiolabeled iodobenzoyl group.

[0148] Antibody-drug conjugates (ADCs) Embodiments of the present invention include immunoconjugates, or antibody-drug conjugates (ADCs), comprising an antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In another embodiment, the present invention further provides methods of using the immunoconjugates. In one embodiment, the immunoconjugate comprises any of the anti-AS-SPIK antibodies described above covalently conjugated to a cytotoxic or detectable agent. ADCs are described, for example, in U.S. Patent No. 8,362,213, the entire disclosure of which is incorporated herein by reference.

[0149] The use of ADCs for the local delivery of cytotoxic or cytostatic drugs, i.e., drugs to kill or inhibit tumor cells in the treatment of cancer, has been reported (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9):1137-1146; Payne, G. (2003) Cancer Cell 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278), allowing targeted delivery of the drug moiety to tumors and their intracellular accumulation therein, whereas systemic administration of these unconjugated drug agents can result in unacceptable levels of toxicity not only to the tumor cells being 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., maximize efficacy and minimize toxicity, of ADCs have focused on the selectivity and drug binding and release properties of polyclonal antibodies (Rowland et al (1986) Cancer Immunol. Immunother., 21:183-87) and monoclonal antibodies (mAbs) (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549).Drug moieties used in ADCs include bacterial protein toxins, e.g., diphtheria toxin, plant protein toxins, e.g., ricin, small molecules, e.g., auristatins, geldanamycin (Mandler et al (2000) J. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), 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 drug moiety can affect cytotoxic and cytostatic mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to become inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0150] The auristatin peptides, auristatin E (AE) and monomethyl auristatin (MMAE), a synthetic analog of dolastatin (WO 02 / 088172), have been used as drug moieties in: (i) the chimeric monoclonal antibody cBR96, specific for Lewis Y in carcinoma; (ii) cAC10, specific for CD30 in hematological tumors (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; US2004 / 0018194); (iii) anti-CD20 antibodies, such as Rituxan for the treatment of CD20-expressing cancers and immune disorders (WO04 / 032828); (iv) anti-EphB2R antibody 2H9 for the treatment of colorectal cancer (Mao et al (2004) Cancer Research 64(3):781-788); (v) E-selectin antibodies (Bhaskar et al (2003) Cancer Res. 63:6387-6394); (vi) trastuzumab (HERCEPTIN®, US2005 / 0238649) and (vi) anti-CD30 antibodies (WO03 / 043583). Variants of auristatin E are disclosed in U.S. Patent No. 5,767,237 and U.S. Patent No. 6,124,431. A monomethyl auristatin E-binding monoclonal antibody is disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented March 28, 2004. The auristatin analogs MMAE and MMAF have been conjugated to various antibodies (US2005 / 0238649).

[0151] Traditional means of attaching drug moieties to antibodies, i.e., covalent linkage, generally result in a heterogeneous mixture of molecules in which the drug moieties are attached to several sites on the antibody. For example, cytotoxic drugs are commonly attached to antibodies via the antibody's often numerous lysine residues, resulting in a heterogeneous mixture of antibody-drug conjugates. Depending on the reaction conditions, the heterogeneous mixture typically contains antibodies with anywhere from 0 to about 8 or more drug moieties attached. Furthermore, within each subgroup of conjugates with a specific integer ratio of drug moiety to antibody, there is a potentially heterogeneous mixture in which the drug moieties are attached to various sites on the antibody. Analytical and preparative methods may be insufficient to separate and characterize the antibody-drug conjugate species in the heterogeneous mixture resulting from the conjugation reaction. Antibodies are large, complex, and structurally diverse biomolecules, often possessing numerous reactive functional groups. Their reactivity with linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration, and cosolvents. Furthermore, the multi-step conjugation process can be irreproducible due to the difficulty in controlling the reaction conditions and characterizing the reactants and intermediates.

[0152] Cysteine ​​thiols are highly reactive at neutral pH, unlike most amines, which become protonated and less nucleophilic near pH 7. Because free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine ​​residues often exist in their oxidized form as disulfide-linked oligomers or with internal disulfide crosslinks. Extracellular proteins generally lack free thiols (Garman, 1997, Non-Radioactive Labeling: A Practical Approach, Academic Press, London, at page 55). Antibody cysteine ​​thiol groups are generally more reactive toward electrophilic coupling reagents, i.e., more nucleophilic, than antibody amine or hydroxyl groups. 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, engineering cysteine ​​thiol groups by mutating various amino acid residues in proteins to cysteine ​​amino acids can be problematic, particularly for unpaired (free Cys) residues or residues that are relatively reactive or prone to oxidation. In concentrated solutions of proteins, whether in the periplasm of E. coli, in culture supernatants, or in partially or fully purified proteins, unpaired Cys residues on the surface of proteins can pair and oxidize to form intermolecular disulfides, thus forming protein dimers or multimers.The formation of disulfide dimers renders the new Cys unreactive for conjugation with drugs, ligands, or other labels. Furthermore, if a protein oxidatively forms an intramolecular disulfide bond between the newly engineered Cys residue and an existing Cys residue, both Cys thiol groups are unavailable for active site participation and interaction. Furthermore, proteins can become inactive or nonspecific due to misfolding or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311:1-9).

[0153] Cysteine-engineered antibodies were engineered as fragment-antibody fragments (ThioFabs) and expressed as full-length IgG monoclonal antibodies (ThioMabs) (Junutula, JR et al. (2008) J Immunol Methods 332:41-52; US2007 / 0092940, the contents of which are incorporated by reference). ThioFab and ThioMab antibodies were linked via a thiol-reactive linker reagent and a drug-linker reagent at the newly introduced cysteine ​​thiol to prepare antibody-drug conjugates (Thio ADCs).

[0154] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0155] Polypeptides Aspects of the invention include compositions comprising a SPIK polypeptide, e.g., an AS-SPIK polypeptide encoded by the nucleic acid sequence of SEQ ID NO:1. The terms "peptide," "polypeptide," and "protein" are used interchangeably herein but typically refer to peptide sequences of various sizes. We sometimes refer to amino acid-based compositions of the invention as "polypeptides" to convey that polypeptides are linear polymers of amino acid residues and to help distinguish them from full-length proteins. Polypeptides according to embodiments of the invention may "consist of" or "comprise" a fragment of an AS-SPIK or NS-SPIK polypeptide, and the invention encompasses polypeptides that constitute or comprise a biologically active variant of an AS-SPIK or NS-SPIK polypeptide. Thus, it will be understood that a polypeptide may comprise only a fragment of an AS-SPIK or NS-SPIK polypeptide (or a biologically active variant thereof) but may also contain additional residues. Biologically active variants will retain sufficient activity to inhibit proteases.

[0156] The bond between amino acid residues can be a conventional peptide bond or another covalent bond (such as an ester or ether bond), and the polypeptide may be modified by amidation, phosphorylation, or glycosylation. Modifications can affect the polypeptide backbone and / or one or more side chains. Chemical modifications can be naturally occurring modifications made in vivo after translation of the mRNA encoding the polypeptide (e.g., glycosylation in a bacterial host) or synthetic modifications made in vitro. Biologically active variants of AS-SPIK or NS-SPIK polypeptides can contain one or more structural modifications resulting from any combination of naturally occurring modifications (i.e., originally made in vivo) and synthetic modifications (i.e., naturally or non-naturally occurring modifications made in vitro). Examples of modifications include, but are not limited to, amidation (e.g., replacement of the free carboxyl group at the C-terminus with an amino group); biotinylation (e.g., acylation of lysine or other reactive amino acid residues with a biotin molecule); glycosylation (e.g., addition of a glycosyl group to either an asparagine, hydroxylysine, serine, or threonine residue to form a glycoprotein or glycopeptide); acetylation (e.g., addition of an acetyl group, generally to the N-terminus of a 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).

[0157] One or more amino acid residues of a biologically active variant may be a non-naturally occurring amino acid residue. Naturally occurring amino acid residues include amino acids originally 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 may also contain amino acid residues that are modified versions of standard residues (e.g., pyrrolysine can be used in place of lysine, and selenocysteine ​​can be used in place of cysteine). Non-naturally occurring amino acid residues are those that are not found in nature but conform to the basic amino acid formula and can be incorporated into peptides. These include D-alloisoleucine (2R,3S)-2-amino-3-methylpentanoic acid and L-cyclopentylglycine (S)-2-amino-2-cyclopentylacetic acid. Additional examples can be found in textbooks or on the World Wide Web (a site currently maintained by the California Institute of Technology shows structures of non-natural amino acids that have been successfully incorporated into functional proteins).

[0158] Alternatively, or in addition, one or more amino acid residues of a biologically active variant may be a naturally occurring residue that is different from a naturally occurring residue not found at the corresponding position in the wild-type sequence. In other words, a biologically active variant may contain one or more, particularly one or two, amino acid substitutions. We may also refer to the substitution, addition, or deletion of an amino acid residue as a mutation of the wild-type sequence. As mentioned, a substitution can replace a naturally occurring amino acid residue with a non-naturally occurring residue or simply a different naturally occurring residue. Furthermore, a substitution may constitute a conservative or non-conservative substitution. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.

