AU2024262099A1 - Diagnosis of late-stage hepatocellular carcinoma - Google Patents

Abstract

Provided herein are methods, systems, and kits for determining late stage liver cancer and monitoring liver cancer status. In particular, methods, systems, and kits are provided that employ the concentration, level, or amount of full-length laminin gamma-2 monomer (LG2m), optionally with the concentration, level, or amount of total LG2m, to determine or identify late stage liver cancer (e.g., hepatocellular carcinoma) in a subject.

Description

DIAGNOSIS OF LATE-STAGE HEPATOCELLULAR CARCINOMA

RELATED APPLICATION INFORMATION

This application claims priority to U.S. Application No. 63/462,640, filed on April 28, 2023, the contents of which are herein incorporated by reference.

FIELD

Provided herein are methods, systems, and kits for determining late stage liver cancer and monitoring liver cancer status. In particular, methods, systems, and kits are provided that employ the concentration, level, or amount of full-length laminin gamma-2 monomer (LG2m), optionally with the concentration, level, or amount of total LG2m, to determine or identify late stage liver cancer (e.g., hepatocellular carcinoma) in a subject.

BACKGROUND

Liver cancer is one of the fastest-growing cancer types in the United States. Hepatocellular carcinoma (HCC) is the most common form of liver cancer, particularly in adults, representing nearly all liver cancers. The detection rate of liver cancer has been improved by the progress in biomarkers and diagnostic imaging. However, while biomarkers are non-invasive and inexpensive, they are unable to monitor the progression and migration of liver cancer, which requires expensive and invasive diagnostic imaging. As such, none of the available non-invasive diagnostic tests are able to accurately diagnose late-stage liver cancers, including HCC.

SUMMARY

Provided herein are methods determining or identifying late stage liver cancer in a subject. In some embodiments, the methods comprise determining or acquiring the concentration, level, or amount of full-length laminin gamma 2 monomer in a biological sample obtained from the subject; comparing the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) to a reference level; and identifying the subject as having late stage liver cancer when the concentration, level, or amount of full-length LG2m is equal to or lower than the reference concentration, level, or amount.

In some embodiments, the concentration, level, or amount of full-length LG2m is determined using an immunoassay. In some embodiments, the immunoassay is a sandwich immunoassay. In some embodiments, the concentration, level, or amount of full-length LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain I/II of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

In some embodiments, the subject has late stage liver cancer when the concentration, level, or amount of full-length LG2m in one of the biological samples is equal to or lower than the reference concentration, level, or amount found in subjects known to have late stage liver cancer.

In some embodiments, the methods further comprise determining or acquiring the concentration, level, or amount of total LG2m in a biological sample obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m; comparing the ratio to a cutoff value; and identifying the subject as having late stage liver cancer when the ratio is lower than the cutoff value.

In some embodiments, total LG2m comprises N-terminal fragments of LG2m comprising domains III-V of LG2m and full-length LG2m. In some embodiments, the methods further comprise determining or acquiring the concentration, level, or amount of the total LG2m. In some embodiments, the concentration, level, or amount of the total LG2m is determined using an immunoassay. In some embodiments, the immunoassay is a sandwich immunoassay. In some embodiments, the concentration, level, or amount of the total LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain III of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

In some embodiments, the late stage liver cancer is stage IV liver cancer. In some embodiments, the late stage liver cancer is metastatic liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma.

In some embodiments, the two or more biological samples are each individually selected from a whole blood sample, a plasma sample, and a serum sample. In some embodiments, the methods further comprise obtaining the biological sample from the subject. In further embodiments, the method comprises treating the subject.

In some embodiments, the methods comprises determining or acquiring the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in a biological sample obtained from the subject; determining or acquiring the concentration, level, or amount of total LG2m in a biological sample obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m; comparing the ratio to a cutoff value; and identifying the subject as having late stage liver cancer when the ratio is lower than the cutoff value.

In some embodiments, total LG2m comprises N-terminal fragments of LG2m comprising domains III-V of LG2m and full-length LG2m. In some embodiments, the methods further comprise determining or acquiring the concentration, level, or amount of the total LG2m. In some embodiments, the concentration, level, or amount of the total LG2m is determined using an immunoassay. In some embodiments, the immunoassay is a sandwich immunoassay.

In some embodiments, the concentration, level, or amount of the total LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain III of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

In some embodiments, the concentration, level, or amount of full-length LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain I/II of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

In some embodiments, the late stage liver cancer is stage IV liver cancer. In some embodiments, the late stage liver cancer is metastatic liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma.

In some embodiments, the subject is diagnosed as having liver cancer, is suspected as having liver cancer, or is diagnosed as having a progressive liver disease.

In some embodiments, the biological sample is a whole blood sample, a plasma sample, or a serum sample. In some embodiments, the methods further comprise obtaining the biological sample from the subject. In some embodiments, the methods further comprises treating the subject.

Also provided herein are methods for monitoring progression of liver cancer in a subject. In some embodiments, the methods comprise obtaining two or more biological samples from the subject, each biological sample separated from the previous sample by a period of time; determining the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the two or more biological samples; comparing the concentration, level, or amount of full-length LG2m to a reference concentration, level, or amount; and identifying the onset of late stage liver cancer when the concentration, level, or amount of full-length LG2m in one of the two or more biological samples is equal to or lower than the reference concentration, level, or amount.

In some embodiments, the concentration, level, or amount of full-length LG2m is determined using an immunoassay. In some embodiments, the immunoassay is a sandwich immunoassay. In some embodiments, the concentration, level, or amount of full-length LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain I/II of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

In some embodiments, the onset of late stage liver cancer is identified when the concentration, level, or amount of full-length LG2m in one of the biological samples is equal to or lower than the reference concentration, level, or amount found in subjects known to have late stage liver cancer.

In some embodiments, the methods further comprise determining or acquiring the concentration, level, or amount of total LG2m in each of the two or more biological samples obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m; comparing the ratio to a cutoff value; and identifying the onset of late stage liver cancer when the ratio in one of the two or more biological samples is lower than the cutoff value.

In some embodiments, the total laminin gamma 2 monomer comprises N-terminal fragments of LG2m comprising domains III-V of laminin gamma 2 monomer and full-length LG2m. In some embodiments, the methods further comprise determining or acquiring the concentration, level, or amount of the total LG2m. In some embodiments, the concentration, level, or amount of the total LG2m is determined using an immunoassay. In some embodiments, the immunoassay is a sandwich immunoassay. In some embodiments, the concentration, level, or amount of the total LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain III of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

In some embodiments, the late stage liver cancer is stage IV liver cancer. In some embodiments, the late stage liver cancer is metastatic liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma.

In some embodiments, the two or more biological samples are each individually selected from a whole blood sample, a plasma sample, and a serum sample. In some embodiments, the methods further comprise obtaining the two or more biological samples from the subject.

In some embodiment, the methods further comprise treating the subject.

Further provided is a kit comprising an antibody, or fragment thereof, which specifically binds to domain I/II of laminin gamma 2 monomer; and an antibody, or fragment thereof, which specifically binds to domain V of laminin gamma 2 monomer. In some embodiments, the kits further comprise an antibody, or fragment thereof, which specifically binds to domain III of laminin gamma 2 monomer.

Further provided are methods for detecting or quantifying full length laminin gamma 2 monomer (LG2m). In some embodiments, the methods comprise incubating the sample with: a first antibody, or fragment thereof, which specifically binds to domain V of laminin gamma 2 monomer; and a second antibody, or fragment thereof, which specifically binds to domain I/II of laminin gamma 2 monomer.

