Anti- enolase 1 (ENO1) antibody and applications thereof

Abstract

This invention relates to antibodies that bind to ENO1 and their applications. These applications encompass the use of such antibodies to treat and diagnose diseases or conditions associated with ENO1 activation and its progression. Specifically, the antibodies of this invention bind to ENO1 on the surface of cancer cells and can be used to reduce cancer cell growth and metastasis and prolong survival. The antibodies of this invention can also be used to detect ENO1, diagnose and prognose cancer, and monitor cancer progression. This invention also provides methods for screening candidate agents for cancer treatment.

Description

[Technical Field]

[0001] Related Application. This application claims the benefit of U.S. Provisional Application No. 63 / 164,137, filed March 22, 2021, pursuant to 35 USC §119, which is incorporated herein by reference in its entirety.

[0002] This invention relates to an antibody that binds to enolase 1 (ENO1) and its applications. Specifically, the antibody of this invention binds to ENO1 on the surface of cancer cells and can effectively reduce cancer cell growth and metastasis and prolong survival time. The antibody of this invention can also be used to detect ENO1, diagnose and prognose cancer, and monitor cancer progression. This invention also provides a method for screening candidate drugs for cancer treatment. [Previous Technology]

[0003] Enolase 1 (ENO1), also known as α-enolase, is a metabolic enzyme involved in pyruvate synthesis. It is also known as a plasminogen receptor and mediates plasmin activation and extracellular matrix degradation. Based on these mechanisms, ENO1 contributes to a variety of cellular and physiological processes, such as proteolysis, extracellular matrix remodeling, metabolism, growth control, tumor metastasis, and allergic reactions.

[0004] Overexpression of ENO1 mRNA or protein has been observed in many different tumor types, including lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer.3,4 In addition to its role as a glycolytic enzyme, ENO1 is also expressed on the cell surface of most tumors, where it forms a multi-protein complex with urokinase-type plasminogen activator receptor (uPAR), integrins, and certain cytoskeletal proteins responsible for adhesion, migration, and proliferation.5 In tumors, ENO1 can regulate intravascular and pericellular fibrinolytic activity to promote cell migration and cancer metastasis.6–8 Furthermore, the concentration of expressed ENO1 can serve as a valuable diagnostic biomarker because it is associated with patient outcomes such as cancer survival and prognosis.9–11 For example, in patients with non-small cell lung cancer (NSCLC), those with tumor cells exhibiting high concentrations of ENO1 have relatively poor disease-free survival and overall survival.12 Recent findings have shown that targeting ENO1 may be a novel approach to overcoming resistance.13 Therefore, tumor-associated antigen (TAA)-ENO1 has become an interesting target for cancer therapy.

[0005] Epithelial-mesenchymal transition (EMT) is a cellular process that is naturally present in a wide range of tissue types and developmental stages.14 However, based on the contributions of EMT to organ fibrosis,15,16 therapy resistance,17 inflammation,18,19 immunosuppression,20,21 and metastatic cell dissemination,22 it is clear that EMT is also important in the pathogenesis of many human diseases and cancers. Furthermore, increasing evidence suggests that EMT is involved in many tumor metastasis processes, including local invasion of tumor cells at the primary tumor site, intravascular infiltration, circulation via vascular distribution, extravasation into the parenchyma of distal tissues, and survival of micrometastatic deposits.23,24 To differentiate cells at the epithelial and mesenchymal axes, researchers typically examine certain cellular characteristics; epithelial cells exhibit epithelial cell-cell junctions, apical-basal polarity, and lack of motility, while mesenchymal cells exhibit higher motility, invasiveness, resistance to apoptosis, and a spindle-shaped morphology lacking apical-basal polarity.25 EMT is typically detected as a morphological shift from an epithelial to a stromal phenotype, involving the loss of epithelial markers (e.g., E-cadherin, α-catenin, and γ-catenin) and the gain of stromal markers (e.g., vimentin, N-cadherin, and fibronectin); this shift is mediated by EMT-activating transcription factors (EMT-TFs) such as SLUG, SNAIL, and TWIST.23,26 Therefore, the pleiotropic role of EMT-TFs in cancer formation, growth, and metastasis is well-known, which is a key reason for their association with poor clinical outcomes in many epithelial tumor types.

[0006] Wnt signaling is a key pathway in embryonic development and adult cell damage and repair processes. After Wnt ligands bind to their receptors, the β-catenin disruption complex inhibits the phosphorylation of cytosolic β-catenin. Unphosphorylated β-catenin accumulates in the cytosolic layer and translocates to the nucleus, where it activates the expression of Wnt target genes. This gene regulation requires integration with the TCF / LEF family of transcription factors and is important in many cases (including cancer progression) for controlling cell proliferation, differentiation, and survival.27,28 In addition to its role in the nucleus, E-cadherin also forms a complex with β-catenin on the cell surface, which helps maintain cell-cell junctions.29 Similar to Wnt ligand binding, negative regulation of E-cadherin can also increase the concentration of β-catenin in the cytoplasm and nuclear compartments, stimulating cell growth and providing a mechanical connection to the EMT.30

[0007] Another important signaling pathway controlling EMT is stimulated by the hepatocyte growth factor receptor (HGFR; also known as c-Met). HGFR and its ligand HGF are involved in many normal and pathological biological processes, such as fetal development of the liver, placenta, muscles, and nervous system.31,32 After birth, activation of the HGF-HGFR pathway appears to be involved in EMT,33 as well as liver, kidney, and epidermal regeneration.31 Importantly, HGFR signaling also promotes tumor growth, invasion, drug resistance, angiogenesis, and especially the generation and maintenance of cancer stem cells. Activation of the HGFR signaling pathway can occur through several different mechanisms, including increased HGF expression, enhanced HGFR protein expression, or by altering other factors or pathways that affect HGFR activation.34 After HGFR activation, phosphorylation of its C-terminal tail generates recruited intracellular transtransferons and multifunctional acceptor sites (PI3K, Src, etc.).35 Therefore, several pathways involving proliferation, survival, cell activity, invasion, and metastasis can be activated through this key messaging pathway.

[0008] Our previous findings suggest that ENO1-targeted liposomes loaded with anticancer drugs have great potential as a targeted drug delivery system for cancer treatment.36 However, the detailed signaling mechanisms by which ENO1 participates in carcinogenicity remain unclear, and to date, no anti-ENO1 drug candidates have undergone clinical trials. Effective anti-ENO1 antibodies are still needed in this field. [Summary of the Invention]

[0009] This article discloses antibodies that bind to ENO1 and their applications. Cells that can be targeted by antibodies include cancer cells. Applications include, but are not limited to, cancer treatment and cancer diagnosis and prognosis. Specifically, antibodies can be used to reduce cancer cell growth and metastasis and prolong survival time. Antibodies can also be used to detect ENO1 and for the diagnosis and prognosis of cancer, as well as to monitor cancer progression in cancer patients.

[0010] Therefore, in one aspect, the present invention provides an anti-ENO1 antibody or an antigen-binding fragment thereof, comprising: (a) a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HC CDR1) containing an amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity-determining region 2 (HC CDR2) containing an amino acid sequence of SEQ ID NO: 4, and a heavy chain complementarity-determining region 3 (HC CDR3) containing an amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LC CDR1) containing an amino acid sequence of SEQ ID NO: 9, a light chain complementarity-determining region 2 (LC CDR2) containing an amino acid sequence of SEQ ID NO: 11, and a light chain complementarity-determining region 3 (LC CDR3) containing an amino acid sequence of SEQ ID NO: 13.

[0011] In some specific embodiments, VH contains the amino acid sequence of SEQ ID NO:15, and / or VL contains the amino acid sequence of SEQ ID NO:16.

[0012] Also provided is a recombinant nucleic acid comprising a nucleotide sequence encoding an anti-ENO1 antibody or antigen-binding fragment as described herein, and a host cell comprising the recombinant nucleic acid.

[0013] Further, a composition comprising an anti-ENO1 antibody or an antigen-binding fragment thereof as described herein and a physiologically acceptable carrier is provided.

[0014] In another aspect, the present invention provides a method for inhibiting ENO1 downstream signaling and / or treating diseases or conditions associated with activation of ENO1 downstream signaling, by administering to an individual in need an effective amount of an anti-ENO1 antibody or its antigen-binding fragment or a composition comprising an anti-ENO1 antibody or its antigen-binding fragment as described herein.

[0015] In some specific embodiments, the disease or symptom is cancer and its metastasis.

[0016] In some specific embodiments, the cancer is selected from the group consisting of: lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer. In one specific example, the cancer is lung cancer.

[0017] In a specific embodiment, the antibody or its antigen-binding fragment as described herein is administered in an amount that effectively inhibits tumor growth and metastasis in individuals in need.

[0018] In a specific embodiment, an antibody or its antigen-binding fragment as described herein is administered in an amount that effectively prolongs the survival time of the individual in need.

[0019] In another aspect, the present invention provides a method for detecting ENO1, comprising: (i) contacting an antibody or an antigen-binding fragment thereof as described herein with cells of an individual in need; and (ii) detecting the binding of the antibody or the antigen-binding fragment thereof to the cells.

[0020] In some embodiments, the method is performed in vitro, wherein the cells are present in a biological sample obtained from an individual. In some embodiments, the method is performed in vivo, wherein the cells are present in the individual.

[0021] In some specific embodiments, an individual is at risk of cancer or is suspected of having cancer.

[0022] In some specific embodiments, the individual has cancer.

[0023] In some specific embodiments, the method for detecting ENO1 as described herein further includes comparing the detection result with a reference concentration and determining the prognosis of an individual with cancer based on the comparison result, wherein an elevated ENO1 concentration indicates a negative prognosis.

[0024] In a specific embodiment, negative prognosis is selected from the group consisting of: reduced survival rate, increased tumor growth, increased risk of metastasis, increased risk of recurrence, and any combination thereof.

[0025] In another aspect, the present invention provides a method for monitoring cancer progression in a patient with cancer, comprising: (a) providing a first biological sample from the patient at a first time point; (b) providing a second biological sample from the patient at a second time point, the second time point being later than the first time point; (c) detecting the concentration of ENO1 in the first and second biological samples, wherein the detection is performed by immunoassay of an anti-ENO1 antibody or its antigen-binding fragment as described herein; and (d) determining cancer progression in the patient based on the ENO1 concentration in the first and second biological samples, wherein an increased ENO1 concentration in the second biological sample compared to the first biological sample indicates cancer progression.

[0026] This invention also describes an anti-ENO1 antibody or an antigen-binding fragment thereof, or a composition comprising the same antibody or an antigen-binding fragment thereof as described herein, for inhibiting ENO1 downstream signaling and / or treating diseases or conditions associated with ENO1 downstream signaling activation. Further disclosed is the use of an anti-ENO1 antibody or an antigen-binding fragment thereof, or a composition comprising the same antibody or an antigen-binding fragment thereof as described herein, for the preparation of an agent for inhibiting ENO1 downstream signaling and / or treating diseases or conditions associated with ENO1 downstream signaling activation. Examples of such diseases or conditions are cancer and its metastases. In some specific embodiments, the cancer is selected from the group consisting of: lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer. In one specific example, the cancer is lung cancer.