[0159] A polypeptide that is a biologically active variant of AS-SPIK can be characterized by the degree to which its sequence is similar or identical to that of the corresponding wild-type polypeptide. For example, the sequence of a biologically active variant may be at least or about 80% homologous (or identical) to the corresponding residues of 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 that has at least or about 80% sequence homology (e.g., at least or about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology) (or the recited percentage of identity) with an AS-SPIK or NS-SPIK polypeptide (SEQ ID NO: 2, 4) or its homolog or ortholog.

[0160] A biologically active variant of an AS-SPIK or NS-SPIK polypeptide will retain sufficient biological activity to be useful in the present methods. A biologically active variant will retain sufficient activity to function as an inhibitor of protease activity. Biological activity can be assessed by methods known to those skilled in the art, including, but not limited to, in vitro cleavage assays or functional assays.

[0161] Polypeptides can be produced by a variety of methods, including, for example, recombinant technology or chemical synthesis. Once produced, the polypeptides 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, can be used. The composition of the final polypeptide may be confirmed by amino acid analysis after digestion of the peptide by standard means, by amino acid sequencing, or by FAB-MS techniques. Salts, including acid salts of polypeptides, esters, amides, and N-acyl derivatives of amino groups can be prepared using methods known in the art, and such peptides are useful in the context of the present invention.

[0162] An AS-SPIK complex is also provided. An AS-SPIK complex according to an embodiment of the present invention comprises an antibody of the present invention described herein that specifically or preferentially binds to AS-SPIK and an AS-SPIK polypeptide or fragment thereof. The antibody may be any of the anti-AS-SPIK antibodies described herein. The AS-SPIK polypeptide or fragment thereof may be an AS-SPIK polypeptide or fragment thereof described herein. In some embodiments, the antibody is an anti-AS-SPIK monoclonal antibody that binds to epitope I or epitope II as described herein. In some embodiments, the AS-SPIK polypeptide is a polypeptide having an amino acid sequence that is at least 98% homologous (or identical) to the amino acid sequence of SEQ ID NO:2. In some embodiments, the AS-SPIK polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO:2.

[0163] As described herein, specific binding of anti-AS-SPIK antibodies, such as antibodies binding to epitope I or epitope II, can form immune complexes with AS-SPIK or AS-SPIK peptides under certain conditions. The complexes can be precipitated from solution for further analysis, for example, by sandwich ELISA testing. Using a 96-well plate with a second anti-SPIK antibody immobilized as a carrier, the immune complexes can be captured by the plate. The amount of AS-SPIK immune complexes formed can then be measured if the antibody in the complex is labeled with a reporter such as horseradish peroxidase (HPR). The AS-SPIK immune complexes can also be captured by agarose beads conjugated with protein A or G for Western blot analysis.

[0164] nucleic acid The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to both RNA and DNA, including DNA (or RNA), including cDNA, genomic DNA, synthetic DNA, and nucleic acid analogs, any of which can encode the polypeptides of the present invention, all of which are encompassed by the present invention. Polynucleotides may have essentially any three-dimensional structure. Nucleic acids may be double-stranded or single-stranded (i.e., sense or antisense). 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, and nucleic acid analogs. In the context of the present invention, nucleic acids can encode fragments of naturally occurring AS-SPIK or NS-SPIK polypeptides or biologically active variants thereof. Non-limiting examples of nucleic acid sequences according to embodiments of the present invention include SEQ ID NOs: 1 and 3, or biologically active fragments or variants thereof.

[0165] An "isolated" nucleic acid may be, for example, a naturally occurring DNA molecule or a fragment thereof, provided that at least one nucleic acid sequence normally found immediately adjacent to the DNA molecule in a naturally occurring genome has been removed or is absent. Thus, an isolated nucleic acid includes, but is not limited to, a DNA molecule that exists as a separate molecule, independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease treatment). An isolated nucleic acid also refers to a DNA molecule incorporated into a vector, a self-replicating plasmid, a virus, or the genomic DNA of a prokaryote or eukaryote. Furthermore, an isolated nucleic acid can include engineered nucleic acids, such as a DNA molecule that is part of a hybrid or fusion nucleic acid. For example, a nucleic acid present among many (e.g., tens, or hundreds to millions) other nucleic acids in a cDNA or genomic library, or a gel slice containing a genomic DNA restriction digest, is not an isolated nucleic acid.

[0166] Isolated nucleic acid molecules can be generated by, for example, polymerase chain reaction (PCR) techniques, which 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 from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Generally, sequence information from the ends of the region of interest or beyond is used to design oligonucleotide primers whose sequences are identical or similar to opposite strands of the template to be amplified. Various PCR methods are also available, which allow site-specific nucleotide sequence modifications to be incorporated into the template nucleic acid.

[0167] Isolated nucleic acids can also be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated 3' to 5' DNA synthesis using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >50-100 nucleotides) containing the desired sequence can be synthesized, each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a double-stranded sequence is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single double-stranded nucleic acid molecule for each oligonucleotide pair, which can then be ligated into a vector. Isolated nucleic acids 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 procedures described above).

[0168] Two nucleic acids or the polypeptides they encode can be said to have a certain degree of homology or identity to each other. For example, an AS-SPIK polypeptide or an NS-SPIK polypeptide and its biologically active variants can be said to exhibit a certain degree of homology or identity. Alignments can be compiled by locating short AS-SPIK or NS-SPIK polypeptide sequences on the Protein Information Research (PIR) site (http: / / pir.georgetown.edu), followed by analysis of "short near-matching sequences" on the NCBI website (http: / / www.ncbi.nlm.nih.gov / blast) using the Basic Local Alignment Search Tool (BLAST) algorithm.

[0169] To determine sequence homology or identity, a query nucleic acid or amino acid sequence may be aligned with one or more subject nucleic acid or amino acid sequences using, for example, a computer program such as BioEdit (version 4.8.5, North Carolina State University), or ALIGN-2, respectively, which allows for full-length alignment of nucleic acid or protein sequences (global alignment), as described above.

[0170] BioEdit calculates the best matches between a query sequence and one or more target sequences, aligns them, and then determines identity, similarity, and difference. To maximize sequence alignment, gaps of one or more residues may be inserted in the query sequence, target sequence, or both. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of superdiagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight change: enabled. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of superdiagonals: 5; and gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalty: on. The output is a sequence alignment that reflects the relationships between the sequences.

[0171] To determine the percent identity between the query and subject sequences, BioEdit divides the number of matches in the best alignment by the number of residues compared (excluding gap positions) and multiplies the result by 100. The output is the percent identity of the subject sequence to the query sequence. Note that percent identity values ​​can be rounded to one decimal place. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, and 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0172] The nucleic acids and polypeptides described herein may also 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 from one species that has been incorporated into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is incorporated into another species via a recombinant nucleic acid construct. An exogenous nucleic acid may also be a sequence that originates from an organism and is reintroduced into the cells of that organism. Exogenous nucleic acids containing native sequences are often distinguishable from naturally occurring sequences by the presence of non-native sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking the native sequence in a recombinant nucleic acid construct. Furthermore, stably transformed exogenous nucleic acids are generally incorporated at locations other than those in which the native sequence is found.

[0173] Recombinant constructs are also provided herein and can be used to transform cells to express AS-SPIK. Recombinant nucleic acid constructs include a nucleic acid encoding an AS-SPIK or NS-SPIK sequence operably linked to a control region suitable for expressing AS-SPIK or NS-SPIK in a particular cell. It will be understood that some 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 two or more nucleotide triplets that serve as codons for that amino acid. For example, the codons in the coding sequence for AS-SPIK or NS-SPIK can be modified using a codon bias table appropriate for a particular organism to obtain optimal expression in that organism.

[0174] Also provided are vectors containing nucleic acids such as those described herein. A "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted to effect replication of the inserted segment. Generally, a vector is capable of replication when associated with appropriate control elements. Suitable vector backbones include those conventionally used in the art, such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term "vector" includes cloning and expression vectors, as well as viral and integrating vectors. An "expression vector" is a vector containing a control region. A wide variety of host / expression vector combinations can be used to express the nucleic acid sequences described herein. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, and retroviruses.

[0175] The vectors provided herein may also include, for example, an origin of replication, a scaffold attachment region (SAR), and / or a marker. A marker gene can confer a selectable phenotype to a host cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin). As described above, expression vectors may include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, CT) sequences, are generally expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including either the carboxyl or amino terminus.

[0176] Additional expression vectors can also include, for example, segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, such as E. coli plasmids colE1, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9, and their derivatives, plasmids such as RP4; phage DNA, such as various derivatives of phage 1, for example NM989, and other phage DNA, such as M13 and filamentous single-stranded phage DNA; yeast plasmids, such as 2μ plasmid or its derivatives, vectors useful in eukaryotic cells, for example, vectors useful in insect or mammalian cells; and vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to use phage DNA or other expression control sequences.

[0177] A vector can also contain a regulatory region. The term "regulatory region" refers to a nucleotide sequence that influences transcription or translation initiation and rate, as well as the stability and / or mobility of the transcription or translation product. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0178] As used herein, the term "operably linked" refers to the positioning of a regulatory region and a transcribed sequence in a nucleic acid so as to affect the transcription or translation of such sequence. For example, to place a coding sequence under the control of a promoter, the translation initiation site of the translation reading frame of a polypeptide is generally positioned 1 to about 50 nucleotides downstream of the promoter. However, the promoter may also be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or as much as about 2,000 nucleotides upstream of the transcription initiation site. A promoter generally comprises at least a core (basal) promoter. A promoter may also comprise at least one control element, such as an enhancer sequence, an upstream element, or an upstream activation region (UAR). The choice of promoter to include depends on several factors, including, but not limited to, efficiency, selectability, 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 positioning promoters and other regulatory regions relative to the coding sequence.

