WO2018085363A2 - Cd147 detection in diagnostic, prognostic and monitoring methods for multiple myeloma - Google Patents

Abstract

The invention generally provides improved compositions and methods for detecting, diagnosing, prognosing, and monitoring a hematological malignancy in a subject. More specifically, the invention provides methods for detecting CD147 in subjects to diagnose, predict survival and/or monitor progression of a hematological malignancy, particularly a multiple myeloma, in a subject.

Description

CD147 DETECTION IN DIAGNOSTIC, PROGNOSTIC AND

MONITORING METHODS FOR MULTIPLE MYELOMA

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. Provisional Application No. 62/416,601, filed on November 2, 2016, and it is herein incorporated by reference in its entirety.

BACKGROUND

1. Technical Field

The compositions and methods of the invention relate generally to detection of biomarkers for the diagnosis, prognosis, and monitoring of cancer. In particular, the invention relates to compositions and methods for detection of CD147 for the diagnosis, prognosis and monitoring of hematological malignancies, such as multiple myeloma.

2. Description of the Related Art

Multiple myeloma is a prevalent blood cancer, representing approximately 1% of all cancers and 2% of all cancer deaths. Although the peak age of onset of multiple myeloma is 65 to 70 years of age, recent statistics indicate both increasing incidence and earlier age of onset. Approximately 100,000 Americans currently have myeloma, and the American Cancer Society estimates that approximately 22,000 new cases of myeloma are diagnosed each year in the United States.

Due to the difficulty in assessing the location of bone marrow (BM)-based malignancies and the heterogeneous involvement of malignant cells within different BM sites, measurement of tumor mass in hematological malignancies such as MM, CLL, and NHL is indirect. Consequently, response to therapy is often difficult to determine. Besides blood and urine monoclonal Ig levels, existing blood markers used to determine MM tumor mass include: hemoglobin, urea nitrogen, calcium, albumin, creatinine, monoclonal protein, beta-2 microglobulin (132M), IL-6, C-reactive protein, soluble IL-6 receptor, lactate dehydrogenase, thymidine kinase, and al-antitrypsin (Kyle, 1994). However, these markers are not produced directly by MM cells and thus, are not reliable. In addition, existing markers are even less useful for monitoring the response of patients to treatment, probably due to their widespread presence in many other not malignant cell types (Jones et al., 2001). Thus, existing markers have not proven to be reliable diagnostic or prognostic indicators of disease progression, survival and/or response to anti-cancer treatments (Kyle, 1994).

Accordingly, the art is deficient in reliable diagnostic, prognostic, and treatment monitoring biomarkers for multiple myeloma and other hematological malignancies. In addition, existing biomarkers do not correlate well with response to anti-cancer treatment, or with the extent or severity of the disease. The present invention meets certain of these needs and offers other related advantages.

BRIEF SUMMARY

The invention generally provides compositions and methods for reliably and reproducibly diagnosing, prognosing and/or monitoring cancer, such as hematological cancers, and particularly multiple myeloma (MM). The levels of CD147 in the supernatants of cultured BMMCs and patient sera can be detected and/or measured and compared against a baseline or control to provide a diagnosis or prognosis in the patient

Therefore, according to one aspect of the invention, there is provided a method of prognosis for the survival of a subject having multiple myeloma (MM) comprising: (a) detecting an amount of CD147 polypeptide or a fragment thereof in a biological sample obtained from a subject; and (b) comparing the amount of CD147 polypeptide or fragment thereof detected in step (a) to a predetermined prognostic cut-off value, wherein an amount of CD147 polypeptide or fragment thereof in the biological sample that exceeds the prognostic cut-off value indicates, for example, refractory disease, a poor outcome, a reduced chance of survival, and the like.

In certain embodiments of this aspect of the invention, the biological sample is a serum sample. In other embodiments, the biological sample is supernatant obtained from culture of the subject's bone marrow mononuclear cells. In still other embodiments, the biological sample is supernatant obtained from culture of the subject's peripheral blood mononuclear cells.

The CD147 polypeptide can be detected using essentially any conventional methodology. For example, in certain embodiments, the CD147 polypeptide or a fragment thereof is detected using a detection system selected from the group consisting of: an immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay ( IA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay.

Accordingly, it will be understood that, in many embodiments of the invention, the CD147 polypeptide or fragment thereof will be detected using an antibody, binding fragment thereof, or any other type of suitable binding agent specific for CD147. In certain specific embodiments, the antibody used in the methods of the invention is a monoclonal or polyclonal antibody that is specific for CD147.

In certain embodiments of the invention, an amount of CD147 polypeptide or fragment thereof that is greater than about 5 ng/mL in the biological sample obtained from the subject having MM indicates a reduced chance of survival for the subject (e.g., a reduced progression free survival (PFS).

In certain embodiments of the invention, an amount of CD147 polypeptide or fragment thereof that is greater than about 6 ng/mL in the biological sample obtained from the subject having MM indicates the presence of refractory disease (RD).