[0027] This invention also discloses an anti-ENO1 antibody or an antigen-binding fragment thereof, or a composition comprising the same antibody or antigen-binding fragment thereof as described herein, for the detection of ENO1, diagnosis and prognosis of cancer, and / or monitoring of cancer progression in patients with cancer. Further disclosed is the use of an anti-ENO1 antibody or an antigen-binding fragment thereof, or a composition comprising the same antibody or antigen-binding fragment thereof as described herein, for the preparation of a kit for the detection of ENO1, diagnosis and prognosis of cancer, and / or monitoring of cancer progression in patients with cancer. The kit comprises an anti-ENO1 antibody or an antigen-binding fragment thereof, or a composition comprising the same antibody or antigen-binding fragment thereof as described herein, and instructions for using the kit to detect ENO1. Specifically, the kit is an immunoassay kit. In some specific embodiments, the cancer is selected from the group consisting of: lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer. In one specific example, the cancer is lung cancer.

[0028] The present invention also provides a method for screening candidate drugs for cancer treatment. Specifically, the method of the present invention includes (i) contacting a drug with cancer cells that express ENO1 on the cell surface of cancer cells; (ii) detecting the binding of the drug to ENO1 on the cell surface of cancer cells and / or measuring the concentration of one or more downstream ENO1 signaling events / components in the cancer cells; and (iii) determining whether the drug is a candidate anticancer drug based on the detection and / or measurement results.

[0029] Details of one or more specific embodiments of the present invention are described below. Other features or advantages of the invention will become apparent from the following detailed description of several specific embodiments and the appended claims.

Implementation Method

[0045] The following description is merely illustrative of various specific embodiments of the invention. Therefore, the specific embodiments or modifications discussed herein should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various changes or equivalent substitutions can be made without departing from the scope of the invention.

[0046] I. Definition

[0047] To provide a clear and easy understanding of the invention, certain terms are first defined. Other definitions are set forth in the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references to objects. Thus, for example, reference to “a component” includes a plurality of that component and equivalents known to those skilled in the art to which this invention pertains.

[0049] The words “comprise” or “comprising” are generally used in the sense of including / including, which means that one or more features, components or parts can exist. The words “comprise” or “comprising” encompass words such as “consists” or “consisting of”.

[0050] As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds. The term "protein" generally refers to a relatively large polypeptide. The term "peptide" generally refers to a relatively short polypeptide (e.g., containing up to 100, 90, 70, 50, 30, 20, or 10 amino acid residues).

[0051] As used herein, the terms "approximately" or "about" refer to the degree of acceptable deviation that would be understood by one of ordinary skill in the art to which this invention pertains, and may vary to some extent depending on the context in which they are used. Specifically, "approximately" or "about" may refer to a value having a range of ±10%, ±5%, or ±3% around the referenced value.

[0052] As used herein, the term “substantially identical” refers to two sequences having 80% or higher, better 85% or higher, better 90% or higher, or even better 95% or higher identity.

[0053] As used herein, the term "antibody" (which may be used interchangeably with the plural form of antibody) refers to an immunoglobulin molecule that has the ability to specifically bind to a particular target antigen molecule. As used herein, the term "antibody" includes not only the complete (i.e., full-length) antibody molecule but also antigen-binding fragments that retain their antigen-binding ability, such as Fab, Fab', F(ab')2, and Fv. These fragments are well known in the art and are frequently used in vitro and in vivo. The term "antibody" also includes chimeric antibodies, humanized antibodies, human antibodies, biantibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of immunoglobulin molecules that contains the desired specific antigen recognition site, including amino acid sequence variants of antibodies, glycosylated variants of antibodies, and covalently modified antibodies.

[0054] A complete or intact antibody comprises two heavy chains and two light chains. Each heavy chain contains a variable region (VH) and a first, second, and third constant region (CH1, CH2, and CH3); and each light chain contains a variable region (VL) and a constant region (CL). The antibody is Y-shaped, with the backbone of the Y consisting of the second and third constant regions of the two heavy chains linked together by disulfide bonds. Each arm of the Y includes a variable region and a first constant region of a single heavy chain that bind to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the two chains are primarily responsible for antigen binding, and each contains three highly variable regions called complementarity-determining regions (CDRs); namely, the heavy (H) chain CDRs including HC CDR1, HC CDR2, and HC CDR3, and the light (L) chain CDRs including LCCDR1, LCCDR2, and LCCDR3. The three CDRs are connected by framework regions (FR1, FR2, FR3, and FR4), which are more conserved than the CDRs and form a scaffold supporting highly variable regions. The constant regions of the heavy and light chains are not responsible for antigen binding but participate in various effector functions. Based on the antibody amino acid sequences of the constant heavy chain domains of immunoglobulins, they can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. The constant heavy chain domains corresponding to different classes of immunoglobulins are respectively named α, δ, ε, γ, and μ.

[0055] As used herein, the terms "antigen-binding fragment" or "antigen-binding domain" refer to a portion or region of the complete antibody molecule responsible for antigen binding. An antigen-binding fragment is capable of binding the same antigen that binds to the parent antibody. Examples of antigen-binding fragments include, but are not limited to: (i) Fab fragments, which may be monovalent fragments consisting of a VH-CH1 chain and a VL-CL chain; (ii) F(ab')2 fragments, which may be divalent fragments consisting of two Fab fragments linked by disulfide bonds at the hinge region; (iii) Fv fragments, which consist of the VH and VL domains of an antibody molecule associated with each other by non-covalent interactions; (iv) single-chain Fv(scFv), which may be a single polypeptide chain consisting of VH and VL domains connected by peptide linkers; and (v) (scFv)2, which may contain two VH domains connected by peptide linkers and two VL domains associated with the two VH structures by disulfide bonds.

[0056] As used herein, the term "chimeric antibody" refers to an antibody containing polypeptides from different sources, such as different species. In some specific embodiments, in a chimeric antibody, the variable regions of the light and heavy chains may mimic the variable regions of antibodies derived from one mammal (e.g., non-human mammals, such as mice, rabbits, and rats), while the constant regions may be homologous to sequences in antibodies derived from another mammal (such as humans).

[0057] As used herein, the term “humanized antibody” refers to an antibody that includes a framework region derived from a human antibody and one or more CDRs derived from non-human (usually mouse or rat) immunoglobulins.

[0058] As used herein, the term "human antibody" refers to substantially the entire sequence of the light and heavy chains, including the complementarity-determining region (CDR), of an antibody derived from a human gene. In some cases, a human antibody may include one or more amino acid residues not encoded by human germline immunoglobulin sequences, for example, by mutations in one or more CDRs or one or more FRs, such as to reduce potential immunogenicity, increase affinity, and eliminate cysteine ​​that may cause undesirable folding.

[0059] As used herein, terms such as "specific binds" or "specifically binding" refer to non-random binding reactions between two molecules, such as the binding of an antibody to an epitope of its target antigen. "Specifically binding" of an antibody to a target antigen or epitope is a well-known term in the art, and methods for determining such specific binding are also well-known in the art. An antibody "specifically binds" a target antigen if it binds to the target antigen with a greater affinity / affinity, more readily, and / or for a longer duration than it binds to other substances. In other words, by reading this definition, it should also be understood that, for example, an antibody that specifically binds to a first target antigen may bind specifically or non-specifically or preferentially to a second target antigen. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, the affinity of binding can be defined according to the dissociation constant (KD). Typically, when used with antibodies, specific binding may involve antibodies that specifically bind (recognize) their targets with a KD value of less than about 10⁻⁷ M, such as about 10⁻⁸ M or lower, such as about 10⁻⁹ M or lower, about 10⁻¹⁰ M or lower, about 10⁻ⁿ M or lower, about 10⁻¹² M or lower, or even lower, and bind to specific targets with an affinity at least 10 times lower, such as at least 100 times lower, for example at least 1,000 times lower or at least 10,000 times lower, equivalent to their affinity for binding to nonspecific antigens (such as BSA or casein).

[0060] As used herein, the terms "nucleic acid" or "polynucleotide" may refer to polymers composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs, including those containing non-naturally occurring nucleotides. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences in which "U" replaces "T" (i.e., A, U, G, C). The term "cDNA" refers to DNA in single-stranded or double-stranded form that is complementary to or identical to mRNA.

[0061] As used herein, the term "complementary" refers to the topological compatibility or matching of the interacting surfaces of two polynucleotides. A first polynucleotide is complementary to a second polynucleotide when the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide-binding partner of the second polynucleotide. Therefore, a polynucleotide of sequence 5′-ATATC-3′ is complementary to a polynucleotide of sequence 5′-GATAT-3′.

[0062] As used herein, the term "encoding" refers to the natural properties of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, having a given RNA transcript (i.e., rRNA, tRNA, and mRNA) sequence or a given amino acid sequence and the resulting biological properties. Thus, if the transcription and translation of mRNA produced by a gene produces a protein in a cell or other biological system, then the gene encodes a protein. Those skilled in the art to which this invention pertains will understand that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. It should also be understood that those skilled in the art to which this invention pertains can use conventional techniques that do not affect the polypeptide sequence encoded by the polynucleotide described herein to perform nucleotide substitutions to reflect the codon usage of any particular host organism in which the polypeptide to be expressed. Therefore, unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" encompasses all nucleotide sequences that are degenerate from one another and encode the same amino acid sequence.

[0063] As used herein, the term "recombinant nucleic acid" refers to a polynucleotide or nucleic acid having a sequence that is not naturally linked together. Recombinant nucleic acids may exist in the form of a vector. A "vector" may contain a given nucleotide sequence of interest and a regulatory sequence. A vector may be used to express a given nucleotide sequence (expression vector) or to maintain a given nucleotide sequence for replication, manipulation, or transfer between different locations (e.g., between different organisms). For the above purposes, the vector may be introduced into a suitable host cell. A "recombinant cell" refers to a host cell in which a recombinant nucleic acid has been introduced. A "transformed cell" is a cell in which a DNA molecule encoding a protein of interest has been introduced by recombinant DNA technology.

[0064] The vector can be of various types, including plastids, cohesive plastids, free gene bodies, F-type cohesive plastids (fosmids), artificial chromosomes, bacteriophages, viral vectors, etc. Typically, in the vector, a given nucleotide sequence is operatively linked to a regulatory sequence such that when the vector is introduced into a host cell, the given nucleotide sequence is expressed in the host cell under the control of the regulatory sequence. The regulatory sequence may include, for example, but not limited to, promoter sequences (e.g., cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, T7 promoter, and alcohol oxidase gene (AOX1) promoter), start codons, origin of replication, enhancers, secretion message sequences (e.g., mating factor messages), stop codons, and other control sequences (e.g., Shine-Dalgarno sequences and termination sequences). Preferably, the vector may further contain marker sequences (e.g., antibiotic resistance marker sequences) for subsequent screening / selection steps. To generate the protein, in a vector, a given nucleotide sequence of interest can be linked to another nucleotide sequence different from the regulatory sequence described above, in order to generate a fusion polypeptide and facilitate subsequent purification steps. This fusion polypeptide includes a tag for purification purposes, such as a His tag.