[0179] Vectors containing AS-SPIK or NS-SPIK nucleic acid sequences may be formulated in a manner that promotes uptake by cells, i.e., prokaryotic or eukaryotic cells, e.g., mammalian cells. Useful vector systems and formulations are described above. In some embodiments, the vector is capable of delivering the composition to a specific cell type. While the present invention is not so limited, other methods of DNA delivery are contemplated, such as chemical transfection using calcium phosphate, DEAE dextran, liposomes, lipoplexes, surfactants, and liquid hydrogen-fluorine-substituted compounds, as well as physical delivery methods such as electroporation, microinjection, ballistic particle, and "gene gun" systems. In some embodiments, the polynucleotides of the present invention may also be used with microdelivery vehicles, such as cationic liposomes, other lipid-containing complexes, and other macromolecular complexes that can mediate delivery of polynucleotides to host cells. Another delivery method is to use a single-stranded DNA-generating vector that can produce an expression product within the cell.

[0180] Another aspect of the present invention is to provide methods for producing the antibodies described herein. Antibodies can be prepared by chemical synthesis, but are generally produced by recombinant DNA technology methods, such as co-expression of all chains that make up a protein in a single recombinant host cell, or co-expression of a heavy chain polypeptide and an antibody, e.g., a human antibody. Furthermore, antibody heavy and light chains can also be expressed using a single polycistronic expression vector. Purification of the individual polypeptides is achieved using standard protein purification techniques, such as affinity (protein A) chromatography, size exclusion chromatography, and / or hydrophobic interaction chromatography.

[0181] Another aspect of the present invention is to provide pharmaceutical compositions comprising one or more proteins of the present invention in admixture with a suitable pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier includes, but is not limited to, an adjuvant, a solid carrier, water, a buffer, or other carrier used in the art to carry therapeutic ingredients, or a combination thereof.

[0182] Therapeutic formulations of proteins (e.g., antibodies) used in accordance with the present invention are prepared for storage, e.g., in the form of a lyophilized formulation or aqueous solution, by mixing the protein having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, and the like. hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0183] How to use The compositions disclosed herein are broadly and diversely useful for the diagnosis and / or treatment of disorders characterized by expression of AS-SPIK. Such disorders include, but are not limited to, cancer, viral infections, and inflammatory disorders. One prominent example is liver cancer. Other non-limiting examples include cancers described herein in connection with the definition of the term "cancer." Accordingly, aspects of the present invention include methods for diagnosing and / or treating cancer (e.g., liver cancer) in subjects with cancer or at risk of developing said cancer. The terms "subject," "patient," and "individual" are used interchangeably herein.

[0184] In some embodiments, the method includes contacting a biological test sample from a subject with an AS-SPIK antibody or antigen-binding fragment to form 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 or is at risk of developing a disorder. In certain embodiments, the method includes contacting the biological test sample with a first antibody or antigen-binding fragment that binds to SPIK to form a SPIK-antibody complex, contacting the SPIK-antibody complex with the AS-SPIK antibody or antigen-binding fragment to form 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 or is at risk of developing a disorder. Some non-limiting examples of antibodies that can be used in such methods are described herein.

[0185] In some embodiments, the method comprises administering a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, or antibody-drug conjugate described herein to a patient suffering from a disease or disorder characterized by expression of AS-SPIK.

[0186] Liver cancer One of the well-known examples of the disorder characterized by the expression of AS-SPIK is liver cancer.Liver cancer encompasses a wide range of conditions that cause liver damage or liver dysfunction.Liver cancer can be caused by, for example, infectious agent, disease, trauma or genetic condition, or the combination of infectious agent, disease, trauma and genetic condition.

[0187] Liver cancers include diseases associated with abnormal cell proliferation, such as primary liver cancers, such as hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma, and hepatoblastoma. Such cancers include cancers at any stage of disease progression, such as very early stage (Barcelona Clinic Liver Cancer (BCLC) system stage 0 and tumor size <2 cm), early stage (BCLC stage A, tumor size between 2 cm and 5 cm), intermediate stage (BCLC stage B, intermediate tumor size >5 cm), late stage (BCLC stage C and D, advanced stage), or metastatic stage (Pons et al., HPB 2005;7(1):35-41), and ICC early stage (stage I, II, and IIIa, tumor size <2 cm), intermediate stage (stage IIIb and IIIc, tumor size ≥2 cm), and late stage (stage IV) (Farges et al., Cancer 2011;117(10):2170-2177).

[0188] Liver cancer can also be caused by infections caused by viruses such as hepatitis B, hepatitis C, and hepatitis D. Regardless of the specific hepatitis virus, such infections can be either acute or chronic.

[0189] Liver cancer can also be caused by liver damage, for example, cirrhosis.Cirrhosis, which is the late scarring or fibrosis of the liver, can be caused by many types of liver disease and conditions.Cirrhosis can be caused by genetic conditions, such as hemochromatosis, cystic fibrosis, Wilson's disease, and autoimmune disorders.Cirrhosis can also be caused by hepatitis virus infection and alcohol consumption.

[0190] Liver cancer can also be caused by other diseases, including, but not limited to, alcoholic liver disease, disorders associated with abnormal fat content in the liver, such as fatty liver, non-alcoholic fatty liver disease, non-alcoholic fatty liver, and liver fibrosis.

[0191] Biological samples A "biological sample," "test sample," or "sample" refers to a sample obtained or derived from a patient. The sample may be, for example, a bodily fluid sample. Exemplary bodily fluid samples include blood, serum, plasma, urine, saliva, semen, stool, 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 a biopsy sample, such as a liver biopsy sample, or a primary cell culture sample or primary culture supernatant prepared from the patient's cells.

[0192] Immunoassays Embodiments of the present invention include diagnostic assays, e.g., diagnostic immunoassays, that can be used to detect the presence or absence of AS-SPIK in a test sample. Immunoassay formats used for detecting AS-SPIK can be configured in a variety of ways. Immunoassays can include both homogeneous and heterogeneous assays, competitive and non-competitive assays, direct and indirect assays, and "sandwich" assays. Useful formats include, but are not limited to, enzyme immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), electrochemiluminescence assay, radioimmunoassay, immunofluorescence, fluorescence polarization, immunoprecipitation, equilibrium dialysis, immunodiffusion, immunoblotting, agglutination, luminescence proximity assay, and turbidimetry.

[0193] 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 the surface of particles. More specifically, the support may include a microplate, beads, a polyvinylidene fluoride (PVDF) membrane, a nitrocellulose membrane, a nylon membrane, a porous membrane, or a non-porous membrane. The composition of the substrate can vary. For example, the substrate or support may include glass, a cellulose-based material, a sintered structure composed of a thermoplastic polymer such as polyethylene, polypropylene, or polyester, a particulate material (e.g., glass or various thermoplastic polymers), or a cast membrane film composed of nitrocellulose, nylon, or polysulfone. In general embodiments, the substrate may be any surface or support to which an antibody or polypeptide can be immobilized, including one or more solid supports (e.g., glass, e.g., glass slides or coated plates, silica, plastic or derivatized plastic, paramagnetic or nonmagnetic metals), 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, synthetic polymers may be used as substrates, including, for example, polystyrene, polypropylene, polyglycidyl methacrylate, aminated or carboxylated polystyrene, polyacrylamide, polyamide, and polyvinyl chloride.

[0194] In some embodiments, the immunoassay format may be a two-antibody "sandwich" assay. A biological sample is contacted with an anti-SPIK antibody of the present invention immobilized on a solid support, e.g., a microtiter plate. The sample and first antibody are incubated under conditions that promote specific binding and the formation of an SPIK-antibody complex. After 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 that is different from the epitope bound by the first antibody. Thus, the first and second antibodies do not competitively inhibit each other's binding to SPIK. In some embodiments, the first antibody can recognize an epitope, i.e., an antigenic determinant, present in both AS-SPIK and NS-SPIK. We sometimes refer to such antibodies as "pan-SPIK" antibodies. Alternatively, the first antibody can recognize an epitope present only in AS-SPIK. In some embodiments, the second antibody can recognize an epitope, i.e., an antigenic determinant, present in both AS-SPIK and NS-SPIK. Alternatively, the second antibody can recognize an epitope present only in AS-SPIK or NS-SPIK. Thus, a sandwich assay can be configured in which the first antibody is a pan-SPIK antibody and the second antibody specifically or preferentially binds to AS-SPIK but does not specifically bind to NS-SPIK. Alternatively, a sandwich assay can be configured in which both the first and second antibodies specifically or preferentially bind to AS-SPIK but do not specifically bind to NS-SPIK.

[0195] Antibody binding can be measured in a variety of ways. For example, the signal generated by the detectable label can be analyzed and, if applicable, quantified using an optical scanner or other image acquisition device and software that allows for measurement of a signal associated with complex formation, such as a fluorescent, luminescent, or phosphorescent signal, or a radioactive signal. Exemplary instruments for measuring detectable signals include, but are not limited to, microplate readers, fluorometers, spectrophotometers, and gamma counters.