In some embodiments, the first antibody, or fragment thereof, and the second antibody, or fragment thereof, are incubated with the sample simultaneously or sequentially in any order. In some embodiments, the first antibody, or fragment thereof, or the second antibody, or fragment thereof, comprises a detectable label. In some embodiments, the antibody, or fragment thereof, not comprising the detectable label is bound to or configured to bind to a solid support. In some embodiments, the methods further comprise assessing the signal from the detectable label.

Additionally provided are systems comprising a sample receiving component configured to receive a sample; a sample analysis component configured to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the sample; and an indicator or display configured to show the presence or concentration, level, or amount of full-length LG2m is equal to or lower than the reference concentration, level, or amount.

In some embodiments, the sample receiving component and the sample analysis component are integrated into a single module of the system. In some embodiments, the sample analysis component comprises one or more assays to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the biological sample.

In some embodiments, the sample analysis component comprises an antibody, or fragment thereof, which specifically binds to domain I/II of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

In some embodiments, the sample analysis components is further configured to measure the concentration, level, or amount of total LG2m in a biological sample obtained from the subject. In some embodiments, the sample analysis component further comprises one or more assays to measure the concentration, level, or amount of total LG2m in a biological sample obtained from the subject.

In some embodiments, the sample analysis component is further configured to calculate a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m. In some embodiments, the indicator or display is further configured to show when the ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m is lower than a cutoff value.

In some embodiments, the sample receiving component and the sample analysis component are integrated into a single module of the system. In some embodiments, the sample receiving component and the sample analysis component are separate components of the system.

In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a whole blood sample, a plasma sample, or a serum sample.

In addition, the disclosure also provides systems comprising a sample receiving component configured to receive a sample; a sample analysis component configured to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the sample and compare the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) from the sample with a reference level concentration, level, or amount; and an indicator or display configured to show when the concentration, level, or amount of full-length LG2m is equal to or lower than the reference concentration, level, or amount.

In some embodiments, the sample receiving component and the sample analysis component are integrated into a single module of the system. In some embodiments, the sample receiving component and the sample analysis component are separate components of the system.

In some embodiments, the sample analysis component comprises one or more assays to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the biological sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a whole blood sample, a plasma sample, or a serum sample.

Other embodiments and embodiments of the disclosure will be apparent in light of the following detailed description and related figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a method for detection of full-length LG2m (top) by an antidomain V antibody and an anti-domain I/II antibody in comparison to current methods which utilize an anti-domain V antibody with an anti-domain III antibody to detect total LG2m (bottom).

FIG. 2 are calibration curves depicting the detection of full-length LG2m using an antidomain V antibody and an anti-domain I/II antibody, full concentration range (left), and expanded the low concentration range (right). FIG. 3 are graphs illustrating the concentration of full-length LG2m using whole LG2m reagents (left) and total LG2m (right), across subjects ranging from normal serum levels to various stages of hepatocellular carcinoma (HCC) serum levels.

FIG. 4 is a graph of the correlation analysis between full-length and total LG2m in normal and HCC samples.

FIG. 5 are graphs of the correlation analysis between full-length and total LG2m in all HCC samples (left) and in HCC stage I-III samples only (right).

FIG. 6 is a graph of the reactivity of the 2E9 and 1B383 antibodies against laminin monomer (LG2m) and laminin trimer (Lm-332).

DETAILED DESCRIPTION

Small liver tumors are hard to detect on a physical exam because most of the liver is covered by the right rib cage. By the time signs and symptoms of liver cancer are appearing, e.g., tumor is physically detectable, the cancer may be advanced. Most diagnostic tests are not sensitive or specific enough to aid in early detection, monitoring, and staging of liver cancer. Liver biopsy is currently the gold standard for liver cancer evaluation, but it has many drawbacks. Since it is an invasive procedure, it has the potential to result in infection, bleeding, and death. Use of non-invasive imaging techniques for the detection and/or diagnosis of primary liver tumors requires integration of multiple, precise, and often challenging steps. Unfortunately, these non-invasive methodologies are often not sufficiently sensitive or specific to detect early liver cancer. Described herein are methods using laminin gamma-2 monomer for diagnosing and discriminating liver cancer, particularly HCC, stages that would be helpful in monitoring liver cancer progression, staging, and aid in the diagnosis of liver disease.

Definitions

The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

“Antibody” and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi-specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, singlechain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001/058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity sake, an antibody against an analyte is frequently referred to herein as being either an “anti -analyte antibody” or merely an “analyte antibody”.

“Antibody fragment” as used herein refers to a portion of an intact antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)) (e.g., comprises the antigen-binding site or variable region). Any antigen- binding fragment of the antibody described herein is within the scope of the present disclosure. The antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N- terminal variable (VH) region and three C-terminal constant (CHI, CH2, and Cm) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda ( ), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

“CDR” is used herein to refer to the “complementarity determining region” within an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted “CDR1,” “CDR2,” and “CDR3,” for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region that binds the antigen. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be referred to as a “molecular recognition unit.” Crystallographic analyses of antigen-antibody complexes have demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as “Kabat CDRs.” Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as “LI,” “L2,” and “L3,” or “Hl,” “H2,” and “H3,” where the “L” and the “H” designate the light chain and the heavy chain regions, respectively. These regions may be referred to as “Chothia CDRs,” which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, FASEB J., 9: 133-139 (1995), and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain aspects use Kabat- or Chothia-defined CDRs.

As used herein, when an antibody, fragment thereof, or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (Ka) of at least 10⁷ M ¹ (e.g., >10⁷ M ¹, >10⁸ M ¹, >10⁹ M ¹, >1O¹⁰ M ¹, >10ⁿ M“ >10¹² M’¹, >10¹³ M’¹, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

“Label” and “detectable label” as used herein refer to a moiety attached to an antibody or an analyte to render the reaction between the antibody and the analyte detectable, and the antibody or analyte so labeled is referred to as “detectably labeled.” A label can produce a signal that is detectable by visual or instrumental means. Various labels include signal-producing substances, such as chromagens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. The moiety, itself, may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling.

Any suitable detectable label as is known in the art can be used. For example, the detectable label can be a radioactive label (such as ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ⁹⁰Y, "Tc, ⁿⁱIn, ¹²⁵1, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶HO, and ¹⁵³Sm), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5 -fluorescein, 6- carboxyfluorescein, 3’6- carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immunopolymerase chain reaction label. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon. A fluorescent label can be used in FPIA (see, e.g., U.S. Patent Nos.5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entireties). An acridinium compound can be used as a detectable label in a homogeneous chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett.\6: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett.4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. LettA 3917-3921 (2004); and Adamczyk et al., Org. Lett.5'. 3779- 3782 (2003)). In one aspect, the acridinium compound is an acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamides are described in Mattingly, J. Biolumin. Chemilumin.6: 107-114 (1991); Adamczyk et al., J. Org. Chem.63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914 (1999); Adamczyk et al., Org. LettA 779-781 (1999); Adamczyk et al., Bioconjugate Chem.11 : 714-724 (2000); Mattingly et al., In Luminescence Biotechnology: Instruments and Applications', Dyke, K. V. Ed.; CRC Press: Boca Raton, pp.77- 105 (2002); Adamczyk et al., Org. Lett.5 3779-3782 (2003); and U.S. Patent Nos.5,468,646, 5,543,524 and 5,783,699 (each of which is incorporated herein by reference in its entirety for its teachings regarding same).

“Laminin gamma-2 monomer,” “LG2m,” “laminin-5 gamma-2 monomer,” “LN-5 gamma-2 monomer,” “gamma-2 monomer,” “gamma-2,” “g-2 monomer” or any of the preceding terms with the “y” symbol in place of the word “gamma” or the letter “g” or “G” are all interchangeable and refer to one of the polypeptide chains, the gamma (y) chain (as opposed to the alpha (a) and beta ( ) chains), that constitutes laminin-5 (also known as “kalinin” and “nicein” among other synonyms) and is identified as of the gamma-2 molecular species (contrasting from the gamma- 1 species).