According to another general aspect of the invention, there is provided a method of monitoring the progression or response to treatment of multiple myeloma (MM),

comprising: (a) detecting an amount of CD147 polypeptide or a fragment thereof in a biological sample obtained from a patient diagnosed with MM at a first time point; (b) detecting an amount of CD147 polypeptide or fragment thereof in a biological sample obtained from the patient at a second time point or following treatment; and (c) comparing the amount detected in step (a) to the amount detected in step (b), wherein an increased amount of CD147 polypeptide or a fragment thereof in the biological sample of (b) as compared to the amount of CD147 polypeptide or a fragment thereof in the biological sample of (a) indicates that said multiple myeloma is progressing, and wherein a decreased amount of CD147 polypeptide or a fragment thereof in the biological sample of (b) as compared to the amount in the biological sample of (a) indicates that said MM is entering remission or responding to treatment.

In certain embodiments of this aspect of the invention, the biological sample is a serum sample. In other embodiments, the biological sample is supernatant obtained from culture of the subject's bone marrow mononuclear cells. In still other embodiments, the biological sample is supernatant obtained from culture of the subject's peripheral blood mononuclear cells.

The CD147 polypeptide can be detected using essentially any conventional methodology. For example, in certain embodiments, the CD147 polypeptide or a fragment thereof is detected using a detection system selected from the group consisting of: an immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay.

Accordingly, it will be understood that, in many embodiments of the invention, the CD147 polypeptide or fragment thereof will be detected using an antibody, binding fragment thereof, or any other type of suitable binding agent specific for CD147. In certain specific embodiments, the antibody used in the methods of the invention is a monoclonal or polyclonal antibody that is specific for CD147.

According to another general aspect of the invention, there is provided a method of diagnosing multiple myeloma (MM), comprising: (a) detecting an amount of CD147 polypeptide or a fragment thereof in a biological sample obtained from a subject; and (b) comparing the amount of CD147 polypeptide or fragment thereof detected in step (a) to a predetermined cut-off value or to an amount detected in a control serum sample, wherein an increased amount of CD147 polypeptide or fragment in the biological sample of (a) as compared to the predetermined cut-off value or amount in the control serum sample of (b) indicates the presence of MM.

In certain embodiments of this aspect of the invention, the biological sample is a serum sample. In other embodiments, the biological sample is supernatant obtained from culture of the subject's bone marrow mononuclear cells. In still other embodiments, the biological sample is supernatant obtained from culture of the subject's peripheral blood mononuclear cells.

The CD147 polypeptide can be detected using essentially any conventional methodology. For example, in certain embodiments, the CD147 polypeptide or a fragment thereof is detected using a detection system selected from the group consisting of: an immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay.

Accordingly, it will be understood that, in many embodiments of the invention, the CD147 polypeptide or fragment thereof will be detected using an antibody, binding fragment thereof, or any other type of binding agent specific for CD147. In certain specific embodiments, the antibody used in the methods of the invention is a monoclonal or polyclonal antibody that is specific for CD147.

In still another general aspect of the present invention, there is provided a kit for detecting, diagnosing, predicting survival, staging, or monitoring multiple myeloma (MM) in a patient, comprising a reagent suitable for determining levels of CD147 polypeptide or a fragment thereof in a biological sample obtained from a patient having or suspected of having MM.

In certain embodiments of this aspect of the invention, the kits will comprise components and/or instructions for obtaining the biological sample from the subject, where the biological sample is a serum sample, supernatant obtained from culture of the subject's bone marrow mononuclear cells or supernatant obtained from culture of the subject's peripheral blood mononuclear cells.

In certain other embodiments, the kits of the invention comprise a detection system selected from the group consisting of: an immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay. I n many embodiment, the reagent suitable for determining levels of CD147 polypeptide or a fragment thereof comprises an antibody (such as a monoclonal or polyclonal antibody), a binding fragment thereof, or any other suitable binding agent specific for CD147. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows sCD147 levels in relation to the clinical status of multiple myeloma patients.

FIG. 2 shows sCD147 levels in relation to the clinical status of multiple myeloma patients not in renal failure.

FIG. 3 shows the relationship between sCD147 levels and serum creatinine levels in multiple myeloma patients.

FIG. 4 shows the progression-free survival of multiple myeloma patients based on their determined sCD147 levels.

FIG. 5 shows the progression-free survival of multiple myeloma patients based on their determined sCD147 levels after adjusting for creatinine.

DETAILED DESCRIPTION

The present invention relates generally to the detection of CD147 levels present in the serum of patients having hematological cancer. Without wishing to be bound to a particular theory, it is believed that because high serum CD147 levels were detected in MM patients having progressive disease compared to patients having responsive disease, and because patients having serum CD147 levels above the median of the population have overall shorter survival rates, serum CD147 can be used to provide valuable prognostic information concerning the survival and therapeutic responsiveness of patients diagnosed with MM.

The practice of the invention will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, im munology, and cell biology that are within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A

Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (I RL Press, Oxford, 1985); Anand,

Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992);

Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); and Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1998).

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present invention, the following terms are defined below.