[0196] Reference sample The level of AS-SPIK in a biological sample can be compared to that of a reference sample. A standard reference level is generally the average AS-SPIK level obtained from a population of individuals. The reference population may include individuals of similar age, body size, ethnic background, or general health status to the individual in question. Thus, the AS-SPIK level in a patient sample can be compared to values ​​obtained from 1) individuals known to have liver cancer, who express AS-SPIK, and whose body fluids contain AS-SPIK; or 2) individuals without liver cancer, whose body fluids contain low levels of AS-SPIK.

[0197] Generally, an elevated level of AS-SPIK can be any level of AS-SPIK higher than either the level of AS-SPIK found in a control sample or the average level of AS-SPIK found in samples from a population of normal healthy individuals without liver cancer (reference value), preferably at least 1, 2, 3, 4, or 5%, more preferably at least 5% higher. A decreased level of AS-SPIK can be any level of AS-SPIK lower than either the level of AS-SPIK found in a control sample or the average level of AS-SPIK found in samples from a population of individuals with liver cancer. Any population size can be used to determine the average level of AS-SPIK found in samples from a population of normal healthy individuals. For example, a population of between 2 and 250 individuals, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, or more individuals, can be used to determine the average level of AS-SPIK in samples from a population of normal healthy individuals, with measurements from larger sample populations being more accurate.

[0198] 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 found in healthy individuals of the same age, ethnic background, or general health as the individual in question. Using this reference chart, any level of AS-SPIK measured in a sample can be classified as low, normal, or elevated compared to a control sample or an average value obtained from a larger population. The term "elevated level" is defined as a level higher than the reference level, preferably at least 2% higher, and more preferably at least 5% higher.

[0199] Alternatively, or in addition, the level of AS-SPIK in a biological sample may be "normalized" to the level of one or more additional biological markers, such as another marker, whose expression is independent of AS-SPIK expression. That is, the level of the additional marker can be assessed in parallel with the level of AS-SPIK, either simultaneously or separately. The additional marker may serve as an internal standard for sample preparation, handling, and storage, as well as for variations in the assay from day to day. The values ​​for the levels of AS-SPIK and the additional marker can be expressed as a ratio, and this ratio may be compared to a similar ratio obtained for a reference sample or population. A useful second marker may be alpha-fetoprotein.

[0200] 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, they may be at different concentrations. In one embodiment, the reference set may include six samples of recombinant AS-SPIK at AS-SPIK concentrations of 50 ng / ml, 30 ng / ml, 8 ng / ml, 3 ng / ml, 1 ng / ml, and 0 ng / ml. 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 may vary. However, those skilled in the art are in a position to determine the average levels of AS-SPIK in various body fluids for each population and determine the respective reference values, ensuring that the levels of AS-SPIK in patients with confirmed liver cancer are sufficiently higher than the reference values, while the levels in patients without liver cancer or healthy individuals are sufficiently lower than the respective reference values. In a preferred embodiment, the reference value is about 5%, more preferably about 7%, and even more preferably about 10% higher than the average level of AS-SPIK found in samples from a population of normal, healthy individuals. The levels of AS-SPIK in the biological sample and the control sample should be measured by the same method so that they are comparable. For example, the absolute value of the AS-SPIK level can be determined from a calibration curve using the recombinant AS-SPIK described above.

[0201] Control sample In some embodiments, the positive control can be a sample of AS-SPIK produced by a eukaryotic cell or cell line. For example, a useful control can be 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, which overexpressed AS-SPIK.

[0202] The methods disclosed herein are useful for detecting liver cancer in patients suspected of having liver cancer or at risk of having liver cancer.The methods can also be used to analyze samples from patients who have been treated for liver cancer, such as hepatocellular carcinoma, to determine whether the patient is likely to be in remission from hepatocellular carcinoma.The methods can also be used to monitor the course of treatment, for example, treatment with therapeutic agents such as small molecule drugs or therapeutic antibodies, chemotherapy, radiotherapy, or surgery, to determine the efficacy of treatment and allow the supervising clinician to modify the treatment as needed.The methods can also be used to detect, monitor, or analyze patients suffering from or at risk of any disorder associated with the regulation, for example, increased, of AS-SPIK levels in biological samples obtained from patients, such as blood or serum samples.

[0203] The methods disclosed herein can be used in conjunction with other standard diagnostic methods, such as serological analysis of liver enzymes or alpha-fetoprotein, ultrasound (ultrasound), computed tomography (CT scan), magnetic resonance imaging (MRI), angiography, laparoscopy, or biopsy.

[0204] manufactured goods For example, compositions described herein for use in detecting, identifying, and quantifying AS-SPIK in biological samples may be packaged in a suitable labeled container. Articles of manufacture, also referred to as "kits," may include antibodies, antigen-binding fragments, and / or antibody-drug conjugates of the present invention, media, purified samples of antigen for use as positive controls, or any combination thereof. A 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 not NS-SPIK. The kit may also include antibodies that bind to both AS-SPIK and NS-SPIK. Suitable buffers for diluting or reconstituting the test sample and antibodies may also be provided. Some components may be provided in dry form and may require reconstitution. The anti-SPIK antibody may be pre-bound to an assay device, e.g., a microplate. Thus, in one embodiment, a kit for detecting, identifying, and quantifying AS-SPIK includes an anti-AS-SPIK antibody and a pan-SPIK antibody. The kit may optionally include a detectable label.

[0205] Accordingly, packaged articles of manufacture (e.g., sterile containers containing one or more compositions described herein packaged for storage, transport, or sale at concentrated or ready-to-use concentrations) and kits containing at least one composition of the invention, e.g., an anti-AS-SPIK antibody, are also within the scope of the invention. An article of manufacture may include a container (e.g., a vial, jar, bottle, bag, etc.) containing one or more compositions of the invention. Additionally, an article of manufacture may further include, for example, packaging materials, instructions for use, a syringe, a delivery device, a buffer, or other control reagents for treating or monitoring a condition requiring diagnosis or treatment.

[0206] Reagents for a particular type of assay may also be provided in the kits of the invention. Thus, the kit may include a collection of beads (e.g., suitable for an agglutination assay or a lateral flow assay) or a plate (e.g., suitable for an ELISA assay). In other embodiments, the kit includes a device such as a lateral flow immunoassay device, an analytical rotor, or an electrochemical, optical, or optoelectronic sensor. The collections of beads, plates, and devices are useful for performing immunoassays. For example, they may be useful for detecting the formation of a first drug-analyte-second drug complex.

[0207] Additionally, the kit may contain various diluents and buffers, labeling conjugates or other agents for detecting specifically bound antigens or antibodies, and other signal-generating reagents such as enzyme substrates, coenzymes, and chromogens. The kit may also contain one or more reference samples at different concentrations, such as purified recombinant AS-SPIK. The kit may also contain a positive control, such as cell supernatant from a cell line overexpressing AS-SPIK. Other components of the kit may include coating reagents, polyclonal or monoclonal capture antibodies or a cocktail of two or more antibodies specific for the antigen or analyte being tested, purified or semi-purified extracts of these antigens as standards, monoclonal detection antibodies, anti-mouse, anti-dog, anti-chicken, or anti-human antibodies conjugated with indicator molecules, a colorimetric indicator chart, disposable gloves, decontamination instructions, an applicator stick or container, a sample preparation cup, etc. In one embodiment, the kit contains buffers or other reagents suitable for forming a reaction medium that allows for the formation of peptide-antibody complexes.

[0208] Such kits provide a convenient and efficient method for clinicians to determine whether a subject has or is at risk of liver cancer.Therefore, in certain embodiments, the kit further comprises instructions for use.The product may also comprise instructions (for example, a printed label or insert or other media (for example, audio or video tape) that explain how to use the product).The instructions may be associated with (for example, attached to) the container and may explain how the assay should be performed, instructions for its use, and other uses.

[0209] The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Although several embodiments have been provided in this disclosure, it will be understood that the disclosed compositions and methods can be embodied in many other specific forms without departing from the spirit or scope of the disclosure. The examples are to be considered illustrative and not restrictive, and are not intended to be limited to the details set forth herein. Various examples of changes, substitutions, and alterations will be apparent to those skilled in the art and can be made without departing from the spirit and scope of the present disclosure. [Example]

[0210] Example 1. AS-SPIK is larger than NS-SPIK The serine protease inhibitor Kazal (SPIK / SPINK1) is a small secreted protein with 79 amino acids (4). Secreted SPIK proteins are typically shorter than the gene SPIK due to the removal of 23 amino acids at the N-terminus, which is thought to act as a signal peptide, resulting in a secreted protein containing only 56 residues (14, 15). However, in hepatoma cells, AS-SPIK is larger than NS-SPIK due to the retention of this additional fragment. This difference in composition between AS-SPIK and NS-SPIK was confirmed by size analysis (gel electrophoresis) and Edman degradation-based protein sequencing. Briefly, AS-SPIK was purified using HPLC from the culture medium of S2-3 cells, a cell line constructed by the present inventors that expresses high levels of AS-SPIK (16), and NS-SPIK was purified from the culture medium of pancreatic cells. 1 μg of each protein was run on a 5–15% gradient SDS-PAGE gel (Invitrogen, Carlsbad, CA). After transfer to a PVDF membrane, the protein was visualized by Coomassie blue staining. Figure 1, which documents the results of this study, shows that the size of NS-SPIK produced by pancreatic cells is approximately 6.5 kD, consistent with published sequence data (SEQ ID NO: 4), confirming that the first 23 amino acids of NS-SPIK are removed during secretion (14, 17). In contrast, the size of AS-SPIK is approximately 10 kD, larger than NS-SPIK and also consistent with the size of the full-length SPIK protein (SEQ ID NO: 2).