“Full-length laminin gamma-2 monomer,” “full-length LG2m,” or any of the preceding terms for referring to LG2m is considered LG2m which contains all of domains I-V, and any variants thereof which are recognized as fidl-length or substantially full-length as compared to the length of wild-type LG2m, including but not limited to variants comprising one or more point mutations, minor substitutions or additions of amino acids to either the N-terminus or the C- terminus, or having an amino acid sequences with at least 60% sequence identity (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity) to a native or wild-type full-length LG2m sequence.

“N-terminal fragments of laminin gamma-2 monomer” refers to forms and variants of LG2m containing domains III-V and lacking all or substantially all of domains I and II.

“Liver cancer” as used herein refers to cancer that originates in the liver. Liver cancer includes hepatocellular carcinoma (HCC) and fibrolamellar carcinoma. In most cases, the cause of liver cancer is usually scarring of the liver (e.g., cirrhosis).

“Liver disease” as used herein refers to damage to or disease of the liver. Such diseases include but are not limited to cirrhosis, alcoholic liver disease, hepatic steatosis, steatohepatitis, nonalcoholic liver disease including nonalcoholic steatohepatitis, liver infections caused by viral infections such as hepatitis B and hepatitis C infections, responses to other pathogens such as schistosomiasis, hereditary haemochromatosis, primary biliary cirrhosis and primary sclerosing cholangitis, reactions to drugs such as methotrexate and congenital disorders such as biliary atresia. Symptoms of liver dysfunction include both physical signs and a variety of symptoms related to digestive problems, blood sugar problems, immune disorders, abnormal absorption of fats, and metabolism problems. In some embodiments, liver disease includes liver fibrosis, liver cirrhosis, and liver cancer.

“Reference level” as used herein refers to a value that is used to assess diagnostic, prognostic, or therapeutic efficacy and that has been linked or is associated herein with various clinical parameters (e.g., stage of liver cancer). It is well-known that reference levels may vary depending on the nature of the assay used to determine the concentration, level, or amount of the analyte (e.g., antibodies employed, reaction conditions, sample purity, etc.) and that assays can be compared and standardized. It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other assays to obtain assay-specific reference levels for those other assays based on the description provided by this disclosure. Whereas the precise value of the reference level may vary between assays, the findings as described herein should be generally applicable and capable of being extrapolated to other assays. The reference level may be based on positive controls from individuals afflicted with late stage liver cancer. Alternatively, the reference level may be based on negative controls from healthy subject (e.g., subjects not diagnosed with late stage liver cancer).

As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen obtained from any source, including biological samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Such examples are not however to be construed as limiting the sample types. Preferably, a sample is a fluid sample such as a liquid sample. Examples of liquid samples that may be assayed include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, and spinal fluid), samples from home, municipal, or industrial water sources, runoff water, or sewage samples; and food samples (e.g., milk, beer, juice, or wine). Viscous liquid, semisolid, or solid specimens may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples. For example, throat or genital swabs may be suspended in a liquid solution to make a sample. Samples can include a combination of liquids, solids, gasses, or any combination thereof (e.g., a suspension of lysed or unlysed cells in a buffer or solution). Samples can comprise biological materials, such as cells, microbes, organelles, and biochemical complexes. Liquid samples can be made from solid, semisolid, or highly viscous materials, such as soils, fecal matter, tissues, organs, biological fluids, or other samples that are not fluid in nature. For example, solid or semisolid samples can be mixed with an appropriate solution, such as a buffer, a diluent, and/or extraction buffer. The sample can be macerated, frozen and thawed, or otherwise extracted to form a fluid sample. Residual particulates may be removed or reduced using conventional methods, such as filtration or centrifugation.

“Test sample,” “sample from a subject,” “biological sample,” and “patient sample” as used interchangeably herein may be a sample of blood, such as whole blood (including for example, capillary blood, venous blood, dried blood spot, etc.), tissue, urine, serum, plasma, amniotic fluid, an anal sample (such as an anal swab specimen), lower respiratory specimens such as, but not limited to, sputum, endotracheal aspirate or bronchoalveolar lavage, nasal mucus, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

A variety of cell types, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, nasal mucus specimens, frozen sections taken for histologic purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum, red blood cells, platelets, an anal sample (such as an anal swab specimen), interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues may also include lymph fluid, cerebrospinal fluid, or any fluid collected by aspiration. A tissue or cell type may be provided by removing a sample of cells from a human and a non-human animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and/or purification may not be necessary. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a capillary blood sample. In some embodiments, the sample is a dried blood spot. In some embodiments, the sample is a serum sample. In yet other embodiments, the sample is a plasma sample. In some embodiments, the sample is an oropharyngeal specimen. In other embodiments, the sample is a nasopharyngeal specimen. In other embodiments, the sample is sputum. In other embodiments, the sample is endotracheal aspirate. In still yet other embodiments, the sample is bronchoalveolar lavage. In still yet other aspects, the sample is nasal mucus. In still yet firrther aspects, the sample is an anal swab specimen.

A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, subject may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., humans and nonhumans) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (e.g., that are identical) as between the sequence of interest and the reference sequence. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(1): 951- 960 (2005), Altschul et al., Nucleic Acids Res., 25(11): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

Determining or Identifying Late Stage Liver Cancer The disclosure provides methods for determining or identifying late stage liver cancer in a subject. Liver cancer stages range from stage I through IV. There are several staging systems for liver cancer, including the TNM system which uses the size of tumors, spread to lymph nodes and metastasis to distal sites to assign an overall stage. Late stage liver cancer may include those cancers with: more than one tumor, a single large tumor, one or more tumors with spread to nearby lymph nodes, or one or more tumors with metastasis to distal sites (e.g., lungs, bones). In some embodiments, the late stage liver cancer is stage IV liver cancer. In some embodiments, the late stage liver cancer is metastatic liver cancer having metastasis to distal sites. In select embodiments, the liver cancer is hepatocellular carcinoma.

In some embodiments, the methods comprise determining or acquiring the concentration, level, or amount of full-length laminin gamma 2 monomer in a biological sample obtained from the subject; comparing the concentration, level, or amount of full-length laminin gamma 2 monomer to a reference level; and identifying the subject as having late stage liver cancer based on the reference concentration, level, or amount.

In some embodiments, the reference concentration, level, or amount is the reference concentration, level, or amount of full-length laminin gamma 2 monomer in a sample or compiled samples from a subject(s) known to be afflicted with late stage cancer. For example, a reference value may be based on the highest value for the concentration, level, or amount of full- length laminin gamma 2 monomer in known subjects with late stage liver cancer, such that values for the concentration, level, or amount of full-length laminin gamma 2 monomer lower than that value are indicative of late stage liver cancer. Alternatively, the reference concentration, level, or amount is the reference concentration, level, or amount of full-length laminin gamma 2 monomer in a sample or compiled samples from a subject(s) known to not have liver cancer.

In some embodiments, the methods further comprise determining or acquiring the concentration, level, or amount of total laminin gamma 2 monomer in a biological sample obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length laminin gamma 2 monomer to the concentration, level, or amount of total laminin gamma 2 monomer; comparing the ratio to a cutoff value; and identifying the subject as having late stage liver cancer when the ratio is lower than the cutoff value.

In some embodiments, the methods comprise determining or acquiring the concentration, level, or amount of full-length laminin gamma 2 monomer in a biological sample obtained from the subject; determining or acquiring the concentration, level, or amount of total laminin gamma 2 monomer in a biological sample obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length laminin gamma 2 monomer to the concentration, level, or amount of total laminin gamma 2 monomer; comparing the ratio to a cutoff value; and identifying the subject as having late stage liver cancer when the ratio is lower than the cutoff value.