[0065] As used herein, the term “treatment” refers to the application of one or more active agents to an individual suffering from a disease, symptoms or condition of a disease, or worsening of a disease, with the aim of curing, healing, alleviating, mitigating, altering, remedying, improving, enhancing or influencing the disease, symptoms or condition of a disease, disability caused by the disease, or worsening or disease tendency.

[0066] As used herein, the term "diagnosis" generally includes determining whether an individual is likely to be affected by a given disease, condition, or functional impairment. Those skilled in the art to which this invention pertains typically make diagnoses based on one or more diagnostic indicators (i.e., markers) whose presence, absence, or quantity indicates the presence or absence of a disease, condition, or functional impairment. It should be understood in the art that a diagnosis does not mean a determination of the presence or absence of a specific disease with 100% accuracy, but rather an increased likelihood of the presence of certain diseases in an individual.

[0067] As used herein, the term "prognosis" generally refers to the prediction of the likely course and outcome of a clinical condition or disease. Patient prognosis is typically determined by assessing factors or signs of the disease that indicate a favorable or unfavorable course or outcome. It should be understood that the term "prognosis" does not necessarily imply the ability to predict the course or outcome of a disease with 100% accuracy. Rather, those skilled in the art to which this invention pertains will understand that the term "prognosis" refers to an increased probability of a given course or outcome occurring; that is, the course or outcome is more likely to occur in patients presenting a given condition compared to those who do not present a condition. It is understood that a positive prognosis generally involves a beneficial clinical outcome or prospect, such as long-term survival without recurrence of the individual's cancer condition, while a negative (poor) prognosis generally involves an unfavorable clinical outcome or prospect, such as cancer recurrence or worsening.

[0068] II. Anti-ENO1 antibody

[0069] This invention is at least in part based on the production of an anti-ENO1 monoclonal antibody (mAb-22) capable of reliably detecting ENO1 on the surface of cancer cells. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of mAb-22, and their complementarity-determining regions (HC CDR1, HC CDR2, and HC CDR3) (LCCDR1, LCCDR2, and LCCDR3) are shown in Table 1 below. Please refer to Example 2.5. The anti-ENO1 antibody of this invention comprises mAb-22 and its functional variants.

[0070] In some specific embodiments, the anti-ENO1 antibody of the present invention is a functional variant of mAb-22, characterized in that it comprises (a) a VH comprising SEQ ID NO: 2 HC CDR1, SEQ ID NO: 4 HC CDR2 and SEQ ID NO: 6 HC CDR3; and (b) a VL comprising SEQ ID NO: 9 LC CDR1, SEQ ID NO: 11 LC CDR2 and SEQ ID NO: 13 HC CDR3, or an antigen-binding fragment thereof.

[0071] In some specific embodiments, the anti-ENO1 antibody of the present invention has (a) a VH comprising SEQ ID NO: 2 HC CDR1, SEQ ID NO: 4 HC CDR2 and SEQ ID NO: 6 HC CDR3; and (b) a VL comprising SEQ ID NO: 9 LC CDR1, SEQ ID NO: 11 LC CDR2 and SEQ ID NO: 13 HC CDR3, which may contain a VH comprising SEQ ID NO: 15 or substantially the same amino acid sequence thereon and a VL comprising SEQ ID NO: 16 or substantially the same amino acid sequence thereon. Specifically, the anti-ENO1 antibody of the present invention comprises VH and VL, wherein the VH comprises an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identity with SEQ ID NO: 15, and the VL comprises an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identity with SEQ ID NO: 16. The anti-ENO1 antibody of the present invention also includes any recombinant (modified) derived antibody encoded by a polynucleotide sequence encoding the relevant VH or VL amino acid sequence described herein.

[0072] The term "substantially identical" may refer to a variant whose relevant amino acid sequence (e.g., in FR, CDR, VH, or VL) is substantially different from that of the reference antibody, such that the variant has substantially similar binding activity (e.g., affinity, specificity, or both) and biological activity relative to the reference antibody. Such variants may include minor amino acid changes. It is understood that peptides may have a limited number of variations or modifications that may be made in specific portions of the peptide unrelated to its activity or function, and still produce variants with acceptable concentrations of equivalent or similar biological activity or function. In some instances, amino acid residue changes are conserved amino acid substitutions in which the chemical structure of the involved amino acid residue is similar to that of another amino acid residue, and the peptide function, activity, or other biological effects have little or no impact on the properties. Generally, relatively more substitutions may be made in the FR region, as opposed to the CDR region, provided that they do not adversely affect the binding function and biological activity of the antibody (e.g., a reduction in binding affinity of more than 50% compared to the original antibody). In some specific embodiments, the sequence identity between the reference antibody and the variant may be approximately 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%, or higher. Variants can be prepared according to methods for altering peptide sequences known to those skilled in the art to which this invention pertains, such as those found in references compiling such methods, such as *Molecular Cloning: A Laboratory Manual*, edited by J. Sambrook et al., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. For example, conserved substitutions of amino acids include substitutions between amino acids in the following groups: (i) A, G; (ii) S, T; (iii) Q, N; (iv) E, D; (v) M, I, L, V; (vi) F, Y, W; and (vii) K, R, H.

[0073] The antibodies described herein may be animal antibodies (e.g., mouse-derived antibodies), chimeric antibodies (e.g., mouse-human chimeric antibodies), humanized antibodies, or human antibodies. The antibodies described herein may also include their antigen-binding fragments, such as Fab fragments, F(ab')2 fragments, Fv fragments, single-chain Fv(scFv), and (scFv)2. Antibodies or their antigen-binding fragments may be prepared using methods known in the art.

[0074] III. Preparation of Antibodies

[0075] Many conventional methods in the art can be used to obtain antibodies or their antigen-binding fragments.

[0076] In some specific embodiments, the antibodies provided herein can be prepared using conventional fusion tumor techniques. Generally, a target antigen, such as a tumor antigen, optionally conjugated with a carrier protein, such as keyhole hemocyanin, and / or mixed with an adjuvant, such as, for example, complete Freund's adjuvant, can be used to immunize a host animal to produce antibodies that bind to the antigen. Lymphocytes secreting monoclonal antibodies are collected and fused with myeloma cells to generate fusion tumors. Fusion tumor colonies formed in this manner are then screened to identify and select those secreting the desired monoclonal antibody.

[0077] In some specific embodiments, the antibodies provided herein can be prepared via recombinant technology. Relatedly, isolated nucleic acids encoding the disclosed amino acid sequences are also provided, as well as vectors containing such nucleic acids and host cells transformed or transfected with such nucleic acids.

[0078] For example, nucleic acids containing nucleotide sequences encoding the variable regions of the heavy and light chains of such antibodies can be selected and colonized into expression vectors (e.g., bacterial vectors, such as E. coli vectors, yeast vectors, viral vectors, or mammalian vectors) using conventional techniques, and any vector can be introduced into suitable cells (e.g., bacterial cells, yeast cells, plant cells, or mammalian cells) to express the antibody. Examples of nucleotide sequences encoding the variable regions of the heavy and light chains of the antibodies described herein are shown in Table 1 below. Examples of mammalian host cell lines are human embryonic kidney cell line (293 cells), young hamster kidney cells (BHK cells), Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (VERO cells), and human liver cells (Hep G2 cells). Recombinant vectors used to express the antibodies described herein typically contain nucleic acids encoding the amino acid sequences of the antibody, which are operatively linked to constitutive or inducible promoters. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of nucleic acids encoding antibodies. Vectors optionally contain selection markers for prokaryotic and eukaryotic systems. In some instances, both the heavy and light chain coding sequences are included in the same expression vector. In other instances, each heavy and light chain of the antibody is selected into a separate vector and generated individually, which can then be cultured under suitable conditions for antibody assembly.

[0079] Recombinant vectors used to express the antibodies described herein typically contain nucleic acids encoding the amino acid sequence of the antibody, which are operatively linked to a constitutive or inducible promoter. Recombinant antibodies can be generated in prokaryotic or eukaryotic expression systems, such as bacterial, yeast, insect, and mammalian cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the nucleic acid encoding the antibody. Vectors optionally contain selection markers for prokaryotic and eukaryotic systems. The resulting antibody protein can be further isolated or purified to obtain a substantially homogeneous formulation for further assays and applications. Suitable purification procedures may include, for example, separation on immunoaffinity or ion exchange columns, ethanol precipitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high-performance liquid chromatography (HPLC), ammonium sulfate precipitation, and gel filtration.

[0080] When a full-length antibody is required, the coding sequences of any VH and VL chains described herein can be linked to the coding sequences of the Fc region of an immunoglobulin, and the resulting genes encoding the heavy and light chains of the full-length antibody can be expressed and assembled in a suitable host cell, such as a plant cell, mammalian cell, yeast cell, or insect cell.

[0081] Antigen-binding fragments can be prepared using conventional methods. For example, the F(ab')2 fragment can be produced by digesting a full-length antibody molecule with pepsin, and the Fab fragment can be prepared by reducing the disulfide bonds of the F(ab')2 fragment. Alternatively, these fragments can be prepared using recombinant techniques by expressing heavy and light chain fragments in suitable host cells and assembling them in vivo or in vitro to form the desired antigen-binding fragment. Single-chain antibodies can be prepared using recombinant techniques by linking nucleotide sequences encoding the variable region of the heavy chain and the variable region of the light chain. Preferably, a flexible linker is incorporated between the two variable regions.

[0082] An antibody may be further modified to conjugate with one or more additional elements at the N-terminus and / or C-terminus of the antibody (such as another protein and / or drug or carrier). Preferably, the antibody conjugated with the additional elements retains the desired binding specificity and therapeutic effect while providing additional properties generated by the additional elements, such as improved solubility, storage or other processing properties, cell permeability, half-life, reduced hypersensitivity, controlled delivery and / or dispensing. Other specific embodiments include conjugation with labels (e.g., dyes or fluorophores) for assaying, detection, tracking, etc. In some specific embodiments, the antibody may be conjugated with additional elements such as peptides, dyes, fluorophores, carbohydrates, anticancer agents, lipids, etc. Additionally, the antibody may attach directly to the surface of liposomes via the Fc region, for example, to form immunoliposomes.

[0083] IV. Composition

[0084] According to the present invention, the anti-ENO1 antibody is formulated into a composition with a pharmaceutically acceptable carrier for delivery and absorption.

[0085] As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition, preferably stabilizes the active ingredient, and is safe for the recipient. The carrier may be a diluent, medium, excipient, or matrix for the active ingredient. Typically, compositions containing an anti-ENO1 antibody as an active ingredient, as described herein, may be in solution form, such as an aqueous solution, e.g., a saline solution, or may be provided in powder form. Suitable excipients also include lactose, sucrose, dextrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterilizing water, syrup, and methylcellulose. The composition may also contain pharmaceutically acceptable excipients close to those required for physiological conditions, such as pH adjusters and buffers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The compositions may be in the form of tablets, pills, powders, lozenges, packets, sugars, elixirs, suspensions, lotions, solutions, syrups, soft gelatin capsules and hard gelatin capsules, suppositories, sterile injections, and packaged powders. The compositions of the present invention can be delivered via any physiologically acceptable route, such as oral, parenteral (e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods. In some specific embodiments, the compositions of the present invention are administered as liquid injectable formulations, which may be provided as ready-to-use dosage forms or as resoluble, stable powders.