[0211] To determine the exact sequence of AS-SPIK, the AS-SPIK band from the gel electrophoresis was excised from the membrane. Alphalyse Inc. (Palo Alto, CA) performed Edman sequence analysis, and the results are shown in Figure 2. The sequence predicted by Edman degradation at the N-terminus of SPIK secreted by S2-3 cells is shown in red. The Edman degradation data indicated that the N-terminus of AS-SPIK is identical to residues 2–6 of SPIK (excluding the first methionine of the initiation codon) (see Figure 2). This suggests that the entire 23 amino acid sequence at the N-terminus of SPIK (starting with (M)K), which is removed in NS-SPIK, was retained in the secreted AS-SPIK. The complete amino acid sequences of both NS-SPIK and AS-SPIK are shown in Figure 3, highlighting this difference.

[0212] Example 2: The three-dimensional structure of AS-SPIK is different from that of NS-SPIK The inclusion of these additional 23 residues at the N-terminus of AS-SPIK, in turn, alters the protein's conformation. This change is visualized by comparing the three-dimensional structures of AS-SPIK and NS-SPIK. The crystal structure of AS-SPIK was determined by a CLIISP protein epitope mapping study (Pepscan, Lelystad, Netherlands), and the crystal structure of NS-SPIK was obtained from a peer-reviewed publication by Hecht et al. (18). Both structures are shown side-by-side in Figure 4. Visual comparison allows identification of three conformational differences between AS-SPIK and NS-SPIK, which are boxed in red. Box I indicates the N-termini of both NS-SPIK and AS-SPIK. The extra 23-residue fragment in AS-SPIK protrudes outward and extends past the body of the protein, whereas the N-terminus of NS-SPIK lacks the additional fragment protruding from the body of the protein. This exposed fragment significantly increases the likelihood that other proteins, such as antibodies, will selectively interact with AS-SPIK but not with NS-SPIK. Box II shows that the first loop in AS-SPIK is flatter and bends differently compared to the corresponding loop in NS-SPIK due to the longer N-terminus of AS-SPIK. This difference results in a larger space between the first loop and the alpha helix in AS-SPIK (Figure 4, Box II), exposing amino acids that are internal and inaccessible in NS-SPIK. Finally, the longer N-terminus of AS-SPIK also changes the relative position and distance between the protein's N-terminus and the alpha helix. The lower position of the N-terminus compared to the alpha helix of AS-SPIK is highlighted in Box III in the crystal structure. These conformational changes and differences in tertiary structure suggest that antibodies with different conformations could be generated to specifically target either form of SPIK.

[0213] Example 3. Development of antibodies that selectively bind to AS-SPIK but not to NS-SPIK Because of the differences between AS-SPIK and NS-SPIK described above, it is possible to develop antibodies that recognize only AS-SPIK but not NS-SPIK. As evidence of this, the inventors developed approximately 20 monoclonal antibodies that bind only to AS-SPIK but not to NS-SPIK in both mice and rabbits. Concurrently, the inventors developed polyclonal antibodies from sheep that bind to both AS-SPIK and NS-SPIK. Briefly, mice and rabbits were immunized with a series of recombinant proteins consisting of 1) a tag, 2) a linker sequence, 3) sequences of various lengths that are subsets of SEQ ID NO: 6, and 4) the common region between AS-SPIK and NS-SPIK (SEQ ID NO: 4). To develop monoclonal antibodies, the animals were injected with recombinant proteins having the above sequences. Blood samples were tested after four injections, and a single clone was established for samples that tested positive. To identify antibodies that bind only to AS-SPIK but not to NS-SPIK, clones were screened by ELISA using plates coated with partially purified AS-SPIK and NS-SPIK. Those that tested positive for AS-SPIK but negative for NS-SPIK were then selected. The cell line with the highest binding affinity for AS-SPIK was expanded to establish hybridomas. The resulting antibodies were then purified from the cell culture medium using a protein G affinity column. To develop polyclonal antibodies in sheep, sheep were immunized with one of the recombinant proteins described above. After four injections, serum was collected, and polyclonal antibodies were purified from the serum.

[0214] Figure 5 shows the binding assay results for eight antibodies from this group of 20 monoclonal antibodies: IM-A1, IM-B10, IM-C6, IM-E2, IM-CA22, IM-CA18, IM-CA46, IM-CA77, and IM-Poly S (a polyclonal antibody derived from sheep). (The sequences of these eight antibodies are shown in Tables 3 and 4 (Figures 15 and 16) and are also shown in U.S. Provisional Patent Application No. 62 / 639,850, PCT Patent Application No. PCT / US19 / 20999, and U.S. Provisional Patent Application No. 62 / 871565, the entire disclosures of which are incorporated herein by reference.) Figure 5 shows that all eight monoclonal antibodies, whether developed in mice or rabbits, strongly bind to AS-SPIK. In contrast, the binding activity to NS-SPIK is at background levels, similar to the negative control (Figure 5, negative control). As expected, polyclonal sheep antibodies strongly bind to both AS-SPIK and NS-SPIK (Figure 5, Poly S), because polyclonal antibodies contain multiple different antibodies that bind to various epitopes, including epitopes that may be common to both NS-SPIK and AS-SPIK. This data confirms that antibodies that selectively bind to AS-SPIK but not NS-SPIK can be developed in multiple animal models.

[0215] As a control, we simultaneously 36 ELNGCTKIYDPV 47 A series of monoclonal antibodies was developed in mice using a peptide containing the sequence: This sequence is the consensus sequence of both AS-SPIK and NS-SPIK (SEQ ID NO: 4) and is colored green in Figure 4 . This resulted in the production of antibody IM-BA2, which binds to both AS-SPIK and NS-SPIK (data not shown). However, using this method, the inventors were unable to generate an antibody that specifically binds to AS-SPIK but not NS-SPIK. This suggests that the epitope specific to AS-SPIK is conformational rather than linear.

[0216] Example 4. Anti-AS-SPIK antibodies are conformation-dependent To further support the notion that the epitope specific to AS-SPIK, but not to NS-SPIK, is conformation-dependent, further studies were completed evaluating antibody binding to various synthetic peptides. Four peptides, each containing sequences from a different region of AS-SPIK, were synthesized by BioMatik (Wilmington, DE). Peptide A contains the entire sequence of AS-SPIK but does not form any disulfide bonds due to inactivation of the cysteine ​​side chains. Peptide B contains the amino acid sequences M1-G1. 50 Peptide C contains the sequence of the AS-SPIK fragment D 23 ~G 50 and peptide D contains the sequence of the AS-SPIK fragment 51 ~C 79The binding activity of our monoclonal antibodies against AS-SPIK and other peptides was tested by ELISA, and the results for IM-CA22 are shown in Figure 6. Similar results were obtained with all other monoclonal anti-AS-SPIK antibodies developed by us, including IM-CA18, IM-CA46, IM-CA77, IM-A1, IM-A6, IM-B10, IM-C6, IM-D3, IM-D5, IM-E2, IM-F5, IM-G6, and IM-G7. Briefly, a 96-well plate was coated with native AS-SPIK and various peptides, and then the plate was reacted with an anti-AS-SPIK antibody (here, IM-CA22) labeled with HRP. After incubating the plate with the substrate TMB, the OD at 450 nm was measured. Figure 6 shows that antibody IM-CA22 recognizes only the native protein and does not recognize any synthetic peptides, such as peptide A, which contains the entire native AS-SPIK sequence. This result further supports the notion that anti-AS-SPIK antibodies are conformation-dependent antibodies. In contrast, our polyclonal antibody Poly S recognizes all synthetic peptides as well as native AS-SPIK (Figure 6, Poly S), suggesting that Poly S contains both linear and conformation-dependent epitopes. However, the conformational differences we identified above, combined with these test results, strongly indicate that the monoclonal antibodies described herein possess conformation-dependent epitopes.