Total laminin gamma 2 monomer comprises N-terminal fragments of laminin gamma 2 monomer comprising domains III-V of laminin gamma 2 monomer and full-length laminin gamma 2 monomer comprising domains I-V. As such, in some embodiments, the methods further comprise determining or acquiring the concentration, level, or amount of the total laminin gamma 2 monomer.

Similar to the reference value, the cutoff value can be based upon ratios of the concentration, level, or amount of full-length laminin gamma 2 monomer to the concentration, level, or amount of total laminin gamma 2 monomer from a subject(s) known to be afflicted with late stage cancer. Alternatively, the cutoff value can be based upon ratios of the concentration, level, or amount of full-length laminin gamma 2 monomer to the concentration, level, or amount of total laminin gamma 2 monomer from subject(s) known to not have liver cancer.

The cutoff value may be provided in a product insert or other publication, or on a website or on a mobile device (e.g., such as through an app). In some embodiments, the cutoff value is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99. In select embodiments, the cutoff value is 0.10. In select embodiments, the cutoff value is 0.11. In select embodiments, the cutoff value is 0.12. In select embodiments, the cutoff value is 0.13. In select embodiments, the cutoff value is 0.14. In select embodiments, the cutoff value is 0.15. In select embodiments, the cutoff value is 0.16. In select embodiments, the cutoff value is 0.17. In select embodiments, the cutoff value is 0.18. In select embodiments, the cutoff value is 0.19. In select embodiments, the cutoff value is 0.20. In select embodiments, the cutoff value isO. 21. In select embodiments, the cutoff value is 0.22. In select embodiments, the cutoff value is 0.23. In select embodiments, the cutoff value is 0.24. In select embodiments, the cutoff value is 0.25. In select embodiments, the cutoff value is 0.26. In select embodiments, the cutoff value is 0.27. In select embodiments, the cutoff value is 0.28. In select embodiments, the cutoff value is 0.29. In select embodiments, the cutoff value is 0.30. In select embodiments, the cutoff value is 0.31. In select embodiments, the cutoff value is 0.32. In select embodiments, the cutoff value is 0.33. In select embodiments, the cutoff value is 0.34. In select embodiments, the cutoff value is 0.35. In select embodiments, the cutoff value is 0.36. In select embodiments, the cutoff value is 0.37. In select embodiments, the cutoff value is 0.38. In select embodiments, the cutoff value is 0.39. In select embodiments, the cutoff value is 0.40. O.In select embodiments, the cutoff value is 0.42. In select embodiments, the cutoff value is 0.43. In select embodiments, the cutoff value is 0.44. In select embodiments, the cutoff value is 0.45. In select embodiments, the cutoff value is 0.46. In select embodiments, the cutoff value is 0.47. In select embodiments, the cutoff value is 0.48. In select embodiments, the cutoff value is 0.49. In select embodiments, the cutoff value is 0.50. In select embodiments, the cutoff value is 0.51. In select embodiments, the cutoff value is 0.52. In select embodiments, the cutoff value is 0.54. In select embodiments, the cutoff value is 0.55. In select embodiments, the cutoff value is 0.56. In select embodiments, the cutoff value is 0.57. In select embodiments, the cutoff value is 0.58. In select embodiments, the cutoff value is 0.59. In select embodiments, the cutoff value is 0.60. In select embodiments, the cutoff value is 0.61. In select embodiments, the cutoff value is 0.62. In select embodiments, the cutoff value is 0.63. In select embodiments, the cutoff value is 0.64. In select embodiments, the cutoff value is 0.65. In select embodiments, the cutoff value is 0.66. In select embodiments, the cutoff value is 0.67. In select embodiments, the cutoff value is 0.68. In select embodiments, the cutoff value is 0.69. In select embodiments, the cutoff value is 0.70. In select embodiments, the cutoff value is 0.71. In select embodiments, the cutoff value is 0.72. In select embodiments, the cutoff value is 0.73. In select embodiments, the cutoff value is 0.74. In select embodiments, the cutoff value is 0.75. In select embodiments, the cutoff value is 0.76. In select embodiments, the cutoff value is 0.77. In select embodiments, the cutoff value is 0.78. In select embodiments, the cutoff value is 0.79. In select embodiments, the cutoff value is 0.80. In select embodiments, the cutoff value is 0.81. In select embodiments, the cutoff value is 0.82. In select embodiments, the cutoff value is 0.83. In select embodiments, the cutoff value is 0.84. In select embodiments, the cutoff value is 0.85. In select embodiments, the cutoff value is 0.86. In select embodiments, the cutoff value is 0.87. In select embodiments, the cutoff value is 0.88. In select embodiments, the cutoff value is 0.89. In select embodiments, the cutoff value is 0.90.

In some embodiments, the subject has been diagnosed with liver cancer. In some embodiments, is suspected of having the liver cancer. In some embodiments, has been diagnosed as having a progressive liver disease In some embodiments, the subject has or has previously had a fatty liver, liver fibrosis, liver inflammation, or another liver condition. In some embodiments, the subject is undergoing or has undergone treatment for a liver disease.

In some embodiments the methods comprise determining or acquiring the values for full- length laminin gamma 2 monomer and/or total laminin gamma 2 monomer (e.g., full-length laminin gamma 2 monomer plus N-terminal fragments of laminin gamma 2 monomer) at single time point.

In some embodiments, the methods comprise determining or acquiring the values for full- length laminin gamma 2 monomer and/or total laminin gamma 2 monomer at an initial time point and one or more additional or subsequent time points. As such, the methods may allow identification of a first diagnosis of late-stage liver cancer or monitoring liver cancer progression in a subject.

The progression (or regression) of the liver cancer may be monitored while the subject is undergoing a treatment (e.g., before commencing the treatment, during a first and/or second time point during the treatment, and/or after completing all or a portion of a therapeutic regimen). Any changes over the time period following treatment can be used to determine whether to initiate or continue the treatment of the disease and determine whether the initial treatment therapy is effective, for example.

In some embodiments, the methods comprise obtaining two or more biological samples from the subject, each biological sample separated from the previous sample by a period of time; determining or acquiring the concentration, level, or amount of full-length laminin gamma 2 monomer in the two or more biological samples; comparing the concentration, level, or amount of full-length laminin gamma 2 monomer to a reference level; and identifying the onset of late stage liver cancer when the concentration, level, or amount of full-length laminin gamma 2 monomer in one of the biological samples is equal to or lower than the reference concentration, level, or amount. In some embodiments, the methods further comprise one or more of: determining or acquiring the concentration, level, or amount of total laminin gamma 2 monomer in each of the biological samples obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length laminin gamma 2 monomer to the concentration, level, or amount of total laminin gamma 2 monomer; comparing the ratio to a cutoff value; and identifying the onset of late stage liver cancer when the ratio in one of the biological samples is lower than the cutoff value.

Generally, for methods in which multiple time points are used to assay for a disease (e.g., monitoring disease progression and/or response to treatment), a second or additional sample from the subject is obtained at a period of time after the first sample has been obtained. Specifically, a second subject sample can be obtained minutes, hours, days, weeks, months, or years after the first subject sample was obtained.

For example, the second, or additional, subject sample can be obtained from the subject at a time period of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 9.5 years, about 10.0 years, or more after the first, or previous, subject sample was obtained.

1. Treatment

In some embodiments, the methods disclosed herein further comprise treating the subject. As such, the methods described herein may be integrated into a treatment method for a subject. For example, in some embodiments, the subject sample(s) is analyzed by the methods described herein, a report of the results is generated, and the subject is treated based on the results (e.g., commence a new treatment, continue existing treatment, change in treatment (e.g., change in intervention type, dose, timing, etc.). Suitable treatments can include administration of a pharmaceutical compound, a vaccine, performing a surgery (e.g., tumor resection), imaging the patient, and/or performing another test (e.g., a biopsy).