[0086] V, Treatment

[0087] According to the present invention, the anti-ENO1 antibody described herein can specifically bind to ENO1 and inhibit downstream ENO1 signaling pathways, including, for example, attenuating HGF-triggered phosphorylation of HGFR and its downstream signaling components, reducing the binding between ENO1 and HGFR, and / or reducing the concentration of SLUG homeostatic protein. Therefore, the anti-ENO1 antibody described herein is beneficial for treating diseases and conditions associated with activation of downstream ENO1 signaling pathways.

[0088] Therefore, this document discloses a method for inhibiting ENO1 downstream signaling and / or treating diseases or conditions associated with ENO1 downstream signaling activation. The method comprises administering to a patient in need an effective amount of an anti-ENO1 antibody as an active ingredient or a composition comprising the antibody. In some specific embodiments, activation of ENO1 downstream signaling is associated with proliferative diseases or conditions (e.g., cancer and its metastases). In some specific embodiments, cancers selected from the group consisting of lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer, which have been observed to exhibit overexpression of ENO1 mRNA or protein. In one specific example, the cancer is lung cancer.

[0089] As used herein, the term "effective amount" refers to the amount of active ingredient that imparts the desired biological effect in the treated individual or cells. Effective amounts can vary for various reasons, such as the route and frequency of administration, the weight and type of the individual receiving the drug, and the purpose of administration. Those skilled in the art to which this invention pertains can determine the dosage in each case based on the disclosure herein, established methods, and their own experience.

[0090] Individuals treated by the methods described herein may be mammals, preferably humans. Mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, mice, and rats. Human individuals requiring treatment may be human patients who have, are at risk of having, or are suspected of having the target disease / condition (such as cancer). Individuals suspected of having any such target disease / condition may exhibit one or more symptoms of the disease / condition. Individuals at risk of the disease / condition may be individuals with one or more risk factors for the disease / condition.

[0091] In some specific embodiments, the individual requiring treatment by the method of the present invention has cells or tissues that express (e.g., overexpress) ENO1. Antibody therapy as described herein can be initiated before or after the first sign of a possible disease, or before or after a disease diagnosis.

[0092] In some specific embodiments, the antibody therapy described herein may be used in combination with one or more cancer therapies known in the art. These known cancer therapies include surgery, radiation, chemotherapy, cell therapy, and any combination thereof.

[0093] VI. Diagnosis and Prognosis

[0094] ENO1 has been observed in many different tumor types and is known to be highly associated with reduced survival and poor prognosis. Therefore, this invention provides a method for detecting ENO1 using an anti-ENO1 antibody as described herein for diagnostic and prognostic purposes. Generally, the detection method described herein comprises (i) contacting the anti-ENO1 antibody described herein or an antigen-binding fragment thereof with cells of an individual in need; and (ii) detecting the binding of the antibody or its antigen-binding fragment to the cells. The cells may be present in vitro, in a biological sample isolated from the individual in need, or within the individual in need. The individual may be at risk of cancer or suspected of having cancer, or may be an individual with cancer who is currently receiving cancer treatment. The detection method described herein can be used to identify individuals who are compliant with cancer therapy and / or to monitor cancer progression or response to therapy.

[0095] In some specific embodiments, when the detection method is performed in vivo, the biological sample can be any type of sample, such as a body fluid sample or a tissue sample in which cancer cells may be present. Examples of such samples include, but are not limited to, blood, urine, saliva, cerebrospinal fluid, ascites, lymph, nipple aspiration fluid, bronchoalveolar lavage fluid, semen, or tissue or live tissue samples from the lungs, breast, cervix, colon, stomach, liver, ovary, pancreas, prostate, skin, and testes. For example, a biological sample is obtained from an individual, and the biological sample is analyzed by immunoassay using the anti-ENO1 antibody described herein to detect the presence or concentration of ENO1 in the sample. Examples of immunoassays include, but are not limited to, Western ink dot assay, enzyme-conjugated immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), immunofluorescence assay (IFA), ELFA (enzyme-conjugated immunofluorescence assay), electrochemiluminescence (ECL), and capillary gel electrophoresis (CGE).

[0096] In some specific embodiments, when performing detection methods in vivo, suitable in vivo imaging techniques can be used throughout the entire living individual as needed. For example, the anti-ENO1 antibody described herein can be detectably labeled and administered to the individual in need, and the bound, detectably labeled antibody can be detected using imaging methods available in the art.

[0097] In some specific embodiments, the detection method can be used for prognostic purposes. Specifically, the method may further include comparing the detection result with an ENO1 reference concentration and determining the individual's prognosis based on the comparison result, wherein an elevated ENO1 concentration indicates a negative prognosis. In some specific embodiments, the reference concentration may refer to a concentration measured in a normal individual or sample, such as disease-free tissue or cells (adjacent non-cancerous / normal tissue). In some specific embodiments, the reference concentration may be a standard value that may represent the mean or median amount of a marker (e.g., ENO1) in a cancer patient population. Typically, such a cancer patient population is selected to match candidate individuals in a context of, for example, age, cancer type, and / or ethnicity. Preferably, such a cancer patient population and candidate individuals are of the same species (e.g., humans).

[0098] In some specific embodiments, a detection method is performed on an individual with cancer to monitor cancer progression. Specifically, biological samples are obtained from cancer patients at different time points, and cancer progression in the patient is determined based on the apparent concentration of ENO1, wherein an increase in ENO1 concentration over time indicates cancer progression.

[0099] In one specific embodiment, the detection method includes (i) providing a first biological sample from a cancer patient at a first time point; (ii) providing a second biological sample from the patient at a second time point, later than the first time point; (c) detecting the concentration of ENO1 in the first and second biological samples, wherein the detection is performed by immunoassay of an antibody or its antigen-binding fragment as described herein; and (iv) determining cancer progression in the patient based on the ENO1 concentration in the first and second biological samples, wherein an increased ENO1 concentration in the second biological sample compared to the first biological sample indicates cancer progression.

[0100] Kits for carrying out the methods of the present invention are also provided. Specifically, the kit comprises an anti-ENO1 antibody or an antigen-binding fragment thereof as described herein, or a composition comprising thereto, and instructions (e.g., written, magnetic tape, VCR, CD-ROM, etc.) for using the kit to detect the presence or quantity of ENO1 in vitro, for example in a biological sample from or in the body of an individual in need. The assay form of the kit may be, for example, a crystalloid or an ELISA.

[0101] VII. Screening methods for anticancer candidate reagents

[0102] According to the present invention, binding to ENO1 on the surface of cancer cells and / or negative regulation of ENO1 downstream signaling are key to inhibiting cancer growth and progression. Therefore, the present invention provides a method for screening candidate agents for cancer therapy, the method being based on binding to ENO1 on the surface of cancer cells and / or negative regulation of ENO1 downstream signaling activity.

[0103] Specifically, the method of the present invention includes (i) contacting an agent with cancer cells that express ENO1 on the cell surface of cancer cells; (ii) detecting the binding of the agent to ENO1 on the cell surface of cancer cells and / or measuring the concentration of one or more downstream ENO1 signaling events / components in cancer cells; and (iii) determining whether the agent is a candidate anticancer drug based on the detection and / or measurement results.

[0104] In some specific embodiments, the detection of the drug binding to ENO1 on the cell surface indicates that the drug has an anticancer effect.

[0105] In some specific embodiments, the measurement results show that the concentration of one or more of the downstream ENO1 messaging events / components in cancer cells is reduced in the presence of the reagent compared to the absence of the reagent, and such measurement results indicate that the reagent has an anticancer effect.

[0106] In some specific embodiments, one or more ENO1 downstream messaging events / components include HGF-triggered phosphorylation of HGFR, association between ENO1 and HGFR, and / or SLUG homeostatic protein.

[0107] In some specific embodiments, the anticancer effect includes inhibiting tumor growth and / or metastasis.

[0108] In some specific embodiments, cancers are selected from the group consisting of: lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer.

[0109] In one specific embodiment, the cancer is lung cancer.

[0110] The drug screening described herein can be performed using in vitro cell models. Various assays can be used for this purpose, such as immunoprecipitation and Western ink dot assay. Subsequently, the functional activity of the reagents can be further analyzed, for example, by cell viability assays and / or cell invasion assays and / or using in vivo animal tumor / metastatic models. The reagents used for screening can be antibodies or small molecule compounds.

[0111] The present invention is further illustrated by the following examples, which are provided for illustrative purposes and not for limitation. Based on the disclosure herein, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed, and the same or similar results can be obtained without departing from the spirit and scope of the invention.

[0112] Example

[0113] ENO1 expression is significantly associated with reduced survival and poor prognosis in many cancer types, including lung cancer. However, the function of ENO1 in oncogenesis remains unknown, and there are currently no approved ENO1-based therapeutics for clinical use. In this study, we found that high expression of ENO1 was present in metastatic lung cancer cell lines and malignant tumors, and it was associated with poor overall survival in lung cancer patients. In vitro functional studies showed that knockdown of ENO1 reduced cancer cell proliferation and invasiveness, and these processes were enhanced by ENO1 overexpression. In addition, ENO1 promoted tumor growth in an orthotopic xenograft model and enhanced lung tumor metastasis in a tail vein injection model. These effects may be mediated by positive regulation of mesenchymal markers (N-cadherin and vimentin) and the epithelial-mesenchymal transition regulator SLUG, as well as simultaneous negative regulation of E-cadherin expression. Mechanistically, ENO1 interacts with the hepatocyte growth factor receptor (HGFR) and activates HGFR and Wnt signaling by increasing phosphorylation of the HGFR and Wnt co-receptor LRP5 / 6. Activation of these signaling responses ultimately positively regulates SLUG and β-catenin, reducing GSK-3β activity, via Src-PI3K-AKT signaling and deactivation of the β-catenin disruption complex. Furthermore, we developed a chimeric anti-ENO1 monoclonal antibody (chENO1-22) that reduces cancer cell proliferation and invasion. chENO1-22 inhibits cancer cell invasion by inhibiting ENO1-mediated GSK3β deactivation, thereby promoting SLUG protein ubiquitination and degradation. Additionally, in a tail vein injection animal model, chENO1-22 inhibited lung tumor metastasis and prolonged survival. In summary, our findings elucidate the molecular mechanism of ENO1 regulation in lung cancer metastasis and introduce a novel antibody for potential therapeutic applications in lung cancer.

[0114] 1. Materials and Methods

[0115] 1.1 Cell Cultures and Reagents

[0116] NL-20, CL1-5, and H460 cells were purchased from the American Type Culture Collection (ATCC) and identified by ATCC based on DNA mapping, cytogenetics analysis, and isoenzyme analysis. Cells were cultured according to the ATCC protocol and passaged for less than 6 months after thawing. NL-20 is an immortalized, non-tumorigenic human bronchial epithelial cell line derived from normal bronchi. NHBE cells purchased from Lonza are normal human bronchial epithelial cells. Hop62, CL1-0, and CL141 cells were generously donated by Dr. Pan-Chih Yang (National Taiwan University). The GSK-3 inhibitor BIO was purchased from Sigma-Aldrich.