[0217] Example 5. Class I and Class II anti-AS-SPIK antibodies To identify potential binding epitopes of anti-AS-SPIK antibodies, we established an ELISA test system to examine whether all of the anti-AS-SPIK antibodies we developed bind to the same epitope. Briefly, we coated a plate with one antibody and then incubated it with native AS-SPIK derived from S2-3 cells. After AS-SPIK was captured on the plate, a second anti-AS-SPIK antibody labeled with HRP was added, followed by the addition of substrate. After color development, the OD value was measured. If a negative result was obtained, this suggests that the capture and signal antibodies bind to the same epitope or epitopes close enough that binding to one inhibits binding to the other. In contrast, if a positive result was obtained, this suggests that these two antibodies bind to different epitopes that are sufficiently distant that they can be simultaneously accessed by different antibodies. Poly S was used as a control. Our data indicate that all of the anti-AS-SPIK antibodies we developed can be divided into two classes: Class I antibodies, including IM-CA22, IM-A1, IM-B10, IM-CA18, IM-D2, IM-D3, IM-D5, and IM-G2, and Class II antibodies, including IM-E2, IM-C6, IM-CA46, IM-CA77, IM-A6, IM-B3, IM-F5, and IM-G6. A defining feature of these antibody classes is that any Class I antibody can work with any Class II antibody, and vice versa. This means that they show positive results in the above-mentioned ELISA tests and can function well as antibody pairs in sandwich ELISAs. Our data also indicate that all of these antibodies bind to one or two distinct conformational epitopes. Table 1 (Figure 13) shows the results of these competitive ELISA tests using four class I antibodies: IM-A1, IM-B10, IM-CA22, and IM-CA18, and four class II antibodies: IM-C6, IM-E2, IM-CA46, and IM-CA77. Briefly, plates were coated with 100 ng / ml of each class II antibody, followed by reaction with AS-SPIK.After washing, a class I signal antibody (labeled with HRP) was added and binding activity was measured. The results are shown in Table 1A (Figure 13). These tests were then repeated using antibodies within the same class for both the capture and signal antibodies. The results for the class I-class I pair are shown in Table 1B (Figure 13) and the results for the class II-class II pair are shown in Table 1C (Figure 13). For all cells, the listed concentrations represent the lowest concentration of signal antibody that still gave a positive result; lower concentrations indicate stronger binding affinity. Based on these results, it is clear that all class I antibodies can work with any class II antibody, but antibodies of the same class almost completely inhibit target binding, even when the signal antibody is present at very high concentrations (500 ng / ml). Finally, not surprisingly, all of the antibodies listed here can work with our polyclonal antibody, Poly S, suggesting that the test system is highly effective.

[0218] Example 6. Epitope I and epitope II of AS-SPIK With the understanding that antibodies within the same class are likely to bind to very similar epitopes, the next step is to determine the precise binding sites for class I and class II anti-AS-SPIK antibodies. To achieve this, we performed epitope mapping using eight monoclonal anti-AS-SPIK antibodies, including four class I antibodies (IM-CA22, IM-CA18, IM-A1, and IM-B10) and four class II antibodies (IM-CA46, IM-CA77, IM-C6, and IM-E2). IM-CA22, IM-CA18, IMCA46, and IMCA77 were generated in mice, and IM-A1, IM-B10, IM-C6, and IM-E2 were generated in rabbits. Epitope mapping was also performed on Poly S, a polyclonal antibody derived from sheep. Precision Epitope Mapping (19) using CLIPS (Chemically Linked Peptides on Scaffolds) peptide arrays was performed by Pepscan (Lelystad, Netherlands).

[0219] The CLIPS technology structurally locks peptides into defined three-dimensional structures. The CLIPS reaction occurs between the bromine group of the CLIPS scaffold and the thiol side chain of a cysteine ​​incorporated into the peptide construct. This reaction is ultrafast, highly specific, and occurs under mild conditions. This precise and simple chemistry is used to convert natural protein sequences into CLIPS constructs with various structures. CLIPS technology is currently commonly used to shape peptide libraries into single, double, or triple loop structures, as well as sheet- and helix-like folds, which allows for the mimicking of discrete binding sites in three-dimensional structures. Therefore, sequence-dependent, conformation-dependent, and discrete conformational epitopes can be determined.

[0220] An array of over 2,400 independent peptides was synthesized and tested for binding by the monoclonal antibodies IM-CA18, IM-CA22, IM-CA46, IM-CA77, IM-A1, IM-B10, IM-C6, and IM-E2, and the polyclonal antibody Poly S. The epitope mapping results were visualized using bar plots, heat maps, and tables, and visualized on structures modeled with the Swiss model using the PDB entry 1HPT.pdb. Potential binding regions within AS-SPIK for each antibody were predicted, including linear, conformational, and discontinuous conformational epitopes. The three-dimensional structures (crystal models) of these AS-SPIK binding regions were also visualized.

[0221] Because AS-SPIK is a small protein with 79 amino acids, only four antigenic regions are required for the construction of a conformation-dependent epitope determined by CLIPS. These regions are highlighted in Figure 7. The first region is G5-A, which contains two parts. 29 The part I (G5~A) shown in light blue 23 ) is only present in AS-SPIK. The yellow part II (D 24 ~A 29 ) is present in both AS-SPIK and NS-SPIK (SEQ ID NO: 4), and the second region is K 31~C 47 and the third region is I, shown in blue. 42 ~N 56 and the fourth region is shown in gray, D 50 ~C 79 However, the CLIPS results suggest that only regions 1, 2, and 4 are directly involved in the binding of AS-SPIK to anti-AS-SPIK antibodies. Table 2 (Figure 14) shows the amino acids that comprise the potential binding regions for anti-AS-SPIK antibodies predicted by CLIPS. The CLIPS study also showed that each antibody binds to at least two separate regions, meaning that all anti-AS-SPIK antibodies described herein bind to discontinuous conformational epitopes. This is consistent with our previous view that anti-AS-SPIK antibodies are exclusively conformational antibodies.

[0222] Example 7. Key residues of epitope I and epitope II In addition to identifying potential binding regions of AS-SPIK for both classes of antibodies, the CLIPS study also assessed the relative importance of amino acids within each region to its binding affinity. Region 1 (7FLLSALALLSLSGNTGADSLGREA) 29 , SEQ ID NO: 7) and region 4 ( 58 CVLCFENRKRQ 68 , SEQ ID NO: 8) constitutes the essential binding site for all class I antibodies. The most important amino acids in region 1 for binding function are: 14 LLSL 17 (SEQ ID NO: 12), and to a lesser extent 24 DS 25 (SEQ ID NO: 13). The key residues in region 4 are: 58 CVLCF 26 (SEQ ID NO: 14). Together, these amino acids constitute a discontinuous conformational epitope for all class I antibodies, termed epitope I (Table 2 (FIG. 14) and FIG. 7). The discontinuous nature of this epitope is further supported by the inhibition studies we performed to confirm these findings (shown in FIG. 8). Here, we 14LLSL 17 We synthesized a short peptide with 9 amino acids containing the region 1 (A). This peptide can inhibit the binding between native AS-SPIK and class I antibodies in this region. However, the inhibition of binding was incomplete, suggesting the existence of a second region of AS-SPIK involved in binding. This is consistent with our hypothesis that anti-AS-SPIK binds to a discontinuous, conformation-dependent epitope that must involve at least two separate regions of AS-SPIK. CLIPS studies also revealed that region 2 (A). 36 LNGCTKIYD 44 , SEQ ID NO: 9) and region 4 ( 64 NRKRQTSILIQ 75 , SEQ ID NO: 10) constitutes the essential binding site for all class II antibodies. The most important amino acids for binding function in region 2 are: 36 LN 37 (SEQ ID NO: 15) and 42 IY 43 (SEQ ID NO: 16). The key residues in region 4 are: 67 Race Queen 68 (SEQ ID NO: 17) and 71 IL 72 (SEQ ID NO: 18). Together, these amino acids constitute a discontinuous conformational epitope for all class II antibodies, called epitope II (Table 2 (FIG. 14) and FIG. 7). The results of CLIIPS studies on the binding epitopes of Poly S indicate that the predominant antibody in Poly S binds to epitope II, similar to other class II antibodies, and can therefore be classified as a class II anti-AS-SPIK antibody.

[0223] Example 8. Consensus sequences within class I and class II antibody CDRs Now that we have defined epitopes I and II and identified examples of antibodies that bind to them, the next step is to identify structural similarities between antibodies within each class. We compared and analyzed the CDRs of four class I antibodies (IM-A (rabbit), IM-B10 (rabbit), IM-CA22 (mouse), and IM-CA18 (mouse)). The consensus sequences of their CDRs were determined using the software BioEdit (North Carolina State University) and are shown in Figure 9. Interestingly, although two antibodies are mouse-derived and two are rabbit-derived, we discovered that at least one amino acid in their CDRs is conserved in all antibodies. The most conserved CDR in these four class I antibodies is CDRL2. Four of the seven amino acids (57%) in this CDR are identical. These conserved residues within each CDR are a defining characteristic of this genus (class I) of antibodies. Similar studies of the class II antibodies IM-C6 (rabbit), IM-E2 (rabbit), IM-CA46 (mouse), and IMCA77 (mouse) have yielded similarly important findings. Figure 10 lists the CDR consensus sequences for these four class II antibodies. The number of conserved amino acids in the CDRs of class II antibodies is greater than that of class I. We discovered that at least two amino acids in each of their CDRs are conserved. The most conserved CDR in these four class II antibodies is CDRL1, with 7 of the 11 amino acids (64%) in this CDR being identical. The conserved residues within each of these CDRs are a defining characteristic of this genus (class II) of antibodies.

[0224] Example 9. Clinical Evaluation An AS-SPIK test kit based on sandwich ELISA (enzyme-linked immunosorbent assay) was used for clinical evaluation. It utilized a monoclonal antibody (IM-CA22) that binds only to AS-SPIK as the capture antibody and an HRP-conjugated polyclonal anti-AS-SPIK antibody (Poly S) as the signal antibody. The mechanism of this kit is shown in Figure 11.

[0225] Study design and sample population: Serum samples were collected from a total of 512 unique study subjects in a prospective, blinded study. These samples were obtained from various research facilities under study protocols approved by the respective Institutional Review Boards (IRBs). Informed patient consent was obtained for all study participants. Of the 512 samples, 164 were from patients with HCC positively diagnosed using biopsy, CT, and / or MRI. This included 81 patients with early-stage HCC (BCLC stages 0 and A). The remaining 348 subjects were part of various control groups, including patients with cirrhosis, non-cirrhotic chronic HBV / HCV, pancreatitis, and healthy subjects.