In some embodiments, the treatment comprises administration of an anti-cancer agent or chemotherapeutic. “Anti-cancer agent” or “chemotherapeutic,” as used herein, refers to any small molecule or other drug used in cancer treatment or prevention. Chemotherapeutics include, but are not limited to, cyclophosphamide, methotrexate, 5 -fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, afinitor, alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, and tucatinib.

In some embodiments, the treating comprises active surveillance. As such, the methods described herein find use in classifying a patient as suitable for active surveillance. During active surveillance the subject is monitored with additional screenings or tests for changes in overall health or changes directly related to disease progression. For example, in some embodiments, the methods comprise collecting or receiving a series of samples over a time period from the subject and detecting the one or more biomarkers in each of the series of samples and comparing any measurable change in the biomarkers over the period of time. In some embodiments, each of the series of samples may be used for diagnosing, detecting, or assigning a risk level for the subject at that given time point, as described in the methods herein, such that monitoring may comprise updating the diagnosis, risk level, and/or risk score, as described above, during the active surveillance period. Any changes over time may be used to predict risk of developing the disease or disorder or to determine whether to initiate or continue any treatment or active surveillance.

2. Subject Sample

Subject samples include, but are not necessarily limited to, bodily fluids such as blood- related samples (e.g., whole blood, serum, plasma, and other blood-derived samples), urine, cerebral spinal fluid, bronchoalveolar lavage, and the like. Another example of a biological sample is a tissue sample. A biological sample may be fresh or stored (e.g., blood or blood fraction stored in a blood bank). The biological sample may be a bodily fluid expressly obtained for the assays of this disclosure or a bodily fluid obtained for another purpose which can be subsampled for the assays of this disclosure. In certain embodiments, the biological sample is whole blood. Whole blood may be obtained from the subject using standard clinical procedures. In other embodiments, the biological sample is plasma. Plasma may be obtained from whole blood samples by known means, including but not limited to, centrifugation (e.g., of anti -coagulated blood), membrane- or filter-based separation, agglutination-based plasma separation, acoustic force, and microfluidics. Such process provides a buffy coat of white cell components and a supernatant of the plasma. In certain embodiments, the biological sample is serum. Serum may be obtained by centrifugation of whole blood samples that have been collected in tubes that are free of anti-coagulant. The blood is permitted to clot prior to centrifugation. The yellowish- reddish fluid that is obtained by centrifugation is the serum. In another embodiment, the sample is urine. The sample may be pretreated as necessary by dilution in an appropriate buffer solution, heparinized, concentrated if desired, or fractionated by any number of methods including but not limited to ultracentrifugation, fractionation by fast performance liquid chromatography (FPLC), or precipitation of apolipoprotein B containing proteins with dextran sulfate or other methods. Any of a number of standard aqueous buffer solutions at physiological pH, such as phosphate, Tris, or the like, can be used. In some embodiments, the biological sample(s) are blood, serum, or plasma sample. In some embodiments, when the methods utilize two or more biological samples, the first, second, or additional samples are the from the same or different biological source. In some embodiments, the two or more biological samples are each individually selected from whole blood, plasma, and serum samples.

In some embodiments, the methods further comprise obtaining the biological sample(s) from the subject. The sample(s) can be obtained using techniques known to those skilled in the art, and the sample(s) may be used directly as obtained from the source or following a pretreatment to modify the character of the sample. Such pretreatment may include, for example, preparing plasma from blood, diluting viscous fluids, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, and the like.

The sample(s) may be obtained in a medical facility, e.g., at an Emergency Room, urgent care clinic, walk-in clinic, a long term care facility, or another appropriate site of medical practice. The sample(s) may be obtained in a home or residential setting (e.g., a senior living or hospice setting), at the site of the suspected myocardial infarction, or during transportation to a medical facility (e.g., ambulance).

3. Assay Methods

The present disclosure is not limited by the type of assay used to determining the concentration, level, or amount of full-length laminin gamma 2 monomer and/or total laminin gamma 2 monomer (e.g., full-length laminin gamma 2 monomer plus N-terminal fragments of laminin gamma 2 monomer). The nature of methods and the test can be any assay known in the art such as, for example, immunoassays, point-of-care assays, clinical chemistry assay, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, a protein assay, a competitive binding assay, a lateral flow assay, a functional protein assay, or chromatography or spectrometry methods, such as high- performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, the assay can be employed in a clinical chemistry format such as would be known by one of ordinary skill in the art.

Full-length laminin gamma 2 monomer, total laminin gamma 2 monomer, and/or N- terminal fragments of laminin gamma 2 monomer can be detected and/or quantified in a sample with the help of one or more separation methods. For example, suitable separation methods may include a mass spectrometry method, such as electrospray ionization mass spectrometry (ESI- MS), ESI-MS/MS, ESI-MS/(MS)ⁿ (n is an integer greater than zero), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI- MS), APCI-MS/MS, APCI-(MS)ⁿ, or atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)ⁿ. Other suitable separation methods include chemical extraction partitioning, column chromatography, ion exchange chromatography, hydrophobic (reverse phase) liquid chromatography, isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), or other chromatographic techniques, such as thin-layer, gas or liquid chromatography, or any combination thereof.

In some embodiments, an immunoassay is employed for determining the concentration, level, or amount of full-length laminin gamma 2 monomer, total laminin gamma 2 monomer, and/or N-terminal fragments of laminin gamma 2 monomer. Examples of such assays include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal- polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, one-step antibody detection assay, homogeneous assay, heterogeneous assay, capture on the fly assay, single molecule detection assay, lateral flow assay, etc.

In some embodiments, concentration, level, or amount of full-length laminin gamma 2 monomer is determined using a sandwich immunoassay. In certain embodiments, the concentration, level, or amount of full-length laminin gamma 2 monomer is determined using an antibody, or fragment thereof, which specifically binds to domain I/II of laminin gamma 2 monomer and an antibody, or fragment thereof, which specifically binds to domain V of laminin gamma 2 monomer.

In some embodiments, concentration, level, or amount of the total laminin gamma 2 monomer is determined using a sandwich immunoassay. In certain embodiments, the concentration, level, or amount of the total laminin gamma 2 monomer is determined using an antibody, or fragment thereof, which specifically binds to domain III of laminin gamma 2 monomer and an antibody, or fragment thereof, which specifically binds to domain V of laminin gamma 2 monomer.

Determining the concentration of full-length laminin gamma 2 monomer and/or N- terminal fragments of laminin gamma 2 monomer by an immunoassay can be adapted for use in a variety of automated and semi-automated systems or platforms (including those wherein the solid phase comprises a microparticle) known in the art. The following adaptations of automated and/or semi-automated systems are included herein as merely exemplary. Specifically, the methods can utilize automated and semi-automated systems or platforms such as those described, e.g., U.S. Patent No. 5,063,081, U.S. Patent Application Publication Nos. 2003/0170881, 2004/0018577, 2005/0054078, and 2006/0160164 and as commercially marketed e.g., by Abbott Laboratories (Abbott Park, IL) as Abbott Point of Care (i-STAT® or i-STAT Alinity, ID Now®, Abbott Laboratories) as well as those described in U.S. Patent Nos. 5,089,424 and 5,006,309, and as commercially marketed, e.g., by Abbott Laboratories (Abbott Park, IL) as ARCHITECT® or the series of Abbott Alinity devices. In certain embodiments, the methods for detecting GP73 are as described in U.S. Patent No. 9,469,686 incorporated herein by reference in its entirety but with particular focus on the assay methods.