The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms "about" or "approximately" when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%. Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements

Reference throughout this specification to "one embodiment," "an embodiment," "another embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment" or

combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used herein, the term "CD147" is intended to generically refer to both the wild-type and variant CD147 polypeptides, unless specifically denoted otherwise. CD147 polypeptides are encoded by the CD147 gene. As it is commonly used in the art, the term "gene" is intended to refer to the genomic region encompassing 5' untranslated region(s) (UTR), exons, introns, and 3' UTR. Individual segments may be specifically referred to, e.g., promoter, coding region, etc. Combinations of such segments that provide for a complete CD147 protein may be referred to generically as a protein coding sequence. The term "CD147 polypeptide" encompasses an amino acid sequence encoded by an open reading frame (ORF) of a known CD147 polynucleotide, including the full-length native polypeptide and fragments thereof, particularly biologically active fragments and/or fragments corresponding to functional domains, e.g., a region or domain having biological activity, etc.; antigenic fragments thereof, and including fusions of the subject polypeptides to other proteins or parts thereof. The nucleic acid and amino acid sequences of CD147 have been previously described and are well known, (see, e.g., NCBI Reference Sequences M 001728.3, P 001719.2). The CD147 polypeptides of the invention can be isolated from a variety of sources, such as from human tissue types or biological samples such as serum, bone, marrow, or tissue.

The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single- and double-stranded and triple helical molecules. "Oligonucleotide" generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art.

The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art. Nucleic acids may be naturally occurring, e.g., DNA or RNA, or may be synthetic analogs, as known in the art. Such analogs may be preferred for use as probes because of superior stability under assay conditions. Modifications in the native structure, including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-0'-5'-S- phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH2-5'-0-phosphonate and 3'-NH-5'-0- phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage.

The terms "polypeptide" and "protein", used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. In various embodiments, CD147 polypeptides are contemplated for use within diagnostic, prognostic, or monitoring compositions and methods disclosed herein. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

A "substantially isolated" or "isolated" substance is one that is substantially free of its associated surrounding materials in nature. By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of the materials with which it is associated in nature. As used herein, an "isolated" can refer to polynucleotides, polypeptides, cells, samples, and antibodies.

Hybridization reactions can be performed under conditions of different

"stringency". Conditions that increase stringency of a hybridization reaction are widely known and published in the art. See, for example, Sambrook et al. (1989). Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C, 37° C, 50° C. and 68° C; buffer concentrations of lOxSSC, 6xSSC, lxSSC, O.lxSSC (where SSC is 0.15 M NaCI and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6xSSC, lxSSC, O.lxSSC, or deionized water. Examples of stringent conditions are hybridization and washing at 50° C. or higher and in O.lxSSC (9 mM NaCI/0.9 mM sodium citrate).

The term "target cell" includes an individual cell, cell from a biological sample, or cell culture. Target cells include progeny of a single target cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. In particular embodiments, target cells include multiple myeloma cells, bone marrow or peripheral blood mononuclear cells or plasma-B cells.

Multiple myeloma is a B-cell malignancy of mature plasma cell morphology characterized by the neoplastic transformation of a single clone of these types of cells. These plasma cells proliferate in BM and may invade adjacent bone and sometimes the blood. Variant forms of multiple myeloma include overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma (see, for example, Braunwald, et al. (eds), Harrison's Principles of Internal Medicine, 15th Edition (McGraw-Hill 2001)).

The detection systems of the invention are based, in part, on the ability of a binding agent to bind CD147. Generally, the invention contemplates the use of a binding agent that specifically binds CD147, resulting in the formation of a detectable complex of CD147 and binding agent. In one embodiment, the invention utilizes two binding agents, a capture binding agent and a detection binding agent, both of which bind to CD147, resulting in the formation of a ternary complex comprising capture binding agent, CD147, and detection binding agent. In another embodiment, the invention utilizes two binding agents, a capture binding agent, and a detection binding agent, both of which bind to CD147, resulting in the formation of a ternary complex comprising capture binding agent, CD147, and detection binding agent.

Any of a variety of binding agents may be used, including, for example, polypeptides, sugars, and nucleic acids. In yet another embodiment, the invention further includes the use of an additional binding agent that binds to the detection binding agent. Such an additional binding agent may be useful, e.g., in detecting bound detection binding agent. Accordingly, one example of such an additional binding agent is antibodies specific for a fragment of an antibody, e.g., an F, fragment, which may be detectably labeled and, therefore used to detect bound detection binding agent, and are particularly useful when the detection binding agent is not itself easily amenable to labeling. In certain embodiments, the binding agent is an antibody specific for bacteria.

The term "binds specifically," in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific polypeptide i.e., epitope of a CD147 polypeptide. Antibody binding to an epitope on a specific polypeptide (also referred to herein as "an epitope") is preferably stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest, e.g., binds more strongly to a specific

CD147epitope than to a different CD147 epitope or non- CD147 epitope. Antibodies which bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less, 5% or less, 1% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g. by use of appropriate controls. In general, antibodies used in compositions and methods of the invention which bind to a specific CD147 polypeptide with a binding affinity of 107 moles/L or more, preferably 108 moles/L or more are said to bind specifically to the specific CD147 polypeptide. In general, an antibody with a binding affinity of 106 moles/L or less is not useful in that it will not bind an antigen at a detectable level using conventional methodology currently used.

In one embodiment, the affinity of specific binding of a CD147 binding agent to CD147 is about 2 times greater than background binding, about 5 times greater than

background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

In another embodiment, the affinity of specific binding is between about 2 to about 1000 times greater than background binding, between about 2 to 500 times greater than background binding, between about 2 to about 100 times greater than background binding, between about 2 to about 50 times greater than background binding, between about 2 to about 20 times greater than background binding, between about 2 to about 10 times greater than background binding, between about 5 to about 100 times greater than background binding, between about 5 to about 50 times greater than background binding, between about 5 to about 20 times greater than background binding, between about 10 to about 100 times greater than background binding, between about 10 to about 50 times greater than

background binding, between about 50 to about 500 times greater than background binding, or any intervening range of affinity.