[0226] Serum levels of AS-SPIK were quantified using an ELISA-based test kit utilizing the monoclonal antibody IM-CA22 (SEQ ID NO: 75 and SEQ ID NO: 76), whose amino acid sequence information is provided herein. AFP, the most commonly used biomarker for HCC, was quantified in all patients by each research institution in their accredited clinical laboratories using an FDA-approved AFP test. Receiver operating curves (ROC) for both AS-SPIK and AFP were constructed to compare their sensitivity, specificity, and area under the curve (AUC). For this analysis, only the intended use population with liver disease (HCC, cirrhosis, and HBV / HCV) was considered. Patients with pancreatitis were evaluated separately to ensure that normal pancreatic SPIK (NS-SPIK) did not interfere with the AS-SPIK test, while healthy patients served as a reference negative control.

[0227] result: A) Serum AS-SPIK is significantly elevated in HCC patients, including those with early-stage HCC. The results showed that the mean serum level of AS-SPIK in all HCC patients (45.2 ng / mL, 95% CI: 40.5 to 49.9) was significantly higher than that in all control groups (p<0.001) (Figure 17; Table 5). For 81 patients with early-stage HCC (BCLC stage 0 and A), the mean serum AS-SPIK concentration was 38.1 ng / mL (95% CI: 32.1 to 44.2), which was significantly different from the serum AS-SPIK levels in all control groups (P<0.001) (Figure 17; Table 5). These results are consistent with previous studies described in Examples 12 and 13 and Figures 15 and 16 of WO2019 / 173503, the entire disclosure of which is incorporated herein by reference.

[0228] B). Performance of AS-SPIK and AFP in distinguishing HCC from other liver diseases. Overall, AS-SPIK demonstrated significantly higher sensitivity and specificity than AFP, as shown in Table 6 (Figure 18). The AUC for AS-SPIK in detecting HCC using liver disease patients as controls was 0.87 (95% CI: 0.83 to 0.91) compared with 0.70 (95% CI: 0.64 to 0.76) for AFP. Using 21.5 ng / mL as the cutoff value for serum AS-SPIK, the sensitivity and specificity of AS-SPIK were 80% and 90%, respectively. In comparison, using the standard 20.0 ng / mL cutoff value for serum AFP, the sensitivity and specificity were only 52% and 86%, respectively, significantly lower than those of AS-SPIK (P<0.05). For early-stage HCC, the AUC for AS-SPIK was 0.84 (95% CI: 0.79 to 0.89) compared with only 0.61 (95% CI: 0.53 to 0.70) for AFP. Using 21.5 ng / mL as a cutoff value, the sensitivity of AS-SPIK in detecting early-stage HCC was 72% and the specificity was 90%, significantly higher than the 42% sensitivity and 86% specificity for AFP (Figure 18; Table 6).

[0229] C) The test can be used for prediction of HCC stage and surveillance / prognosis. In this study, we observed a correlation between AS-SPIK levels and the stage of cancer progression (Figure 19; Table 7). The mean AS-SPIK level for patients in the early-stage group was 38.1 ng / mL (95% CI: 32.1-44.1), while the mean AS-SPIK level for patients in the late-stage group was 52.2 ng / mL (95% CI: 45.3-59.1). Comparing these values, we found a statistically significant difference between the two groups (P<0.05), indicating a correlation between stage-specific HCC progression and increasing AS-SPIK levels (Figure 19; Table 7).

[0230] This is further supported by the observation that mean AS-SPIK values ​​were consistently higher for later and more advanced stages when classified by BCLC stage. Patients in the very early stage group (BCLC stage 0) had the lowest mean AS-SPIK value of only 33.7 ng / mL, while patients in end-stage D had the highest mean AS-SPIK value of 65.6 ng / mL. However, analysis strictly based on BCLC classification leads to excessive stratification and uneven sample size, especially for difficult-to-recruit groups such as BCLC stage 0 (very early stage) and stage D (end-stage) groups. Therefore, this result was not statistically significant (P > 0.05) (Figure 20; Table 8). These results suggest that AS-SPIK may have potential as a tool for monitoring HCC progression.

[0231] D) AS-SPIK detection kit that detects only AS-SPIK in patient serum and does not detect NS-SPIK Because AS-SPIK is a liver cancer-specific isoform of SPIK containing an additional fragment at the N-terminus, we evaluated whether NS-SPIK (pancreatic SPIK) interferes with the AS-SPIK test described herein. Figure 12, panel A, shows that two monoclonal antibodies, IM-CA22 (which recognizes only AS-SPIK) and IM-BA2 (which recognizes both AS-SPIK and NS-SPIK), were used to coat plates and then reacted with either AS-SPIK or NS-SPIK. While IM-CA22 and the AS-SPIK test kit recognize only AS-SPIK, IM-BA2 detects and confirms the presence of both AS-SPIK and NS-SPIK. Figure 12, panel B, shows the serum levels of AS-SPIK in 24 pancreatitis patients from a clinical trial, who were expected to have elevated levels of pancreatic SPIK. The data show that AS-SPIK levels in these patients (7.4 ng / mL) were similar to those in healthy patients (7.4 ng / mL, P>0.99) and significantly lower than those in HCC patients in the clinical trial (45.2 ng / mL, P<0.001). Figure 12, panel C, confirms the observation that elevated serum levels of NS-SPIK in pancreatitis patients do not interfere with the AS-SPIK detection kit described herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

Table 1-8

Table 1-9

[0232] References: 1. Yu NC,Chaudhari V,Raman SS,Lassman C,Tong MJ,Busuttil RW,Lu DS. CT and MRI improve detection of hepatocellular carcinoma, compared with ultrasound alone,in patients with cirrhosis. Clin Gastroenterol Hepatol 2011;9:161-167. 2. Bruix J,Sherman M. Management of hepatocellular carcinoma. Hepatology 2005;42:1208-1236. 3. Lok AS,Heathcote EJ,Hoofnagle JH.Management of hepatitis B:2000--summary of a workshop. Gastroenterology 2001;122:2092-2093. 4. Greene LJ,Pubols MH,Bartelt DC. Human pancreatic secretory trypsin inhibitor. Methods Enzymol 1976;45:813-825. 5. Stenman UH. Tumor-associated trypsin inhibitor. Clin Chem 2002;48:1206-1209. 6. Greene LJ. Pancreatic exocrine secretory proteins. J Surg Oncol 1975;7:151-154. 7. Lee YC,Pan HW,Peng SY,Lai PL,Kuo WS,Ou YH,Hsu HC. Overexpression of tumour-associated trypsin inhibitor (TATI) enhances tumour growth and is associated with portal vein invasion,early recurrence and a stage-independent prognostic factor of hepatocellular carcinoma. Eur J Cancer 2007;43:736-744. 8. Lu X,Lee M,Tran T,Block T.High level expression of apoptosis inhibitor in hepatoma cell line expressing Hepatitis B virus. Int J Med Sci 2005;2:30-35. 9. Ohmachi Y,Murata A,Matsuura N,Yasuda T,Yasuda T,Monden M,Mori T,et al.Specific expression of the pancreatic-secretory-trypsin-inhibitor(PSTI)gene in hepatocellular carcinoma. Int J Cancer 1993;55:728-734. 10. Hirota M,Ohmuraya M,Baba H.The role of trypsin,trypsin inhibitor,and trypsin receptor in the onset and aggravation of pancreatitis. J Gastroenterol 2006;41:832-836. 11. Playford RJ,Hanby AM,Quinn C,Calam J.Influence of inflammation and atrophy on pancreatic secretory trypsin inhibitor levels within the gastric mucosa. Gastroenterology 1994;106:735-741. 12. Kobayashi K,Horiuchi M,Saheki T.Pancreatic secretory trypsin inhibitor as a diagnostic marker for adult-onset type II citrullinemia. Hepatology 1997;25:1160-1165. 13. Lu F,Lamontagne J,Sun A,Pinkerton M,Block T,Lu X. Role of the inflammatory protein serine protease inhibitor Kazal in preventing cytolytic granule granzyme A-mediated apoptosis. Immunology 2011;134:398-408. 14. Bartelt DC,Shapanka R,Greene LJ.The primary structure of the human pancreatic secretory trypsin inhibitor. Amino acid sequence of the reduced S-aminoethylated protein. Arch Biochem Biophys 1977;179:189-199. 15. Kikuchi N,Nagata K,Yoshida N,Tanaka T,Yamamoto M,Saitoh Y.Purification and complete amino acid sequence of canine pancreatic secretory trypsin inhibitor. FEBS Letters 1985;191:269-272. 16. Lu X,Lamontagne J,Lu F,Block TM. Tumor-associated protein SPIK / TATI suppresses serine protease dependent cell apoptosis. Apoptosis 2008;13:483-494. 17. Horii A,Kobayashi T,Tomita N,Yamamoto T,Fukushige S,Murotsu T,Ogawa M,et al.Primary structure of human pancreatic secretory trypsin inhibitor(PSTI)gene. Biochem Biophys Res Commun 1987;149:635-641. 18. Hecht HJ,Szardenings M,Collins J,Schomburg D.Three-dimensional structure of a recombinant variant of human pancreatic secretory trypsin inhibitor(Kazal type). J Mol Biol 1992;225:1095-1103. 19. Timmerman P,Puijk WC,Meloen RH.Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology. J Mol Recognit 2007;20:283-299.