Other methods of detection include the use of or adaption for use on a nanopore device or nanowell device, e.g., for single molecule detection. As used herein the term “single molecule detection” refers to the detection and/or measurement of a single molecule of an analyte in a test sample at very low levels of concentration (such as pg/mL or femtogram/mL levels). A number of different single molecule analyzers or devices are known in the art and include nanopore and nanowell devices. Examples of nanopore devices are described in PCT International Application WO 2016/161402, which is hereby incorporated by reference in its entirety. Examples of nanowell device are described in PCT International Application WO 2016/161400, which is hereby incorporated by reference in its entirety.

The methods are not limited by the method of acquiring the concentration, level, or amount of full-length laminin gamma 2 monomer and/or N-terminal fragments of laminin gamma 2 monomer. In some embodiments, the methods comprise receiving the values from a testing lab, from an analytical testing system, and/or from a hand-held or point of care testing device. In select embodiments, the methods comprise receiving the values from an analytical testing system. In some embodiments, the methods comprise receiving the values from a hand-held or point of care testing device. “Point-of-care device” refers to a device used to provide medical diagnostic testing at or near the point-of-care (namely, outside of a laboratory), at the time and place of patient care (such as in a hospital, physician’s office, urgent or other medical care facility, a patient’s home, a nursing home and/or a long-term care and/or hospice facility). Such point-of-care devices can also include portable, desktop sized devices. Examples of point-of-care devices include those produced by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT®, i-STAT® Alinity, ID Now®), Universal Biosensors (Rowville, Australia) (see US 2006/0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles, USA).

Detecting Full length Laminin Gamma 2 Monomer

The disclosure also provides methods detecting or quantifying (e.g., determining an amount, quantity, concentration and/or level) full length laminin gamma 2 monomer (LG2m) in a sample. In some embodiments, the methods comprise incubating the sample with a first antibody, or fragment thereof, which specifically binds to domain V of laminin gamma 2 monomer and a second antibody, or fragment thereof, which specifically binds to domain I/II of laminin gamma 2 monomer.

In some embodiments, the first antibody and the second antibody are incubated with the sample simultaneously. In some embodiments, the first antibody and the second antibody are incubated with the sample sequentially in any order.

In some embodiments, the first antibody comprises a detectable label. In some embodiments, the second antibody comprises a detectable label. In some embodiments, the antibody lacking a detectable label is bound to or configured to bind to a solid support. In some embodiments, the methods further comprise assessing the signal from the detectable label as a measure of the presence or quantification of the full length LG2m.

The methods detecting or quantifying full length laminin gamma 2 monomer (LG2m) in a sample may adapted in view of known assays for analyzing analytes. Examples of well-known assays include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal- monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), competitive inhibition immunoassay (e.g., forward and reverse), enzyme multiplied immunoassay technique (EMIT), a competitive binding assay, bioluminescence resonance energy transfer (BRET), one-step antibody detection assay, homogeneous assay, heterogeneous assay, capture on the fly assay, etc. Full length LG2m may be detected or quantified using the disclosed first and second antibodies in an immunoassay. The presence or amount of full length LG2m can be determined by the detection of specific binding to the disclosed antibodies in various types of immunoassays, such as sandwich immunoassay (e.g., monoclonal- monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, MN)). Other methods that can be used include a chemiluminescent microparticle immunoassay, in particular one employing the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, IL), as an example.

Specific immunological binding of the antibodies to fiill length LG2m can be detected via direct labels, such as fluorescent or luminescent tags, metals and radionuclides attached to the antibody or via indirect labels, such as alkaline phosphatase or horseradish peroxidase.

The use of immobilized antibodies or antibody fragments thereof may be incorporated into the immunoassay. The antibodies may be immobilized onto a variety of solid supports. The solid support can be any solid support, such as, but not limited to, a magnetic particle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffolding molecule, a film, a filter paper, a disc, and a chip. In those embodiments where the solid support is a bead, the bead may be a magnetic bead or a magnetic particle. Magnetic beads/particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO/Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO.Fe2O3). Beads can have a solid core portion that is magnetic and is surrounded by one or more non-magnetic layers. Alternately, the magnetic portion can be a layer around a non-magnetic core. An assay strip can be prepared by coating the antibody or plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

In some embodiments, the methods comprise a sandwich immunoassay. A sandwich immunoassay measures the amount of antigen (e.g., full length LG2m) between two layers of antibodies (e.g., a capture antibody and a detection antibody. The capture antibody and the detection antibody bind to different epitopes on the antigen. Desirably, binding of the capture antibody to an epitope does not interfere with binding of the detection antibody to an epitope. As described herein the first antibody or the second antibody may be the capture antibody with the alternate being the detection antibody. A sample suspected of containing full length LG2m may be contacted with at least one first capture antibody (or antibodies) and at least one second detection antibodies either simultaneously or sequentially.

Optionally, prior to contacting the test sample with the at least one first capture antibody, the at least one first capture antibody can be bound to a solid support, as described above, which facilitates the separation of the complex of the capture antibody/antigen (e.g., full length LG2m) complex. After incubating for a sufficient length of time under conditions which favor formation of the capture antibody/antigen (e.g., full length LG2m) complex, the complex can be contacted with a detection antibody. Alternatively, the detection antibody can be incubated with the sample simultaneously with the capture antibody. Preferably, the detection antibody contains a detectable label. The detectable label can be bound to the at least one second antibody prior to, simultaneously with or after the formation of the capture antibody/analyte/detection antibody complex. Any detectable label known in the art can be used.

Single molecule detection assays and methods, such as the use of a nanopore device or nanowell device, can also be used. Examples of nanopore devices are described in PCT International Application WO 2016/161402, which is hereby incorporated by reference in its entirety. Examples of nanowell device are described in PCT International Application WO 2016/161400, which is hereby incorporated by reference in its entirety. Other devices and methods appropriate for single molecule detection can also be employed.

Lateral flow assays are generally provided in a device comprising a lateral flow test strip (e.g., nitrocellulose or filter paper), a sample application area (e.g., sample pad), a test results area (e.g., a test line), an optional control results area (e.g., a control line), and an analytespecific binding partner that is bound to a detectable label (e.g., a colored particle or an enzyme detection system). See, e.g., U.S. Patent Nos. 6,485,982; 6,187,598; 5,622,871; 6,565,808; and 6,809,687; and U.S. Patent Publication No. 2004/0184954, each of which is incorporated herein by reference.

Kits

The disclosure also provides a kit comprising an antibody, or fragment thereof, which specifically binds to domain I/II of laminin gamma 2 monomer; and an antibody, or fragment thereof, which specifically binds to domain V of laminin gamma 2 monomer. In some embodiments, the kits further comprise an antibody, or fragment thereof, which specifically binds to domain III of laminin gamma 2 monomer. The kit may further comprise quality control reagents. “Quality control reagents” in the context of immunoassays and kits described herein, include, but are not limited to, calibrators, controls, and sensitivity panels. A calibrator or standard typically is used (e.g., one or more, such as a plurality) in order to establish calibration (standard) curves for interpolation of the concentration of an analyte, such as an antibody or an analyte. Alternatively, a single calibrator, which is near a predetermined positive/negative cutoff, can be used. Multiple calibrators (e.g., more than one calibrator or a varying amount of calibrator(s)) can be used in conjunction to comprise a sensitivity panel.

The kit may also include a detectable label that can be or is conjugated to an antibody. The detectable label can for example be a direct label, which may be an enzyme, oligonucleotide, nanoparticle, chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin. Kits may optionally include any additional reagents needed for detecting the label.

The kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co- factors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls.

The kit may farther comprise pretreatment reagents. Pretreatment reagents include, for example, lysis, precipitation and/or solubilization reagents, as used in a diagnostic assays as described herein. The pretreatment reagent optionally can comprise: (a) one or more solvents and salt, (b) one or more solvents, salt, and detergent, (c) detergent, (d) detergent and salt, or (e) any reagent or combination of reagents appropriate for cell lysis and/or solubilization of the desired analyte (e.g., LG2m).