Accordingly, specific binding occurs between a binding agent and CD147 where there is an interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction. In a particular embodiment, specific binding is characterized when one member of a pair substantially binds to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. In another particular embodiment, specific binding is characterized when one member of a pair substantially binds to one or more particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. In another particular embodiment, specific binding is characterized when one member of a pair substantially binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs.

Generally speaking, the binding affinity of a binding agent of the invention (A) to CD147 (B) can be generally expressed by the chemical equilibrium constant Kd resulting from the following reaction: [A]+[B]-[AB]. The chemical equilibrium constant Kd is then given by: Kd=[A]x[B]/[AB]. Whether the binding of a binding agent is specific or not can be judged from the difference between the binding affinity (Kd value) of the binding agent to CD147, versus the binding to another polypeptide.

Kd values and differences in Kd values can be measured using, for example, in vitro or in vivo binding assays and/or assays on other materials such as a polystyrene microtitre plate or a specialized surface in an analytical biosensor. In one embodiment, the difference between the Kd value of a binding agent to CD147, versus the binding to an undesired polypeptide is 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 50 fold, 100 fold, 1000 fold, or more.

In another embodiment, the Kd value is less than 10"4 M, less than 10"5 M, less than 10"6 M, less than 10"7 M, less than 10"8 M, less than 10"9 M, less than 10"10 M and could be 10"11 M, less than 10"12 M, less than 10"13 M, less than 10"14 M, less than 10"15 M or less.

In another embodiment, the Kd value is between about 10"4 M and about 10"15 M, between about 10"4 M and about 10"12 M, between about 10"4 M and about 10"10 M, between about 10"6 M and about 10"15 M, between about 10"6 M and about 10"12 M, between about 10"6 M and about 10"10 M, between about 10"8 M and about 10"15 M, between about 10"8 M and about 10"12 M, between about 10"8 M and about 10"10 M, between about 10"7 M and about 10"10 M, or any intervening range of affinity

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. In one embodiment, the monoclonal antibody is an anti- CD147 monoclonal antibody.

Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991), for example.

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567;

Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Methods of making chimeric antibodies are known in the art.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F (ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.

For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and no more than 3 in the L chain. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992). The humanized antibody includes a PRIMATIZED antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest. Methods of making humanized antibodies are known in the art.

Human antibodies can also be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1): 86-95 (1991).

"Functional fragments" of the binding antibodies of the invention are those fragments that retain binding to antigen with substantially the same affinity as the intact full chain molecule from which they are derived.

An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or

nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present.

Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The terms "detectably labeled antibody" refers to an antibody (or antibody fragment which retains binding specificity for a CD147 polypeptide or epitope), having an attached detectable label. The detectable label is normally attached by-chemical conjugation, but where the label is a polypeptide, it could alternatively be attached by genetic engineering techniques. Methods for production of detectably labeled proteins are well known in the art. Detectable labels may be selected from a variety of such labels known in the art, including, but not limited to, haptens, radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate. Various detectable label/substrate pairs (e.g., horseradish

peroxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin)), methods for labeling antibodies, and methods for using labeled antibodies are well known in the art (see, for example, Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)).

In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support. In one embodiment, the antibody is an anti-CD147 polyclonal antibody.

A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived there from and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, urine, cerebral spinal fluid, biological fluid, and tissue samples. The sample may be pretreated as necessary by dilution in an appropriate buffer solution or concentrated, if desired. Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, preferably at physiological pH can be used. Biological samples can be derived from patients using well known techniques such as venipuncture, lumbar puncture, fluid sample such as saliva or urine, or tissue biopsy and the like.

As used herein, the terms "correlated with" or "associated with" refer to the levels of CD147 in a biological sample of a subject that has a statistically significant correlation with a physiologic state, e.g., disease status or extent of the disease, response to treatment, and survival. The strength of the correlation between CD147 levels and the presence or absence of a particular physiologic state may be determined by a statistical test of significance. Methods for determining the strength of a correlation between the expression level of a differentially-expressed gene and a particular physiologic state by assigning a statistical score to the correlation are reviewed in Holloway et al. (2002) Nature Genetics Suppl. 32:481-89, Churchill (2002) Nature Genetics Suppl. 32:490-95, Quackenbush (2002) Nature Genetics Suppl. 32: 496-501; Slonim (2002) Nature Genetics Suppl. 32:502-08; and Chuaqui et al. (2002) Nature Genetics Suppl. 32:509-514; each of which is herein incorporated by reference in its entirety.

A "conjugate" refers to any molecule, e.g., antibody bound or joined covalently or non-covalently to another molecule, e.g., a hapten, small molecule, or label, including fusion proteins and as well as molecules that contain both amino acid or protein portions and nonprotein portions. Conjugates may be synthesized by a variety of techniques known in the art including, for example, solid phase synthesis, solution phase synthesis, organic chemical synthetic techniques or a combination of these techniques. The choice of synthesis will depend upon the particular molecule to be generated.