Claims

1. 1. An isolated antibody that specifically binds to a conformational epitope of an AS-SPIK protein and does not specifically bind to an NS-SPIK protein, wherein the conformational epitope of the AS-SPIK protein is one or more amino acids selected from the group consisting of L14, L15, S16, L17, D24 and S25 of SEQ ID NO: 2; one or more amino acids selected from the group consisting of C58, V59, L60, C61, and F62 of SEQ ID NO:2; The isolated antibody comprising:

2. 2. The isolated antibody of claim 1, wherein the conformational epitope comprises amino acids L14, L15, S16, and L17 of SEQ ID NO:

2.

3. 2. The isolated antibody of claim 1, wherein the conformational epitope comprises amino acids L60 and C61 of SEQ ID NO:

2.

4. 2. The isolated antibody of claim 1, wherein the conformational epitope comprises amino acids L14, L15, S16, L17, L60, and C61 of SEQ ID NO:

2.

5. 3. The isolated antibody of claim 2, wherein the conformational epitope further comprises amino acids D24 and S25 of SEQ ID NO:

2.

6. 4. The isolated antibody of claim 3, wherein the conformational epitope further comprises amino acids C58, V59 and F62 of SEQ ID NO:

2.

7. 2. The isolated antibody of claim 1, wherein the conformational epitope comprises amino acids L14, L15, S16, L17, D24, S25, C58, V59, L60, C61 and F62 of SEQ ID NO:

2.

8. a CDRH1 sequence comprising S6; and / or a CDRH2 sequence comprising I2, G5, G6, Y10, and K16; and / or a CDRH3 sequence comprising G4 and Y7; and / or a CDRL1 sequence comprising Q4 and S9; and / or a CDRL2 sequence comprising A2, S3, T4 and S7; and / or CDRL3 sequence comprising Q1, Q2, Y4 and S5 The isolated antibody of any one of claims 1 to 7, comprising:

9. a CDRH1 sequence comprising S6; and a CDRH2 sequence comprising I2, G5, G6, Y10, and K16; and a CDRH3 sequence comprising G4 and Y7; a CDRL1 sequence comprising Q4 and S9; a CDRL2 sequence comprising A2, S3, T4 and S7; a CDRL3 sequence comprising Q1, Q2, Y4 and S5; 9. The isolated antibody of claim 8, comprising:

10. 1. An isolated antibody that specifically binds to a conformational epitope of an AS-SPIK protein and does not specifically bind to an NS-SPIK protein, wherein the conformational epitope of the AS-SPIK protein is one or more amino acids selected from the group consisting of L36, N37, I42 and Y43 of SEQ ID NO: 2; one or more amino acids selected from the group consisting of R67, Q68, I71 and L72 of SEQ ID NO: 2; The isolated antibody comprising:

11. 11. The isolated antibody of claim 10, wherein the conformational epitope comprises amino acids L36 and N37 of SEQ ID NO:

2.

12. 11. The isolated antibody of claim 10, wherein the conformational epitope comprises amino acids 142 and Y43 of SEQ ID NO:

2.

13. 11. The isolated antibody of claim 10, wherein the conformational epitope comprises amino acids L36, N37, I42 and Y43 of SEQ ID NO:

2.

14. 11. The isolated antibody of claim 10, wherein the conformational epitope comprises amino acids R67, Q68, I71 and L72 of SEQ ID NO:

2.

15. 11. The isolated antibody of claim 10, wherein the conformational epitope comprises amino acids L36, N37, I42, Y43, R67, Q68, I71 and L72 of SEQ ID NO:

2.

16. The antibody a CDRH1 sequence comprising Y3, S7 and W9; and / or a CDRH2 sequence comprising A1, I2, G4, G6, and Y10; and / or a CDRH3 sequence comprising R1 and D7; and / or a CDRL1 sequence comprising A2, S3, Q4, I6, Y9, L10, and S11; and / or a CDRL2 sequence comprising A2, S3, L5 and S7; and / or 16. The isolated antibody of any one of claims 10 to 15, comprising a CDRL3 sequence comprising Q1, Q2, and T5.

17. The antibody a CDRH1 sequence comprising Y3, S7 and W9; a CDRH2 sequence comprising A1, I2, G4, G6, and Y10; and a CDRH3 sequence comprising R1 and D7; a CDRL1 sequence comprising A2, S3, Q4, 16, Y9, L10 and S11; and a CDRL2 sequence comprising A2, S3, L5 and S7; a CDRL3 sequence comprising Q1, Q2, and T5; 17. The isolated antibody of claim 16, comprising:

18. The antibody of any one of claims 1 to 17, which is multispecific.

19. 19. The antibody of claim 18, which is bispecific.

20. 20. The antibody of claim 19, having binding affinity for effector cells.

21. 20. The antibody of claim 19, having binding affinity for a T cell antigen.

22. 22. The antibody of claim 21, wherein the T cell antigen comprises a CD3 protein.

23. The antibody according to any one of claims 1 to 22, which is a monoclonal antibody.

24. The antibody according to any one of claims 1 to 22, which is a CAR-T type.

25. An immunoconjugate comprising an antibody according to any one of claims 1 to 23 covalently linked to a cytotoxic agent.

26. 26. The immunoconjugate of claim 25, wherein the cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, a radioisotope, and a nucleolytic enzyme.

27. An immunoconjugate having the formula Ab-(LD)p, wherein: (a) Ab is an antibody according to any one of claims 1 to 23; (b) L is a linker; (c) D is a drug moiety; (d) the immunoconjugate, wherein p is an integer ranging from 1 to 8.

28. 28. The immunoconjugate of claim 27, wherein D is selected from the group consisting of maytansinoids, auristatins, and dolostatins.

29. 28. The immunoconjugate of claim 27, wherein L comprises one or more linkers selected from the group consisting of 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4-(2-pyridyldithio)butyric acid-N-hydroxysuccinimide ester (SPDB), and N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB).

30. A pharmaceutical composition comprising an antibody or immunoconjugate according to any one of claims 1 to 29.

31. 31. A method for the treatment of a disorder characterized by expression of AS-SPIK, comprising administering to a subject an antibody or immunoconjugate according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 30.

32. Use of an antibody or immunoconjugate according to any one of claims 1 to 29 in the preparation of a medicament for the treatment of a disorder characterized by expression of AS-SPIK.

33. The antibody or immunoconjugate of any one of claims 1 to 29 for use in the treatment of a disorder characterized by expression of AS-SPIK.

34. 34. The method or use according to any one of claims 31 to 33, wherein the disorder is a liver disorder.

35. 35. The method or use of claim 34, wherein the liver disorder is hepatocellular carcinoma.

36. 35. The method or use of claim 34, wherein the liver disorder is intrahepatic cholangiocarcinoma.

37. 35. The method or use of claim 34, wherein the liver damage is a viral infection.

38. 35. The method or use of claim 34, wherein the liver damage is an inflammatory liver damage.

39. 39. The method or use of claim 38, wherein the inflammatory liver disorder is cirrhosis.

40. A polynucleotide encoding the antibody of any one of claims 1 to 24.

41. A vector comprising the polynucleotide of claim 40.

42. A host cell comprising the vector of claim 41.

43. 42. A method for producing an antibody or immunoconjugate according to any one of claims 1 to 29, comprising growing a host cell according to claim 42 under conditions permissive for expression of said antibody, and isolating said antibody from said cell.

44. 1. A diagnostic method for determining whether a subject has or is at risk for developing a disorder characterized by expression of AS-SPIK, comprising: (a) contacting a biological test sample from the subject with an AS-SPIK antibody of any one of claims 1 to 23 to form an AS-SPIK-antibody complex; (b) detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; and (c) comparing the concentration of the AS-SPIK-antibody complex to a reference value to determine whether the subject has or is at risk of developing the disorder.

45. 1. A diagnostic method for determining whether a subject has or is at risk for developing a disorder characterized by expression of AS-SPIK, comprising: (a) contacting a biological test sample from the subject with a first antibody or antigen-binding fragment that specifically binds to SPIK to form a SPIK-antibody complex; (b) contacting the SPIK-antibody complex with an AS-SPIK antibody or antigen-binding fragment of any one of claims 1 to 23 to form an AS-SPIK-antibody complex; (c) detecting the concentration of the AS-SPIK-antibody complex in the biological test sample; and (d) comparing the concentration of the AS-SPIK-antibody complex with a reference value to determine whether the subject has or is at risk for developing the disorder. The method comprising:

46. 46. ​​The diagnostic method of claim 44 or 45, wherein the antibody or antigen-binding fragment comprises a detectable label.

47. 46. ​​The diagnostic method of claim 44 or 45, wherein the disorder is a liver disorder.

48. 48. The diagnostic method of claim 47, wherein the liver disorder is selected from the group consisting of hepatocellular carcinoma, intrahepatic cholangiocarcinoma, viral infection of the liver, inflammatory disorders of the liver, and cirrhosis.

49. A kit comprising an antibody or immunoconjugate according to any one of claims 1 to 29.

50. 50. The kit of claim 49, further comprising an antibody that specifically binds to SPIK.

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