[0117] 1.2 Reverse Transcription-PCR and Western Ink Dot Method

[0118] Perform reverse transcriptase-PCR and immunoblotting as previously described 37. The following primary antibodies were used in the immunochromatographic assay: anti-ENO1 (11204-1-AP; Proteinch), anti-E-cadherin (24E10; Cell Signaling Technology), anti-Vimentin (D21H3; Cell Signaling Technology), anti-N-cadherin (610920; BD Biosciences), anti-SNAIL (C15D3; Cell Signaling Technology), anti-TWIST (GTX127310; GeneTex), anti-SLUG (C19G7; Cell Signaling Technology), anti-GSK-3β (27C10; Cell Signaling Technology), phosphate-GSK-3β (Y216) (612312; BD Biosciences), phosphate-GSK-3β (Ser9) (5B3; Cell Signaling Technology), anti-non-phosphate (active) β-catenin (D13A1; Cell Signaling Technology), and anti-β-catenin (D10A8; Cell...). Signaling Technology), phosphate-Src family (Tyr416) ​​(D49G4; Cell Signaling Technology), phosphate-PI3 kinase p85 (Tyr458) / p55 (Tyr199) (Cell Signaling Technology), phosphate-Akt (Ser473) (Cell Signaling Technology), anti-FLAG (F3165; Sigma-Aldrich) and anti-Myc (9E10; Santa Cruz Biotechnology), and anti-HGFR (D1C2; Cell Signaling Technology), and appropriately use wasabi peroxidase (HRP) conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology).

[0119] 1.3 Flow Cytometry

[0120] Flow cytometry was performed on a Beckman Dickinson LSR-II flow cytometer (Cornell Biotechnology; Flow Cytometery Core Facility) equipped with 407, 488, and 633 nm lasers. Typical monochromatic analysis was performed. Curve fitting and data analysis were performed using GraphPad Prism (v 6.0). P-values ​​were obtained by unpaired Student's t-test. Cell counting was performed using Countess II (Invitrogen).

[0121] 1.4 Tissue microarrays and immunohistochemistry

[0122] The tissue microarray was obtained from a commercial source (SuperBioChips). A total of 9 normal lung samples, 40 primary lung cancer samples, and 10 metastatic lung cancer cells were tested. The primary antibody used for staining targeted anti-ENO1 (Abcam Biotechnology). Independent moderators unaware of the identification information graded the IHC messages as 0 (no manifestation), 1 (weak manifestation), 2 (moderate manifestation), and 3 (strong manifestation). The area of ​​positive staining was also assessed as a percentage.

[0123] 1.5 Functional analysis of target proteins

[0124] Lentiviral virus (pLKO.1) containing shRNA and either an empty pLKO.1 vector or a pLKO-luciferase control group was generated and used to infect cancer cells according to standard procedures. Stable transfectants were established by purine selection as previously described.38 In addition to the overexpression of the gene product after transfection, the function of the target gene was evaluated by functional assays. Changes in cell morphology, proliferation rate, and invasiveness were evaluated using the previously described protocol.38

[0125] 1.6 Cell viability

[0126] Cells were collected by trypsinization and resuspended in RPMI containing 10% FBS. Cells were then seeded at a density of 3000 cells per well in 96-well plates and cultured overnight. Each drug concentration was used for four replication cycles; the control group received PBS buffer. After adding WST-1, the reagents were incubated in the 96-well plates for 1 hour. Absorbance at 450 nm was monitored.

[0127] 1.7 Matricella Invasion Assay Transwell Cell Invasion Assay

[0128] The matrix gel invasion assay was performed using a 24-well transwell disc (Costar) with a polycarbonate filter (8 μm pore size). The matrix gel was diluted to 1 mg / ml with serum-free medium at 4°C on ice and added to the upper chamber of the disc. After incubation at 37°C for 30 minutes, a cell suspension of 1 × 10⁵ cells / ml was added. For the lower chamber, 800 μl of medium with 10% FBS was added. After 16–24 hours, the cells were fixed with 95% methanol for 5 minutes. Crystal violet (0.1%) was used to stain the cells. The transwell discs were then observed under a microscope.

[0129] 1.8 In vivo in situ lung tumor and metastasis model

[0130] Female non-obese diabetic / severe combined immunodeficiency (NOD-SCID) mice were used to establish orthotopic lung tumor and metastatic lung cancer xenograft models. For the orthotopic xenograft model, 1 × 10⁵ CL1-5 cells stably expressing luciferase and infected with shENO1 or the control group shCtrl were injected orally into the lung parenchyma of NOD-SCID mice. For the metastatic model, 1 × 10⁶ CL1-5 cells stably expressing luciferase and infected with shENO1 or the control group shCtrl were injected into mice via tail vein injection. All animal studies were approved by the Laboratory Animal Care and Use Committee of the National Defense Medical Center, Taipei, Taiwan.

[0131] 1.9 Immunofluorescence Analysis

[0132] Cells were grown to the appropriate confluence concentration as shown. At the end of the experiment, cells were fixed at 4°C for 20 minutes by adding paraformaldehyde (PFA). The PFA was removed, and the cells were washed three times with PBS and blocked at 37°C with 2 ml imaging blocking buffer (3% BSA in PBS) for 1 hour. The plates were washed twice with PBS and then treated with primary antibody at room temperature for 1 hour. The primary antibody was removed, and the plates were washed twice with PBS. Secondary antibody was then added for 1 hour. The plates were washed once with PBS containing 1 mg / ml DAPI, followed by one wash with PBS. Samples were stored at 4°C until imaging was performed.

[0133] 1.10 TOP / FOP Wnt Activity Assay

[0134] CL1-5 cells (2 × 10⁴ cells per well) were seeded in 24-well dishes. Plastids were transfected into the cells using Lipofectamine 3000 (Invitrogen). Luciferase reporters (TOPflash or FOPflash) or co-transfected with pGL4.74 hRluc-TK constructs (Reniform luciferase). Luciferase activity was measured using a dual luciferase reporter assay system (Promega, Madison, WI, USA). Transcriptional activity was determined by the expression of firefly luciferase normalized to Reniform luciferase concentration at 48 hours post-transfection. Measurements were performed using a dual luciferase reporter assay kit (Promega).

[0135] 1.11 Phosphate-RTK Array Analysis

[0136] According to the manufacturer's protocol, the relative concentrations of tyrosine phosphorylation of 49 different RTKs were determined using a Human Phospho-RTK Array Kit (R&D Systems). Briefly, after blocking with array buffer 1 for 1 hour, the array was incubated overnight at 4°C with 300 µg of protein lysate. The array was washed and co-incubated with an anti-phospho-tyrosine-HRP detection antibody. Signals were generated using a chemical reagent mixture and captured using a chemiluminescence imaging system (GE ImageQuant LAS4000). The intensity of each phospho-RTK array signal was quantified using ImageJ software; the relative intensity of the average signal from each pair of duplicated spots was determined relative to the negative control spots.

[0137] 1.12 GST dropdown

[0138] In the GST pull-down analysis, GST-ENO1 with His-HGFR bound to glutathione beads or GST with beads alone (control group) were incubated in pull-down buffer (Pierce™ GST Protein Interaction Pull-Down Kit) at 4°C for 1 hour.

[0139] 1.13 Immunoprecipitation (IP) - Western Ink Dot Method

[0140] The protein lysate was cultured with anti-FLAG agarose beads (A2220; Sigma-Aldrich, USA) and anti-Myc agarose beads (C3956; Sigma-Aldrich, USA) for co-IP experiments, or with anti-SLUG (C19G7; Cell Signaling Technology, Inc., USA) for ubiquitin (Ub) assays. The following primary antibodies were used in the immunoblotting assay: anti-ENO1 (11204-1-AP; Proteintech), anti-SLUG (C19G7; Cell Signaling Technology), anti-FLAG (F3165; Sigma-Aldrich), anti-Myc (9E10; Santa Cruz Biotechnology), anti-ubiquitin (P4D1; Cell Signaling Technology), and anti-HGFR (D1C2; Cell Signaling Technology). Rabbit anti-mouse or goat anti-rabbit secondary antibodies conjugated with wasabi peroxidase (HRP) were used as appropriate (Santa Cruz Biotechnology).

[0141] 1.14 Production of monoclonal antibodies against tumor antigens

[0142] Following standard procedure 39, with some modifications 40–42, the antitumor antigen mAb was generated. In short, the spleen of immunized mice was removed, and spleen cells were fused with NSI / 1-Ab4-1 (NS-1) myeloma cells. The spleen cells and myeloma cells were washed twice with serum-free DMEM medium. The final precipitate was mixed in a 15 ml conical tube, and 1 ml of 50% (v / v) polyethylene glycol (GIBCO BRL) was added over 1 minute while gently stirring. The mixture was diluted by slowly adding 1 ml of DMEM twice (1 minute each time), followed by slowly adding 8 ml of serum-free DMEM medium over 2 minutes. The mixture was centrifuged, and the fused cell pellet was resuspended in DMEM medium supplemented with 15% FBS, hypoxanthine-aminopterin-thymidine (HAT) medium, and fusion tumor selection factor, and then dispensed into 96-well tissue culture dishes. Fusomotor colonies were screened by ELISA to secrete mAbs that bind to tumor antigens. Selected cell lines were then subselected using limiting dilutions. Isotype analysis of the final fusomotor cell lines was performed using an isotype kit. Ascites was induced in pristane-primed BALB / c mice. Fusomotor cell lines were grown in DMEM medium containing 10% heat-deactivated FBS. Monoclonal antibodies were purified by protein G agarose 4B gel affinity chromatography. ELISA and Western blot assays were used to measure antibody activity and specificity.

[0143] 1.15 ELISA

[0144] Cell cultures (96-well) were fixed with 2% paraformaldehyde and blocked with 1% bovine serum albumin (BSA). Cells were added to the plates and cultured for 1 hour. The plates were then washed with PBS containing 0.1% (w / v) Tween-20 (PBST 0.1), followed by co-culturing with wasabi peroxidase-conjugated anti-mouse IgG (115-035-062; Jackson ImmunoResearch Laboratories) for 1 hour. After washing, the plates were co-cultured with the receiving solution (o-phenylenediamine dihydrochloride; P6787, Sigma). The reaction was terminated by adding 3N HCl, and the signal was detected at 490 nm using a microdisk reader.

[0145] 1.16 Immunogold Marker

[0146] CL1-5 cells were fixed with 4% formaldehyde and processed for cryosectioning. Sections were blocked with BSA and then labeled with ENO1 using chENO1 antibody-22 (40 µg / ml) or NHIgG. After fixation, samples were stained and examined under a transmission electron microscope.

[0147] 1.17 Statistical Analysis

[0148] Data are presented as mean ± SEM. Student's t-test was used for comparisons between the two groups. Statistical analysis was performed using GraphPad Prism software version 5.0. All statistical tests were two-tested, and a p-value < 0.05 was considered statistically significant.