The terms "individual," "subject," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets. The term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal herein is human. B. Methods of Diagnosis, Prognosis and Monitoring

The present inventors have discovered that CD147 levels are increased in the serum of MM patients compared to normal healthy subjects. Accordingly, particular embodiments of the invention provide methods and compositions for the diagnosis of MM, prognosis of survival in patients diagnosed with MM, as well as monitoring the response of the disease to treatment, based upon the level of CD147 observed in a biological sample obtained from a patient, including, e.g., a patient's bloodstream, serum, bone marrow, or tissue. A variety of methods of determining CD147 levels are known and available in the art. In certain embodiments, these involve the use of a CD147 binding agent, such as a CD147 specific antibody. As discussed elsewhere herein, there are a variety of assay formats known to those of ordinary skill in the art and suitable for using a binding agent to detect polypeptide markers in a sample. E.g., ELISA assays, lateral flow assays, etc.; see also, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

In general, MM is diagnosed by the presence of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or higher levels of CD147 as compared to those in a normal control subject. In general, methods of diagnosing MM comprise: (a) detecting an amount of CD147 in a biological sample, e.g., serum, obtained from a subject; and (b) comparing the amount detected in step (a) to a predetermined cut-off value or to an amount detected in a control biological sample, wherein an increased amount of CD147 in the biological sample of (a) as compared to the predetermined cut-off value or amount in the control biological sample of (b) indicates the presence of MM, and diagnosing the subject with MM.

In one embodiment, a method of prognosis for the survival of a subject having

MM is provided. In general, a subject diagnosed with and/or treated for MM and having an amount of serum CD147 detected that is more than a median value of serum CD147 detected in a population of subjects being treated for MM has a poorer chance of survival compared to the subjects in the population having less than or equal to the median serum CD147 levels. In particular embodiments, a subject having serum CD147 levels greater than the median value in a population being treated for MM has a reduced chance of survival of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or more.

In general, a method of prognosis for the survival of a subject having MM comprises: (a) detecting an amount of CD147 in a biological sample, e.g., serum, obtained from a subject; and (b) comparing the amount detected in step (a) to a the median value of serum CD147 levels in a population of subjects being treated for MM (e.g., a prognostic cut-off value), wherein an increased amount of CD147 in the biological sample of (a) as compared to the median value or corresponding amount in the treated population of (b) indicates a reduced chance of survival for the subject having MM.

Being able to predict the reduced chance of survival is advantageous because it allows the clinician to change the therapeutic course in the hopes of increasing the chance of survival of the subject. As such, the methods of the present invention offer important advantages in facilitating a determination by a medical practitioner of a suitable treatment plan for a subject having MM.

In a certain embodiment, a method of monitoring the progression or response to treatment of MM is provided. A method of monitoring the progression or response to treatment of MM comprises: (a) detecting an amount of CD147 in a biological sample, e.g., serum, obtained from a subject diagnosed with MM at a first time point; (b) detecting an amount of CD147 in a biological sample obtained from the subject at a second time point or following treatment; and (c) comparing the amount detected in step (a) to the amount detected in step (b), wherein an increased amount of CD147 in the biological sample of (b) as compared to the amount in the biological sample of (a) indicates that said MM is progressing, and wherein a decreased amount of CD147 in the biological sample of (b) as compared to the amount in the biological sample of (a) indicates that said MM is entering remission or responding to treatment.

In various embodiments of methods of detecting CD147, a biological sample is selected from the group consisting of: serum, bone marrow, and tissue. In particular embodiments, mRNA levels are determined, while in other preferred embodiments, polypeptide levels are determined. In one embodiment, detection is performed using one or more primers specific for CD147. In another preferred embodiment, detection is performed using an antibody specific for CD147.

In one embodiment, the presence or absence of MM in a patient may be determined by (a) contacting a biological sample obtained from a patient with a CD147 binding agent; (b) detecting in the sample a level of CD147 polypeptide that binds to the binding agent; and (c) comparing the level of CD147 polypeptide with a predetermined cut-off value or with the value obtained from a normal control subject. In certain embodiments, the cut-off value for the detection of a MM is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without MM.

In particular embodiments, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for MM. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for MM.

In one embodiment, the assay involves the use of a CD147 binding agent immobilized on a solid support to bind to and remove the CD147 polypeptide from the remainder of the sample. The bound CD147 polypeptide may then be detected using a detection reagent that contains, for example, a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the CD147 polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an antiimmunoglobulin, protein G5 protein A or a lectin.

In a related embodiment, the assay is performed in a lateral flow or strip test format, as discussed elsewhere herein, wherein the CD147 binding agent, e.g., antibody, is immobilized on a membrane, such as nitrocellulose. In the lateral flow test, CD147 polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the CD147 binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which CD147 binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. Concentration of second binding agent at the area of immobilized antibody indicates the presence of MM.