The kit may comprise instructions for using the antibodies or fragments thereof described herein, e.g., for detecting or quantifying fall length LG2m or determining or identifying late stage liver cancer.

The kit may be supplied in a solid (e.g., lyophilized) or liquid form. The various components of the kit of the present disclosure may optionally be contained within different containers (e.g., vial, ampoule, test tube, flask, or bottle) for each individual component (e.g., amplification oligonucleotides, probe oligonucleotides, or buffer). Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Other containers suitable for conducting certain steps of the amplification/detection assay may also be provided. The individual containers are preferably maintained in close confinement for commercial sale.

Systems

The disclosure also provides a systems for measuring the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in a sample, for example, as described in the above disclosed methods. The systems may comprise a sample receiving component, a sample analysis component, and an indicator or display to communicate the results of the sample analysis.

In some embodiments, the system comprises a sample receiving component configured to receive the sample. The sample receiving component may be configured to receive any type of sample, as described above, or may further comprise reagents/components necessary to process the sample prior to analysis. In some embodiments, the sample receiving component is integrated with the sample analysis component (e.g., in a single module of the system).

The sample analysis component is configured to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the sample. In some embodiments, the sample analysis component is further configured to compare the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) from the sample with a reference level concentration, level, or amount.

The sample analysis component may comprise one or more assays to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the biological sample. Assays or tests encompass any or all of the reagents and instrumentation necessary to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the biological sample. In some embodiments, the sample analysis component comprises an antibody, or fragment thereof, which specifically binds to domain I/II of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

In some embodiments, the sample analysis component further comprises one or more assays to measure the concentration, level, or amount of total LG2m in a biological sample obtained from the subject. As above, the assays or tests encompass any or all of the reagents and instrumentation necessary to measure a concentration, level, or amount of total laminin gamma 2 monomer (LG2m) in the biological sample. Accordingly, in some embodiments, the sample analysis component is further configured to calculate a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m. In some embodiments, the system comprises an indicator or display configured to show the presence or concentration, level, or amount of full-length LG2m. In some embodiments, the system comprises an indicator or display to show when the concentration, level, or amount of full-length LG2m is equal to or lower than the reference concentration, level, or amount. In some embodiments, the indicator or display shows the presence or concentration, level, or amount of full-length LG2m and shows when the concentration, level, or amount of full-length LG2m is equal to or lower than the reference concentration, level, or amount.

In some embodiments, the indicator or display is further configured show the presence or concentration, level, or amount of total LG2m. In some embodiments, the indicator or display is further configured to show when the ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m is lower than a cutoff value.

The indicator or display may use any manner of visual or audible means to display the result including but not limited to text, graphs, charts, heat maps, other image based methods, color indications, beeping, and the like. The indicator or display may be configured to transmit the results to another device connected wirelessly or integral to the present system. For example, the indicator or display may transmit the result to a clinical device, a patient record, a patient device, a data storage repository, or similar.

The systems of the present invention may be configured to work with a wide variety of sample types, analysis methods, and detection systems, as described elsewhere herein. The systems may be used to perform analysis of full-length LG2m and/or total LG2m in an automated or a semi-automated manner. The systems optionally may include disposable/consumable components that are utilized for the analysis.

Thus, using the systems, kits, and methods as described herein, full-length LG2m and/or total LG2m present in a sample (e.g., biological sample) can be measured, and in some instances, a patient can be diagnosed (e.g., with late stage liver cancer).

EXAMPLES

The following examples are for purposes of illustration only and are not intended to limit the scope of the claims.

METHODS AND MATERIALS

2H2 Monoclonal Antibody. 2H2 mAb was a mouse monoclonal antibody described in N. Koshikawa et al., Cancer Research (2008) 68(2):530-536. The 2H2 mAb was purified from the supernatant of the cultured hybridoma by a Protein G column. 2E9 Monoclonal Antibody. 2H2 mAb was obtained using a part of LG2m DI/II (853- 1178 aa) as an antigen. 2E9 reacts with DI/II of LG2m but does not react with Lm-332-g2 chain (FIG. 6).

IB 383 Monoclonal Antibody. 1B383 mAb was determined by epitope mapping to bind to an epitope of LG2m in Domain III at amino acid positions 460-516. 1B383 recognizes both LG2m and Lm-332-g2 chain (FIG. 6).

EXAMPLE 1

An immunoassay was established using an anti-LG2m domain I/II mAb, 2E9, and an anti-LG2m domain V mAb, 2H2, to measure recombinant full length LG2m. The measurement range was from 20 pg/mL to 20,000 pg/mL (FIG. 2).

Concentration of full-length LG2m in commercially available hepatocellular cancer (HCC) and normal serum was measured as described above. The same samples were measured using a sandwich assay between 2H2 and an anti-LG2m domain III mAb, 1B383, which has the capability to measure full-length LG2m andN-terminal fragments comprising domains III-V (hereinafter referred to as “totally LG2m”). Full-length LG2m concentration increased with cancer stage by stage III, however at stage IV levels dropped to those seen in normal serum (FIG. 3, left). Total LG2m concentration increased in cancer stages I-III, similarly to full-length LG2m. However, unlike full-length LG2m, total LG2m concentration at stage IV was also higher than normal (FIG. 3, right). Thus, total LG2m concentration in all HCC stages was higher than normal.

Good correlation was obtained (R²=0.8312) between the two immunoassay measurements when comparing all HCC and serum samples (FIG. 4). Correlation analysis between full-length LG2m and total LG2m was performed with only the cancer samples. Good correlation was obtained (R²=0.8286), but two samples were significantly out of correlation (FIG.5, left). Both samples were stage IV (stage IV samples are shown in red). A second correlation analysis was performed only including stage I-III cancer samples; excluding stage IV samples. The correlation of stage I-III was better than that of the cancer samples when including stage IV (R²=0.9799; FIG. 5, right).

Those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this disclosure. Accordingly, all such modifications and alternative are intended to be included within the scope of the disclosure as defined in the following claims. Those skilled in the art should also realize that such modifications and equivalent constructions or methods do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims

We claim:

1. A method for determining or identifying late stage liver cancer in a subject diagnosed with liver cancer comprising: determining or acquiring the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in a biological sample obtained from the subject; comparing the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) to a reference level; and identifying the subject as having late stage liver cancer when the concentration, level, or amount of full-length LG2m is equal to or lower than the reference concentration, level, or amount.

2. The method of claim 1, wherein the concentration, level, or amount of full-length LG2m is determined using an immunoassay.

3. The method of claim 2, wherein the immunoassay is a sandwich immunoassay.

4. The method of claim 2 or 3, wherein the concentration, level, or amount of full-length LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain I/II of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

5. The method of any of claims 1-4, wherein the subject has late stage liver cancer when the concentration, level, or amount of full-length LG2m in one of the biological samples is equal to or lower than the reference concentration, level, or amount found in subjects known to have late stage liver cancer.

6. The method of any of claims 1-5, wherein the method further comprises: determining or acquiring the concentration, level, or amount of total LG2m in a biological sample obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m; comparing the ratio to a cutoff value; and identifying the subject as having late stage liver cancer when the ratio is lower than the cutoff value.

7. The method of claim 6, wherein total LG2m comprises N-terminal fragments of LG2m comprising domains III-V of LG2m and full-length LG2m.

8. The method of claim 7, further comprising determining or acquiring the concentration, level, or amount of the total LG2m.

9. The method of claim 8, wherein the concentration, level, or amount of the total LG2m is determined using an immunoassay.