[0149] 2. Result

[0150] 2.1 ENO1 is highly expressed in metastatic cancer cell lines and patients, and its high expression is associated with poor overall survival in lung cancer patients.

[0151] To assess the correlation between ENO1 expression and lung cancer malignancy, we measured the ENO1 protein concentration in a group of lung cancer cells and clinical NSCLC specimens. Compared to non-metastatic cell lines (CL1-0 and Hop62), ENO1 protein concentration was elevated in highly metastatic lung cancer cell lines (CL1-5 and CL141), and only weak expression was detected in normal human bronchial epithelial (NHBE) cells or NL-20 immortalized human bronchial epithelial cells (Fig. 1A). Furthermore, according to flow cytometry analysis using a chimeric ENO1 antibody (Fig. 9) (Fig. 1B), the surface expression of ENO1 was positively regulated in CL1-5 cells compared to NHBE or NL-20 cells. Additionally, we performed immunogold labeling assays to confirm ENO1 protein localization. ENO1 was expected to be detected on the cell surface, in the cytoplasm, and in the nucleus of CL1-5 cells (Fig. 1C). In clinical tumor samples from NSCLC patients, increased ENO1 expression was associated with increased TNM and metastatic stages, but ENO1 mRNA and protein were not abundant in normal lung tissue (Figs. 1D to 1F and Fig. 10). Furthermore, survival analysis based on ENO1 protein expression concentration showed that NSCLC patients with high ENO1 expression had significantly lower survival rates compared to those with low ENO1 expression (Fig. 1G). Additionally, according to the Kaplan-Meier plotter database (KM plotter, http: / / kmplot.com / analysis / ) (Fig. 1H) and the TCGA database (Fig. 1I), high ENO1 mRNA expression was strongly associated with poorer overall survival in NSCLC patients. In conclusion, these findings indicate a strong correlation between high ENO1 expression and lung cancer malignancy.

[0152] 2.2 ENO1 enhances the viability and invasiveness of lung cancer cells

[0153] To investigate the biological function of ENO1 in lung cancer cells in vitro, we established a constitutive ENO1 knockdown system. CL1-5 or CL-141 cells were infected with lentiviruses carrying one of two small hairpin RNAs (shRNAs) carrying different coding sequences of ENO1 (shENO1-1, shENO1-2) or a control non-targeting shRNA (shLacZ) (Fig. 2A). Knockout of ENO1 significantly reduced cell viability (Fig. 2B) and cell invasion (Fig. 2C). We also stably overexpressed ENO1 in CL1-0 cells, which typically exhibited low ENO1 expression (Fig. 2D), and found that ENO1 overexpression significantly enhanced cell viability (Fig. 2E) and simultaneously increased the number of invading cells (Fig. 2F). In summary, these results indicate that ENO1 increases the viability and invasion of lung cancer cells in vitro.

[0154] 2.3 ENO1 promotes tumorigenesis and metastasis of lung cancer in xenograft models

[0155] Tumor growth, invasion, and metastasis are key steps in tumor progression. First, to investigate the role of ENO1 in in vivo tumorigenesis, we established an orthotopic xenograft model. One hundred thousand CL1-5 cells stably expressing luciferase and infected with shENO1 or shCtrl were orthotopically injected into the lung parenchyma of NOD-SCID mice. Using luciferase-based bioluminescence to detect tumor size, we found that mice injected with ENO1 knockdown cells had lower luciferase signals than mice injected with shCtrl-infected cells, indicating a lower tumor growth rate in ENO1 knockdown tumors (Figures 3A and 3B). Lung tissue was collected 21 days post-implantation, and pulmonary nodules were detected using hematoxylin and eosin staining.

[0156] To further test whether ENO1 may affect lung cancer metastasis, we investigated the formation of pulmonary nodules in NOD-SCID mice that were intravenously injected with CL1-5 cells stably expressing luciferase and infected with shENO1 or shCtrl. Lungs were collected 31 days post-injection, and the number of pulmonary nodules was counted. As shown in Figures 3C and 3D, a stronger luciferase signal was observed in the lungs of mice injected with control shCtrl cells compared to the ENO1 knockdown group. Furthermore, fewer metastatic pulmonary nodules were generated by ENO1 knockdown cells compared to control cells (Figure 3E). In conclusion, these data demonstrate that ENO1 plays an important role in promoting the metastatic behavior of tumor-forming lung cancer cells in a xenograft model.

[0157] 2.4 ENO1 enhances cancer cell invasion via HGFR- and WNT-driven epithelial-mesenchymal transition.

[0158] Previous studies have shown that EMT is associated with cancer invasion, metastasis, and progression. Compared to control cells exhibiting fibroblast-like stromal features, CL1-5 cells with ENO1 knockdown showed a more epithelial-like phenotype (Fig. 4A). Therefore, we decided to map the expression profiles of EMT-related genes in ENO1 knockdown and control cells. We found that ENO1 knockdown inhibited the expression of stromal markers N-cadherin and vimentin, as well as the protein concentration of the EMT regulator SLUG, but simultaneously enhanced the expression of E-cadherin (Fig. 4B). Positive regulation of SLUG expression mediated by ENO1 was also observed in CL1-5 cells by immunofluorescence. In contrast, ectopic expression of ENO1 in CL1-0 cells increased the concentrations of N-cadherin, vimentin, and SLUG, but simultaneously decreased E-cadherin. Interestingly, ENO1 did not affect SLUG mRNA concentration (Fig. 4C). Treatment with MG132 (a 26S proteasome inhibitor) increased the steady-state concentration of SLUG protein, indicating that protein concentration is primarily controlled by degradation (Fig. 4D). Knockdown of ENO1, compared to control cells, led to an increase in the concentration of ubiquitinated SLUG (Fig. 4E), and, based on the protein concentration after cyclohexylamine treatment, it shortened the half-life of SLUG protein (Figs. 4F and 4G). Based on these results, we conclude that ENO1 regulates the stability of SLUG protein but not its expression.

[0159] Since the Wnt signaling antagonist GSK-3β is known to affect SLUG protein switching via phosphorylation and ubiquitin-mediated proteolysis, we hypothesized that ENO1 stabilizes SLUG by regulating GSK-3β. Indeed, after treatment with a GSK3β inhibitor, SLUG was positively regulated in ENO1-knockdown cells; simultaneously, the active form of β-catenin also increased (Fig. 5A). Interestingly, we found that ENO1 knockout enhanced GSK-3β (Fig. 5A) and also reduced Wnt signaling activity, as measured by TOP / FOP luciferase reporter assay (Fig. 5B). Confocal immunofluorescence showed that ENO1 knockdown also reduced nuclear β-catenin concentration (Fig. 5C). To further test whether β-catenin and SLUG are involved in ENO1-induced cell invasion, we overexpressed constitutively active β-catenin (β-catenin Δ45) or a vector control group. We found that overexpression of β-catenin Δ45 restored the invasive ability of ENO1-knockdown cells, but did not affect the invasion of control cells (Fig. 5D). Additionally, we overexpressed mutant SLUG (Slug-4SA; SLUG protein in which the four serine residues targeted by GSK-3β are replaced by alanine) or wild-type Slug (Slug-WT)43. We found that overexpression of mutant Slug restored the invasive ability and cell proliferation in ENO1-knockdown cells, but wild-type Slug did not. Next, we analyzed the SLUG protein concentration in a group of lung cancer cells and found that the SLUG protein concentration was increased in highly metastatic lung cancer cell lines (CL1-5 and CL141) compared to non-metastatic cell lines (CL1-0 and Hop62) and normal human bronchial epithelial (NHBE) cells. This performance group is reminiscent of the performance of ENO1 in cell lines (Figure 1A). Patient samples were stratified into low ENO1, high ENO1, and SLUG groups, and survival rates in each group were analyzed. The analysis showed that patients with high ENO1 and high SLUG performance had the worst overall survival (Figure 5E). In conclusion, these data support the idea that ENO1 promotes malignant cell invasion via the GSK3β-SLUG message axis.

[0160] Since the phenotype of ENO1-expressing cells is typically caused by receptor tyrosine kinase (RTK) activation, we further investigated whether any RTKs might be involved in ENO1 regulation of the GSK3β-SLUG axis. Human phosphate-RTK arrays were treated with lysate from CL1-5 cells infected with shLacZ and shENO1, revealing extreme differences in HGFR phosphorylation concentrations between groups. This result was confirmed by Western ink dot assay (ENO1 knockout reduces HGFR phosphorylation), which was also used to probe known downstream signals. HGFR phosphorylation occurred concurrently with activation of the Src-PI3K-AKT pathway and deactivation of the GSK-3β disruption complex, as well as with stable SLUG and β-catenin in CL1-5 and CL141 cells (Fig. 5F). Furthermore, purified recombinant ENO1 rescued shENO1 knockdown phenotypes, such as HGFR and WNT downstream signaling (Fig. 5F), and also rescued shENO1 knockdown-inhibitory cell invasion ability (Fig. 5G). HGF is known to be an important transactivator of standard Wnt signaling, an effect mediated by HGFR-stimulated GSK-3β-dependent LRP5 / 6 phosphorylation.44 Our data show that ENO1 enhances Wnt signaling (Figs. 5B to 5D and Fig. 5F). Therefore, we tested whether ENO1 activates Wnt signaling via LRP5 / 6 phosphorylation. Indeed, knocking down ENO1 reduced LRP5 / 6 phosphorylation, but did not affect LRP5 / 6 protein expression (Fig. 5F).

[0161] Surprisingly, we also found that ENO1 is essential for HGFR activation, as knockdown of ENO1 prevents HGF from activating HGFR and its downstream signaling pathways (Fig. 6A). Therefore, we attempted to define the mechanism by which ENO1 induces HGFR phosphorylation. We performed a GST pull-down assay, in which the GST-ENO1 fusion protein or GST alone was co-cultured with purified His-labeled HGFR to examine the direct interaction between purified ENO1 and HGFR proteins. The pull-down results showed that, under our experimental conditions, His-HGFR bound to GST-ENO1, rather than to GST alone (Fig. 6B). Next, we used the non-cellular permeable cross-linking agent DTSSP to further confirm the binding of ENO1 to HGFR on the cell surface by membrane protein extraction and IP-Western ink dot assay (Fig. 6C). Furthermore, we performed co-immunoprecipitation (Co-IP) of exogenous (Fig. 6D) and endogenous ENO1 and HGFR to verify the interaction between ENO1 and HGFR (Fig. 6E). To further confirm the interaction, we also generated several Myc-tagged HGFR truncated mutants, as shown in the upper part of Fig. 6F. Co-IP assays showed that interaction with ENO1 requires the SEMA domain corresponding to amino acids 52–496 of HGFR (Fig. 6F). The interaction between ENO1 and HGFR was evident. Therefore, we propose that ENO1 may activate HGFR through direct protein-protein interactions, and that ENO1-induced phosphorylation of LRP5 / 6 may depend on HGFR activation. We also found that ENO1-induced HGFR activation is essential for the stability of LRP5 / 6-mediated SLUG and β-catenin; inhibition of HGFR activity in ENO1-expressing cells reduces LRP5 / 6 phosphorylation (Fig. 6G), and knockdown of both LRP5 and LRP6 reduces the stability of ENO1-induced SLUG and β-catenin (Fig. 11). In summary, these data demonstrate that ENO1 enhances cancer cell invasion by regulating HGFR and WNT-driven EMT.