The invention provides similar methods for staging or monitoring the progression of MM, as well as determining response to treatment. Since serum CD147 levels correlate with severity or extent of the disease, levels associated with particular stages are determined and compared to those observed in a subject's serum to determine the stage of the subject's disease. Similarly, disease progression and response to treatment or therapy is monitored by comparing CD147 levels in a subject's serum (or other biological sample) at different time points during the course of the disease or before and after a treatment regimen. In particular embodiments, CD147 serum levels are elevated in MM patients, and the levels of CD147 correlate with disease stage, i.e., CD147 levels are higher in progressive MM and become lower in response to treatment or entering remission. Thus, the present invention provides a rapid and reliable method of detecting, diagnosing, prognosis, staging, and monitoring progression or response to treatment of MM, using a serum sample obtained from the subject's bloodstream. In particular embodiments, the method is practiced by ELISA assay, lateral flow assay, or strip test assay using an antibody specific for CD147. The invention further provides systems and kits for detecting, diagnosing, prognosing, staging, or monitoring multiple myeloma, which comprise reagents suitable or determining CD147 levels in a biological, e.g., serum, sample obtained from a subject. In one embodiment, the kit includes reagents for performing ELISA, lateral flow, or strip test assays such as an antibody specific for CD147. Detection systems and kits of the invention are described in further detail below.

C. Detection Systems and Kits

In various embodiments, the present invention provides detection systems and kits for MM. A detection system or kit of the present invention may be used for diagnosis, prognosis, or monitoring of MM patients in a biological sample, e.g., serum, of a subject. The diagnostic kit could include the method for the detection of antigen-antibody reaction in addition to the material. The detection method is preferably selected from the group consisting of flow cytometry, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), radioimmunoassay ( IA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assays and strip assay. The reactivity of the antigen recognition material could be confirmed using device detecting an enzyme reaction, fluorescence, luminescence, or radiation. In one embodiment, the diagnosis, prognosis, or monitoring of MM can be made with a flow cytometry kit, immunohistochemistry kit, ELISA kit or lateral flow or strip kit including the anti-CD147 antibody or an antigen binding fragment thereof.

In one embodiment, a kit or system may comprise one or more or all of the following components: 1) one or more standards comprised of one or more of the biomarker(s) of the invention, such as CD147; 2) a binding agent, such as an antibody or a plurality of antibodies, that are specific for the biomarker(s) that are to be assayed for using the kit; 3) written instructions; 4) diluents for samples and the standards; 5) a wash buffer; 6) color reagents; 7) stop solution; and 8) a carrier, such as an antibody carrier, for example, a lateral flow device, or a microplate with bound antibody, or polystyrene beads.

In one embodiment, the detection system or kit used to diagnose, prognose, or monitor MM is a quantitative ELISA (enzyme-linked immunosorbent assay) that determines the concentration or concentrations of the biomarker or biomarker(s) in accordance with methods embodied by the invention. The principle of the assay is to use the quantitative sandwich enzyme immunoassay technique wherein a monoclonal or polyclonal antibody selective for a biomarker is pre-coated onto a carrier such as a microplate into its wells. The standards and sample are then pipetted into the wells and any of the biomarker that is present is bound to this immobilized antibody. Next, the wells are washed with washing buffer, and an enzyme- linked monoclonal or polyclonal antibody that is specific for the biomarker is added to the wells. Washing is again performed, then a substrate solution is added to the wells. Color subsequently develops in proportion to the amount of polypeptide of the invention that is bound in the first step. The color development is stopped using a stop solution, and the intensity of the color is measured by a microplate reader.

In other embodiments, the diagnosis, prognosis, or monitoring of MM may be carried out using, for example, a lateral flow assay. Such lateral flow assays have the potential to be a cost-effective, fast, simple, and sensitive method, for instance for on-site screening assays. The lateral flow assay comprises a carrier that allows a lateral flow to occur wherein either the sample or the detection reagent is displaced form one location on the carrier to another. There are many formats of lateral flow assays suitable for use in a method embodied by the invention, and the skilled person will readily know how to select and optimize a particular format. An example of a lateral flow test strip of the invention comprises, for example, the following components: sample pad; an absorbent pad onto which the test sample is applied; a conjugate or reagent pad that contains antibodies specific to the target analyte and conjugated to colored particles (usually colloidal gold particles, or latex microspheres); a reaction membrane, typically a hydrophobic nitrocellulose or cellulose acetate membrane onto which anti-target analyte antibodies are immobilized in a line across the membrane as a capture zone or test line (a control zone may also be present, containing antibodies specific for the conjugate antibodies); and a wick or waste reservoir, a further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it.

There are a number of variations on lateral flow technology. The capture zone on the membrane may contain immobilized antigens or enzymes depending on the target analyte rather than antibodies. It is also possible to apply multiple capture zones to create a multiplex test. For example, in particular embodiments, test strips able to detect CD147 and separately in the same sample additional biomarkers of multiple myeloma, e.g., β2Μ, IL-6, C-reactive protein, and serum monoclonal protein are contemplated. Lateral flow immunoassays are simple to use by untrained operators and generally produce a result within 15 minutes. They are very stable and robust, have a long shelf life and do not usually require refrigeration. They are also relatively inexpensive to produce. These features make them ideal for use at the point- of-care and for testing samples in the field, as well as in the laboratory.

While most lateral flow immunoassays are only capable of providing a qualitative result, it is possible to obtain some degree of quantification by measuring the amount of conjugate bound to the capture zone. This can be done using a dedicated reader to measure the intensity of the colored test line. For example, the Neogen Corporation has developed the Accuscan™ lateral flow reader for use with its range of Reveal^® assay kits and Charm Sciences also supplies a reader for its Rosa^® range of test strips. More sophisticated techniques, such as fluorescent dye labeled conjugates, have also been developed to improve the quantitative potential of lateral flow assays.