10. The method of claim 9, wherein the immunoassay is a sandwich immunoassay.

11. The method of any of claims 8-10, wherein the concentration, level, or amount of the total LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain III of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

12. The method of any of claims 1-11, wherein the late stage liver cancer is stage IV liver cancer.

13. The method of any of claims 1-12, wherein the late stage liver cancer is metastatic liver cancer.

14. The method of any of claims 1-13, wherein the liver cancer is hepatocellular carcinoma.

15. The method of any of claims 1-14, wherein the biological sample is a whole blood sample, a plasma sample, or a serum sample.

16. The method of any of claims 1-15, further comprising obtaining the biological sample from the subject.

17. The method of any of claims 1-16, further comprising treating the subject.

18. A method for determining or identifying late stage liver cancer in a subject comprising: determining or acquiring the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in a biological sample obtained from the subject; determining or acquiring the concentration, level, or amount of total LG2m in a biological sample obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m; comparing the ratio to a cutoff value; and identifying the subject as having late stage liver cancer when the ratio is lower than the cutoff value.

19. The method of claim 18, wherein total LG2m comprises N-terminal fragments of LG2m comprising domains III-V of LG2m and full-length LG2m.

20. The method of claim 18 or 19, wherein the concentration, level, or amount of N-terminal fragments of LG2m, the concentration, level, or amount of full-length LG2m, or both is determined using an immunoassay.

21. The method of claim 20, wherein the immunoassay is a sandwich immunoassay.

22. The method of claim 21, wherein the concentration, level, or amount of total LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain III of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

23. The method of any of claims 18-22, wherein the concentration, level, or amount of full- length LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain I/II of LG2m and an antibody, or fragment thereof, which specifically binds to domain V ofLG2m.

24. The method of any of claims 18-23, wherein the late stage liver cancer is Stage IV liver cancer.

25. The method of any of claims 18-24, wherein the late stage liver cancer is metastatic liver cancer.

26. The method of any of claims 18-25, wherein the liver cancer is hepatocellular carcinoma.

27. The method of any of claims 18-26, wherein the subject is diagnosed as having liver cancer, is suspected as having liver cancer, or is diagnosed as having a progressive liver disease.

28. The method of any of claims 18-27, wherein the biological sample is a whole blood sample, a plasma sample, or a serum sample.

29. The method of any of claims 18-28, further comprising obtaining the biological sample from the subject.

30. The method of any of claims 18-28, further comprising treating the subject.

31. A method for monitoring liver cancer progression in a subject comprising: obtaining two or more biological samples from the subject, each biological sample separated from the previous sample by a period of time; determining the concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the two or more biological samples; comparing the concentration, level, or amount of full-length LG2m to a reference concentration, level, or amount; and identifying the onset of late stage liver cancer when the concentration, level, or amount of full-length LG2m in one of the two or more biological samples is equal to or lower than the reference concentration, level, or amount.

32. The method of claim 31, wherein the concentration, level, or amount of full-length LG2m is determined using an immunoassay.

33. The method of claim 32, wherein the immunoassay is a sandwich immunoassay.

34. The method of any of claims 31-33, wherein the concentration, level, or amount of full- length LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain T/IT of LG2m and an antibody, or fragment thereof, which specifically binds to domain V ofLG2m.

35. The method of any of claims 31-34, wherein the onset of late stage liver cancer is identified when the concentration, level, or amount of full-length LG2m in one of the biological samples is equal to or lower than the reference concentration, level, or amount found in subjects known to have late stage liver cancer.

36. The method of any of claims 31-35, further comprising: determining or acquiring the concentration, level, or amount of total LG2m in each of the two or more biological samples obtained from the subject; calculating a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m; comparing the ratio to a cutoff value; and identifying the onset of late stage liver cancer when the ratio in one of the two or more biological samples is lower than the cutoff value.

37. The method of claim 36, wherein total laminin gamma 2 monomer comprises N-terminal fragments of LG2m comprising domains III-V of laminin gamma 2 monomer and full-length LG2m.

38. The method of claim 36 or 37, further comprising determining or acquiring the concentration, level, or amount of the total LG2m.

39. The method of claim 37 or 38, wherein the concentration, level, or amount of total LG2m is determined using an immunoassay.

40. The method of claim 39, wherein the immunoassay is a sandwich immunoassay.

41. The method of claim 40, wherein the concentration, level, or amount of the total LG2m is determined using an antibody, or fragment thereof, which specifically binds to domain III of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

42. The method of any of claims 31-41, wherein the late stage liver cancer is Stage IV liver cancer.

43. The method of any of claims 31-42, wherein the late stage liver cancer is metastatic liver cancer.

44. The method of any of claims 31-43, wherein the liver cancer is hepatocellular carcinoma.

45. The method of any of claims 31-44, wherein the two or more biological samples are each individually selected from a whole blood sample, a plasma sample, and a serum sample.

46. The method of any of claims 31-45, fiirther comprising obtaining the two or more biological samples from the subject.

47. The method of any of claims 31 -46, further comprising treating the subject.

48. A kit comprising: an antibody, or fragment thereof, which specifically binds to domain I/II of laminin gamma 2 monomer; and an antibody, or fragment thereof, which specifically binds to domain V of laminin gamma 2 monomer.

49. The kit of claim 48, wherein the kit further comprises an antibody, or fragment thereof, which specifically binds to domain III of laminin gamma 2 monomer.

50. A method for detecting or quantifying full length laminin gamma 2 monomer (LG2m) in a sample comprising: incubating the sample with: a first antibody, or fragment thereof, which specifically binds to domain I/II of laminin gamma 2 monomer; and a second antibody, or fragment thereof, which specifically binds to domain V of laminin gamma 2 monomer.

51. The method of claim 50, wherein the first antibody, or fragment thereof, and the second antibody, or fragment thereof, are incubated with the sample simultaneously or sequentially in any order.

52. The method of claim 50 or 51, wherein the first antibody, or fragment thereof, or the second antibody, or fragment thereof, comprises a detectable label.

53. The method of claim 52, wherein the antibody, or fragment thereof, not comprising the detectable label is bound to or configured to bind to a solid support.

54. The method of claim 53, further comprising assessing the signal from the detectable label.

55. A system comprising: a sample receiving component configured to receive a sample; a sample analysis component configured to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the sample; and an indicator or display configured to show the presence or concentration, level, or amount of full-length LG2m.

56. A system comprising: a sample receiving component configured to receive a sample; a sample analysis component configured to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the sample and compare the concentration, level, or amount of frill-length laminin gamma 2 monomer (LG2m) from the sample with a reference level concentration, level, or amount; and an indicator or display configured to show when the concentration, level, or amount of full-length LG2m is equal to or lower than the reference concentration, level, or amount.

57. The system of claim 55 or 56, wherein the sample receiving component and the sample analysis component are integrated into a single module of the system.

58. The system of any of claims 55-57, wherein the sample analysis component comprises one or more assays to measure a concentration, level, or amount of full-length laminin gamma 2 monomer (LG2m) in the biological sample.

59. The system of any of claims 55-58, wherein the sample analysis component comprises an antibody, or fragment thereof, which specifically binds to domain I/II of LG2m and an antibody, or fragment thereof, which specifically binds to domain V of LG2m.

60. The system of any of claims 56-59, wherein the sample analysis component further comprises one or more assays to measure the concentration, level, or amount of total LG2m in a biological sample obtained from the subject.

61. The system of any of claims 56-60, wherein the sample analysis component is further configured to calculate a ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m.

62. The system of any of claims 56-61, wherein the indicator or display is further configured to show when the ratio of the concentration, level, or amount of full-length LG2m to the concentration, level, or amount of total LG2m is lower than a cutoff value.

63. The system of any of claims 55-62, wherein the sample is a biological sample.

64. The system of claim 63, wherein the biological sample is a whole blood sample, a plasma sample, or a serum sample.