[0162] 2.5 Generation and Characterization of Anti-ENO1 Therapeutic Monoclonal Antibodies

[0163] Although numerous reports have shown that ENO1 performance is significantly associated with reduced survival and poor prognosis in many cancer types (such as lung cancer12), no therapeutic antibodies against ENO1 for clinical use have yet been approved. Therefore, we developed an anti-ENO1 mAb for cancer research and treatment. We identified four candidate anti-ENO1 mAbs using mouse fusion tumor screening. Our results show that all four anti-ENO1 mAbs exhibited high binding activity against CL1-5 cells as determined by cell ELISA (Figure 9A). Importantly, flow cytometry experiments demonstrated that anti-ENO1 mAb-22 (ENO1-22) reliably detected ENO1 on the surface of CL1-5 cells (Figure 9B). We identified the amino acid sequences in the VH and VL domains of the anti-ENO1 mAb-22 antibody derived from fusion tumor cells (Table 1). Table 1 V H domain FW1 CDR1 FW2 CDR2 QVQLQQSGPVVRPGVSVKISCKGSGYTFT (SEQ ID NO: 1) DYAMH (SEQ ID NO: 2) WVKQSHAKSLEWIG (SEQ ID NO: 3) VISTYNGNTNYNQKFKG (SEQ ID NO: 4) FW3 CDR3 FW4 KATMTVDKSSSTAYMELARLTSEDSAIYYCAR (SEQ ID NO: 5) SPLRY (SEQ ID NO: 6) WGQGTTLTVSS (SEQ ID NO: 7) V L domain FW1 CDR1 FW2 CDR2 QIVLSQSPAILSASPGEKVTMTC (SEQ ID NO: 8) RASSSVSYMH (SEQ ID NO: 9) WYQQKPGSSPKPWIY (SEQ ID NO: 10) ATSNLAS (SEQ ID NO: 11) FW3 CDR3 FW4 GVPARFSGSGSGTSYSLTISRVEAEDAATYYC (SEQ ID NO: 12) QQWSSNPLT (SEQ ID NO: 13) FGAGTKLELK (SEQ ID NO: 14) Full-length amino acid sequences of heavy and light chains Heavy chain QVQLQQSGPEVVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVISTYNGNTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARSPLRYWGQGTTLTVSS (SEQ ID NO: 15) Light chain QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 16)

[0164] Next, we selected the variable domain of the anti-ENO1 mAb-22 antibody into a human IgG Fc vector to generate chimeric ENO1-22 (chENO1-22). We confirmed the binding activity of chENO1-22, finding that the antibody retained a high affinity for live CL1-5 cells (Fig. 9C), and that ENO1 binding specificity was confirmed in ENO1-KD cells (Fig. 9D). In addition, we performed an immunogold labeling assay to confirm the localization of the ENO1 protein. ENO1 was expected to be detected on the cell surface, cytoplasm, and nucleus of CL1-5 cells (Fig. 1C); then, the binding specificity of ENO1 was verified by knocking down CL1-5 cells (Fig. 7A) or overexpressing ENO1 in CL1-0 cells (Fig. 7B).

[0165] Next, we evaluated the anticancer potential of chENO1-22, including its antiproliferative and antiinvasive effects. chENO1-22 inhibited tumor cell growth (Fig. 7C) and cancer cell invasion (Fig. 7D) in vitro, but mENO1-37, which is not detectable in the surface form of ENO1, failed to inhibit cancer cell invasion (Fig. 7D). We then attempted to test whether the anticancer effect of chENO1-22 was due to its inhibition of downstream ENO1 signaling pathways. Indeed, chENO1-22 treatment inhibited the concentrations of downstream signals of ENO1-HGFR, including SLUG and active β-catenin (Fig. 7E). In addition, chENO1-22 treatment attenuated the phosphorylation of HGF-triggered HGFR and its downstream signaling components (Fig. 7F). These results are similar to shENO1 knockdown. mAb also reduced the association between ENO1 and HGFR, as detected by co-IP of endogenous proteins (Fig. 7G). Treatment with chENO1-22 reduced SLUG homeostatic protein concentration, while treatment with MG132 increased it (Fig. 7H). Furthermore, chENO1-22 shortened the half-life of SLUG protein, as assessed with cyclohexylimine treatment (Fig. 7I). These data suggest that the inhibition of lung cancer invasion by chENO1-22 may be a result of suppressed ENO1-HGFR axis signaling, leading to rapid degradation of SLUG protein.

[0166] 2.6 chENO1-22 antibody reduces in vivo lung cancer cell metastasis

[0167] Next, we wanted to further analyze the therapeutic effect on tumor metastasis in vivo. Therefore, we established a metastatic xenograft model by injecting CL1-5 luciferase cells into NOD-SCID mice via tail vein injection. The injected mice were then treated with either normal human IgG (NHIgG) as a control group or chENO1-22 (10 or 20 mg / kg). Four weeks after mAb treatment, regardless of the dose of 10 or 20 mg / kg, weaker luciferase signals than NHIgG were observed in the lungs of mice receiving chENO1-22 (Figures 8A and 8B). Therefore, chENO1-22 treatment significantly inhibited lung tumor metastasis. In addition, compared with mice treated with NHIgG, mice treated with chENO1-22 maintained normal body weight (Figure 12) and showed improved survival (Figure 8C).

[0168] Based on these findings, we propose a model in Figure 8D of how ENO1 promotes cell invasion through the HGFR-Src-PI3K-AKT-GSK3β-SLUG and WNT-GSK3β-SLUG signaling axes.

[0169] 3. Discussion

[0170] Our findings show that ENO1 protein is highly expressed in NSCLC cells but weakly expressed in normal lung tissue. Furthermore, high ENO1 expression is positively correlated with TMN stage and adverse outcomes in lung cancer patients. These findings suggest that ENO1 is a potentially valuable diagnostic biomarker and / or therapeutic target for lung cancer. The invasion-metastasis cascade can be simplified to two key steps: (i) cancer cells spread from the primary tumor to distant tissues, and (ii) micrometastases are formed by the invasion of cancer cells into distant organs.22 The expression of shRNA targeting ENO1 inhibits the invasive ability of lung cancer cells in vitro and disrupts the formation of micrometastases and metastatic migration of tumor cells in vivo. Therefore, ENO1 may play an important role in lung cancer metastasis. Additionally, shRNA targeting ENO1 inhibits lung cancer growth in an orthotopic tumor model, suggesting that ENO1 may also be involved in lung cancer progression. In conclusion, these findings confirm that ENO1 can serve as a therapeutic target for lung cancer.

[0171] In this study, we reveal some novel features of how ENO1 participates in oncogenic activity. Although previous studies have shown that ENO1 can regulate intravascular and pericellular fibrinolytic activity to promote cell migration and cancer metastasis, the detailed signaling mechanisms remain unclear. In this study, we show that a direct protein-protein interaction between ENO1 and HGFR stimulates HGFR signaling. Furthermore, it is previously known that HGFR induces GSK3-dependent LRP5 / 6 phosphorylation, and we further found that ENO1 triggers HGFR activity to activate the LRP5 / 6-GSK3β-β-catenin standard Wnt pathway. Therefore, we reveal that ENO1 regulates the complex Wnt- / β-catenin pathway via the Wnt co-receptor LRP5 / 6-GSK3β signaling. Additionally, we found that the interaction between ENO1 and HGFR triggers the HGFR-Src-PI3K-AKT-GSK3β signaling axis. Interestingly, these two pathways (Wnt and AKT) coordinate to stabilize β-catenin and SLUG to promote EMT.

[0172] Altered cadherin morphology is a recognized characteristic of EMT. Loss of E-cadherin represents a key step in acquiring invasiveness, while an increase in N-cadherin indicates a pro-invasive phenotype; this "cadherin transition" is associated with poor clinical outcomes in many cancers.45 In the current study, we show that knockdown of ENO1 inhibits mesenchymal transition by reducing mesenchymal genes (N-cadherin, vimentin, and SLUG) and positively regulating the epithelial gene E-cadherin. These results reveal ENO1's involvement in previously undescribed molecular mechanisms of lung cancer progression. Slug (an EMT-TF) has been shown to transcriptionally suppress E-cadherin expression and promote cancer invasion and metastasis in various cancer types.46,47 Previous studies have shown that the SLUG-E-cadherin axis is associated with cancer metastasis and poor clinical outcomes in multiple types of NSCLC.46,48 This suggests that SLUG is crucially involved in lung cancer progression. Indeed, our study shows that knockdown of SLUG reduces lung cancer cell invasion and that it is involved in ENO1-promoted cell invasion. In addition, we found that NSCLC patients in the ENO1+ and SLUG+ groups had the worst overall survival (P = 0.022; Figure 5D), which demonstrates the important role of ENO1 and SLUG in NSCLC outcomes.