A detection system in kit form can include, for example, in an amount sufficient for at least one assay and an antibody composition or monoclonal antibody composition that binds a serological biomarker for MM, as a packaged reagent. Instructions for use of the packaged reagent are also typically included.

A detection system in kit form can also include, for example, a means for combining the test sample with a buffering system (Reagent 1) containing viscosity controllers and stabilizers into a reaction vessel and mixing the solution. A detection system in kit form can also include a means for reading the a parameter of the reaction vessel with sample and buffer, and further means for combining the test sample and buffer mixture with a fluorescence- labeled ligand (Reagent 2) to said biological substance in the reaction vessel, mixing the solution to produce an assay solution. Furthermore, Reagent 2 may be delivered to the reaction vessel without further dilution volume of the assay solution. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits an antibody composition or monoclonal antibody composition. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide or antibody have been operatively affixed.

"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.

In particular embodiments, a detection system of the present invention further includes a label or indicating means capable of signaling the formation of a complex containing a polypeptide or antibody molecule of the present invention.

"Complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are

immunoreaction products.

As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis, et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.

In certain embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as

diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-azino-di-(3-ethyl- benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 1241, 1251, 1281, 1321 and 51Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 1251. Another group of useful labeling means are those elements such as 11C, 18F, 150 and 13N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such lllindium or 3H.

The linking of labels, i.e., labeling of, polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795, which are all incorporated herein by reference. The detection systems or kits of the present invention can be used in an "ELISA" format to detect, for example, the presence or quantity of CD147 in a body fluid sample such as the bloodstream, serum, bone marrow, or tissue, etc. "ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen or antibody present in a sample. Thus, for example, a polypeptide, antibody molecule composition or monoclonal antibody molecule composition of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems. The reagent is typically affixed to the solid matrix by adsorption from an aqueous medium although other modes of affixation, well known to those skilled in the art, can be used.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include cross-linked dextran; agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifying reagent of any detection system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this detection assay system.

The packaging materials discussed herein in relation to detection systems are those customarily utilized in diagnostic systems. Such materials include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic-foil laminated envelopes and the like. In one embodiment, a detection system of the present invention is useful for assaying for the presence of, for example, CD147. Such a system comprises, in kit form, a package containing an antibody to, for example, CD147. All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES

Example 1

Serum CD147 Levels are Increased in Multiple Myeloma Patients and Elevated Levels are Associated with Refractory Disease and Shortened Progression Free Survival

Serum CD147 (sCD147) levels were analyzed in relation to renal function, response to treatment and progression free survival (PFS) among multiple myeloma patients. Serum samples were collected from 152 multiple myeloma patients who were treated at a single clinic. Levels of sCD147 were analyzed using an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). Mann-Whitney analysis was used to analyze the relationship between sCD147 and clinical status. A subset of patients (n=62) had their sCD147 levels measured immediately prior to starting a new treatment regimen. Using Kaplan-Meier analysis, PFS was compared between patients with sCD147 levels above or below 5.00 ng/mL. Levels of sCD147 were measured from patients at the start of treatment who achieved a complete response (CR) and among those with progressive disease (PD) without achieving any response (refractory disease (RD)). In addition, sCD147 levels were also assessed among patients in CR and those with PD at the time of their sample blood draw. The Spearman correlation coefficient was used to correlate levels of serum creatinine with sCD147. Previous studies have shown that median sCD147 levels in healthy donors is about 2.5 ng/mL and invariably below 4.5 ng/mL, which is consistent with our observations in samples from healthy donors (all <4.50 ng/mL). At the time of sampling, the median level of sCD147 among patients with MM who were in CR (n=49, median=3.69 ng/mL) was not different compared with the median level of among patients with PD (n=74, median=3.86 ng/mL;

P=0.3525) (FIG. 2). Overall, patient's sCD147 levels assessed prior to the initiation of treatment were not different among those achieving CR (n=16, median=3.79 ng/mL) compared with those showing RD (n=46, median=4.06 ng/mL; P=0.3895). Consistent with data obtained on pts with AKI from other causes, levels of sCD147 were directly correlated with serum creatinine levels in MM pts (n=136, Spearman correlation coefficient <0.0001) (FIG. 3).

Among pts without renal failure, only 1/52 (2%) of pts in CR at the time of blood draw showed a sCD147 level > 6.00 ng/mL (6.39 ng/mL) whereas 21/97 (22%) of pts with RD showed a level above this threshold (range, 6.01-17.20 ng/m L). Notably, patients with sCD147 levels drawn prior to the start of treatment that were above the maximum value for all normal donors (sCD147 levels >5.00 ng/mL; n=34) showed a much shorter PFS (median=2.11 months) compared with those with levels below this level (n=81; median=4.64 months; P=.0204) (FIG. 4). When PFS was determined among MM pts without renal failure, there remained a difference among those with levels > 5.00 ng/mL compared with those with levels below this threshold (5.3 vs 2.1 months; P=0.04) (FIG. 5).

These findings confirm that CD147 represents a valuable prognostic indicator for

MM. Notably, sCD147 levels > 6.00 ng/mL predict for RD, and baseline levels elevated above the threshold of healthy donors (> 5.00 ng/mL) also predict for a markedly shorter PFS among all MM pts or those without renal failure. This demonstrates that sCD147 levels > 5.00 ng/mL at baseline predict poor outcome for MM pts and that CD147 represents a new prognostic factor for MM patients and possibly those with other hematologic malignancies.

I n general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. A method of prognosis for the survival of a subject having multiple myeloma (MM) comprising: (a) detecting an amount of CD147 polypeptide or a fragment thereof in a biological sample obtained from a subject; and (b) comparing the amount of CD147 polypeptide or fragment thereof detected in step (a) to a predetermined prognostic cut-off value, wherein an amount of CD147 polypeptide or fragment thereof in the biological sample that exceeds the cut-off value indicates a reduced chance of survival for the subject having MM.

2. The method of claim 1, where the biological sample is a serum sample.

3. The method of claim 1, where the biological sample is supernatant obtained from culture of the subject's bone marrow mononuclear cells.

4. The method of claim 1, where the biological sample is supernatant obtained from culture of the subject's peripheral blood mononuclear cells.

5. The method of claim 1, where the CD147 polypeptide or a fragment thereof is detected using a detection system selected from the group consisting of: an

immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay.

6. The method of claim 1, where CD147 polypeptide or fragment thereof is detected using an antibody or binding fragment thereof specific for CD147 polypeptide or a fragment thereof.

7. The method of claim 6, where the antibody is a monoclonal or polyclonal antibody.

8. The method of claim 1, where an amount of CD147 polypeptide or fragment thereof greater than about 5 ng/mL indicates a reduced chance of survival for the subject having MM.

9. The method of claim 1, where an amount of CD147 polypeptide or fragment thereof greater than about 6 ng/mL indicates the presence of refractory disease in the subject having MM.

10. A method of monitoring the progression or response to treatment of multiple myeloma (MM), comprising: (a) detecting an amount of CD147 polypeptide or a fragment thereof in a biological sample obtained from a patient diagnosed with MM at a first time point; (b) detecting an amount of CD147 polypeptide or fragment thereof in a biological sample obtained from the patient at a second time point or following treatment; and (c) comparing the amount detected in step (a) to the amount detected in step (b), wherein an increased amount of CD147 polypeptide or a fragment thereof in the biological sample of (b) as compared to the amount of CD147 polypeptide or a fragment thereof in the biological sample of (b) indicates that said multiple myeloma is progressing, and wherein a decreased amount of CD147 polypeptide or a fragment thereof in the biological sample of (b) as compared to the amount in the biological sample of (a) indicates that said MM is entering remission or responding to treatment,

11. The method of claim 10, where the biological sample is a serum sample.

12. The method of claim 10, where the biological sample is supernatant obtained from culture of the subject's bone marrow mononuclear cells.

13. The method of claim 10, where the biological sample is supernatant obtained from culture of the subject's peripheral blood mononuclear cells.

14. The method of claim 10, where the CD147 polypeptide or a fragment thereof is detected using a detection system selected from the group consisting of: an immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay.

15. The method of claim 10, where CD147 polypeptide or fragment thereof is detected using an antibody or binding fragment thereof specific for CD147 polypeptide or a fragment thereof.

16. The method of claim 15, where the antibody is a monoclonal or polyclonal antibody.

17. A method of diagnosing multiple myeloma (MM), comprising: (a) detecting an amount of CD147 polypeptide or a fragment thereof in a biological sample obtained from a subject; and (b) comparing the amount of CD147 polypeptide or fragment thereof detected in step (a) to a predetermined cut-off value or to an amount detected in a control serum sample, wherein an increased amount of CD147 polypeptide or fragment in the biological sample of (a) as compared to the predetermined cut-off value or amount in the control serum sample of (b) indicates the presence of MM.

18. The method of claim 17, where the biological sample is a serum sample.

19. The method of claim 17, where the biological sample is supernatant obtained from culture of the subject's bone marrow mononuclear cells.

20. The method of claim 17, where the biological sample is supernatant obtained from culture of the subject's peripheral blood mononuclear cells.

21. The method of claim 17, where the CD147 polypeptide or a fragment thereof is detected using a detection system selected from the group consisting of: an immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay.

22. The method of claim 17, where CD147 polypeptide or fragment thereof is detected using an antibody or binding fragment thereof specific for CD147 polypeptide or a fragment thereof.

23. The method of claim 22, where the antibody is a monoclonal or polyclonal antibody.

24. A kit for detecting, diagnosing, predicting survival, staging, or monitoring multiple myeloma (MM) in a patient, comprising a reagent suitable for determining levels of CD147 polypeptide or a fragment thereof in a biological sample obtained from a patient having or suspected of having MM.

25. The kit of claim 24, where the biological sample is a serum sample.

26. The kit of claim 24, where the biological sample is supernatant obtained from culture of the subject's bone marrow mononuclear cells.

27. The kit of claim 24, where the biological sample is supernatant obtained from culture of the subject's peripheral blood mononuclear cells.

28. The kit of claim 24, where the kit comprises a detection system selected from the group consisting of: an immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), lateral flow assay, or strip assay.

29. The kit of claim 24, where the reagent suitable for determining levels of CD147 polypeptide or a fragment thereof comprises an antibody or a binding fragment thereof specific for CD147.

30. The kit of claim 29, where the antibody is a monoclonal or polyclonal antibody.

31. The method of any one of claims 1-23, wherein the results of the method facilitate a determination by a medical practitioner of a suitable treatment plan for the subject having MM.