[0173] We also demonstrated that ENO1 is located on the cell membrane of lung cancer cells, which was confirmed by flow cytometry and immunogold assay. This observation suggests that ENO1 may be a targetable therapeutic agent in lung cancer. Subsequently, we developed a therapeutic mAb against ENO1 and chENO1-22, which, when used in a lung cancer xenograft model, reduced tumor growth and blocked tumor metastasis. The potential mechanism of action of chENO1-22 is to inhibit ENO1-HGFR signaling. Since chENO1-22 reduced tumor progression and metastatic metastasis in this study, this suggests that this mAb can be translated into clinical applications. References: 1. Gatenby, RA & Gillies, RJ Why do cancers have high aerobic glycolysis?Nat Rev Cancer4, 891-899 (2004). 2. Hsiao, KC, et al.Surface alpha-enolase promotes extracellular matrix degradation and tumor metastasis and represents a new therapeutic target.PLoS One8, e69354 (2013). 3. Diaz-Ramos, A., Roig-Borrellas, A., Garcia-Melero, A. & Lopez-Alemany, R. alpha-Enolase, a multifunctional protein: its role on pathophysiological situations. J Biomed Biotechnol2012, 156795 (2012). 4. Capello, M., Ferri-Borgogno, S., Cappello, P. & Novelli, F. alpha-Enolase: a promising therapeutic and diagnostic tumor target.FEBS J278, 1064-1074 (2011). 5. Saldanha, RG, et al.Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy.J Proteome Res6, 1016-1028 (2007). 6. Fu, Q.F., et al.Alpha-enolase promotes cell glycolysis, growth, migration, and invasion in non-small cell lung cancer through FAK-mediated PI3K / AKT pathway.J Hematol Oncol8, 22 (2015). 7. Zhan, P., et al.alpha-enolase promotes tumorigenesis and metastasis via regulating AMPK / mTOR pathway in colorectal cancer.Mol Carcinog56, 1427-1437 (2017). 8. Sun, L., et al.Alpha-enolase promotes gastric cancer cell proliferation and metastasis via regulating AKT signaling pathway.European journal of pharmacology845, 8-15 (2019). 9. Takashima, M., et al.Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma: association with tumor progression as determined by proteomic analysis.Proteomics5, 1686-1692 (2005). 10. Tomaino, B., et al.Circulating autoantibodies to phosphorylated alpha-enolase are a hallmark of pancreatic cancer.J Proteome Res10, 105-112 (2011). 11. Tsai, S.T., et al.ENO1, a potential prognostic head and neck cancer marker, promotes transformation partly via chemokine CCL20 induction.Eur J Cancer46, 1712-1723 (2010). 12. Chang, G.C., et al.Identification of alpha-enolase as an autoantigen in lung cancer: its overexpression is associated with clinical outcomes.Clin Cancer Res12, 5746-5754 (2006). 13. Qian, X., et al.Enolase 1 stimulates glycolysis to promote chemoresistance in gastric cancer.Oncotarget8, 47691-47708 (2017). 14. Wang, H. & Unternaehrer, J.J. Epithelial-mesenchymal Transition and Cancer Stem Cells: At the Crossroads of Differentiation and Dedifferentiation.Dev Dyn248, 10-20 (2019). 15. Baulida, J. Epithelial-to-mesenchymal transition transcription factors in cancer-associated fibroblasts.Mol Oncol11, 847-859 (2017). 16. Yao, L., et al.Paracrine signalling during ZEB1-mediated epithelial-mesenchymal transition augments local myofibroblast differentiation in lung fibrosis.Cell Death Differ26, 943-957 (2019). 17. Mir, N., Jayachandran, A., Dhungel, B., Shrestha, R. & Steel, J.C. Epithelial-to-Mesenchymal Transition: A Mediator of Sorafenib Resistance in Advanced Hepatocellular Carcinoma.Curr Cancer Drug Targets17, 698-706 (2017). 18. Lovisa, S., Genovese, G. & Danese, S. Role of Epithelial-to-Mesenchymal Transition in Inflammatory Bowel Disease.J Crohns Colitis13, 659-668 (2019). 19. Yan, L., Xu, F. & Dai, C.L. Relationship between epithelial-to-mesenchymal transition and the inflammatory microenvironment of hepatocellular carcinoma.J Exp Clin Cancer Res37, 203 (2018). 20. De Matteis, S., et al.Advances in Molecular Mechanisms and Immunotherapy Involving the Immune Cell-Promoted Epithelial-to-Mesenchymal Transition in Lung Cancer.J Oncol2019, 7475364 (2019). 21. Nieto, M.A., Huang, R.Y., Jackson, R.A. & Thiery, J.P.Emt: 2016.Cell166, 21-45 (2016). 22. Brabletz, T., Kalluri, R., Nieto, M.A. & Weinberg, R.A. EMT in cancer.Nat Rev Cancer18, 128-134 (2018). 23. Nieszporek, A., Skrzypek, K., Adamek, G. & Majka, M. Molecular mechanisms of epithelial to mesenchymal transition in tumor metastasis.Acta Biochim Pol66, 509-520 (2019). 24. Lambert, A.W., Pattabiraman, D.R. & Weinberg, R.A. Emerging Biological Principles of Metastasis.Cell168, 670-691 (2017). 25. Zhang, Y. & Weinberg, R.A. Epithelial-to-mesenchymal transition in cancer: complexity and opportunities.Front Med12, 361-373 (2018). 26. Loh, C.Y., et al.The E-Cadherin and N-Cadherin Switch in Epithelial-to-Mesenchymal Transition: Signaling, Therapeutic Implications, and Challenges.Cells8(2019). 27. Baarsma, H.A., Konigshoff, M. & Gosens, R. The WNT signaling pathway from ligand secretion to gene transcription: molecular mechanisms and pharmacological targets.Pharmacol Ther138, 66-83 (2013). 28. Volckaert, T. & De Langhe, S.P.Wnt and FGF mediated epithelial-mesenchymal crosstalk during lung development.Dev Dyn244, 342-366 (2015). 29. Heuberger, J. & Birchmeier, W. Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling.Cold Spring Harb Perspect Biol2, a002915 (2010). 30. Chang, Y.W., et al.Diverse Targets of beta-Catenin during the Epithelial-Mesenchymal Transition Define Cancer Stem Cells and Predict Disease Relapse.Cancer research75, 3398-3410 (2015). 31. Parikh, R.A., Wang, P., Beumer, J.H., Chu, E. & Appleman, L.J. The potential roles of hepatocyte growth factor (HGF)-MET pathway inhibitors in cancer treatment.Onco Targets Ther7, 969-983 (2014). 32. Uehara, Y., et al.Placental defect and embryonic lethality in mice lacking hepatocyte growth factor / scatter factor.Nature373, 702-705 (1995). 33. Tsarfaty, I., et al.The Met proto-oncogene mesenchymal to epithelial cell conversion.Science263, 98-101 (1994). 34. Maroun, C.R. & Rowlands, T.The Met receptor tyrosine kinase: a key player in oncogenesis and drug resistance.Pharmacol Ther142, 316-338 (2014). 35. Nakashiro, K., et al.Phenotypic switch from paracrine to autocrine role of hepatocyte growth factor in an androgen-independent human prostatic carcinoma cell line, CWR22R.Am J Pathol165, 533-540 (2004). 36. Wu, C.H., Kuo, Y.H., Hong, R.L. & Wu, H.C. alpha-Enolase-binding peptide enhances drug delivery efficiency and therapeutic efficacy against colorectal cancer.Sci Transl Med7, 290ra291 (2015). 37. Tsao, C.M., et al.SOX1 functions as a tumor suppressor by antagonizing the WNT / beta-catenin signaling pathway in hepatocellular carcinoma.Hepatology56, 2277-2287 (2012). 38. Lin, C.W., et al.Epithelial cell adhesion molecule regulates tumor initiation and tumorigenesis via activating reprogramming factors and epithelial-mesenchymal transition gene expression in colon cancer.J Biol Chem287, 39449-39459 (2012). 39. Kohler, G. & Milstein, C.Continuous cultures of fused cells secreting antibody of predefined specificity.Nature256, 495-497 (1975). 40. Chen, Y.C., et al.Generation and characterization of monoclonal antibodies against dengue virus type 1 for epitope mapping and serological detection by epitope-based peptide antigens.Clin Vaccine Immunol14, 404-411 (2007). 41. Liu, I.J., Chiu, C.Y., Chen, Y.C. & Wu, H.C. Molecular mimicry of human endothelial cell antigen by autoantibodies to nonstructural protein 1 of dengue virus.J Biol Chem286, 9726-9736 (2011). 42. Tung, K.H., et al.CHC promotes tumor growth and angiogenesis through regulation of HIF-1alpha and VEGF signaling.Cancer Lett331, 58-67 (2013). 43. Kao, S.H., et al.GSK3beta controls epithelial-mesenchymal transition and tumor metastasis by CHIP-mediated degradation of Slug.Oncogene33, 3172-3182 (2014). 44. Koraishy, F.M., Silva, C., Mason, S., Wu, D. & Cantley, L.G.Hepatocyte growth factor (Hgf) stimulates low density lipoprotein receptor-related protein (Lrp) 5 / 6 phosphorylation and promotes canonical Wnt signaling. J Biol Chem289, 14341-14350 (2014). 45. Hazan, RB, Qiao, R., Keren, R., Badano, I. & Suyama, K. Cadherin switch in tumor progression. Ann NY Acad Sci1014, 155-163 (2004). 46. Shih, JY & Yang, PC The EMT regulator slug and lung carcinogenesis.Carcinogenesis32, 1299-1304 (2011). 47. Shih, JY, et al. Res11, 8070-8078 (2005). 48. Chang, TH, et al. Slug confers resistance to the epidermal growth factor receptor tyrosine kinase inhibitor. Am J Respir Crit Care Med183, 1071-1079 (2011). [Simplified Explanation of the Diagram]

[0030] The foregoing content and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the accompanying drawings show specific embodiments that are currently preferred. However, it should be understood that the invention is not limited to the precise configurations and means shown.

[0031] In the diagram:

[0032] Figures 1A to 1I: ENO1 is highly expressed in metastatic cancer cell lines and is associated with poorer overall survival in lung cancer patients. (Figure 1A) ENO1 expression was analyzed in NHBE, NL-20, and human lung cancer cell lines using Western ink dot assay with anti-ENO1 antibody. α-Tubulin was used as an internal control. (Figure 1B) The binding of chimeric ENO1 mAb (chENO1) or normal human IgG (NHIgG) to live NHBE, NL-20, or CL1-5 cells was assessed by flow cytometry using phycoerythrin (PE) conjugated goat anti-mouse IgG. X-axis: PE fluorescence intensity; Y-axis: number of events. (Figure 1C) Immunogold labeling of ENO1 in CL1-5 cells. Cells were fixed with 4% formaldehyde and processed for cryosectioning. Sections were blocked with BSA and then labeled with chENO1-22 (40 µg / ml) or NHIgG for ENO1. After fixation, samples were stained and examined under a transmission electron microscope. (Figure 1D) Analysis of ENO1 mRNA expression in NSCLC tumor tissue (T) and adjacent non-cancerous tissue (N) from the TCGA database. (Figure 1E) Representative images showing ENO1 immunohistochemical staining in full-spectrum (original magnification, 40×; scale bar = 200 μm) samples at the lung cancer stage. (Figure 1F) Immunohistochemical staining data are expressed as the percentage of area stained with ENO1. The median for each group is represented by a horizontal line. (Figure 1G) Overall survival analysis of ENO1 protein expression based on prognostic information for lung cancer patients from tissue microarray data. OS was analyzed using a Kaplan-Meier plotter (KM plotter) (Figure 1H) and the TCGA database (I). Hazard ratios (HR) with 95% confidence intervals and log-rank p-values ​​are provided from the web. **P < 0.01 and ***P < 0.001.

[0033] Figures 2A to 2F: In vitro identification of the molecular function of ENO1 in lung cancer cells. (Figure 2A) ENO1 expression in CL1-5 or CL-141 cells infected with lentiviruses carrying control shRNA (shLacZ) or ENO1 shRNA (shENO1-1 and shENO1-2) was analyzed by Western blotting. α-Tubulin was used as an internal control. Cell proliferation (WST-1) was measured using ENO1 knockdown of CL1-5 or CL-141 cells (Figure 2B) and CL1-0 cells (Figure 2E). Absorbance values ​​are expressed as mean ± SEM from four independent experiments. Matrix gel invasion was measured using ENO1 knockdown of CL1-5 or CL-141 cells (Figure 2C) and CL1-0 cells (Figure 2F). The number of invading cells was counted, and the results are expressed as mean ± SEM from three independent experiments. (*P < 0.5 and ***P < 0.001, Student's t-test) (Figure 2D) The expression of ENO1 in AS2-ENO1-FLAG transfected CL1-0 cells or empty vector was analyzed by Western ink dot assay using anti-FLAG antibody.

[0034] Figures 3A to 3E: ENO1 knockdown inhibits tumor growth and lung tumor metastasis in a mouse xenograft model. CL1-5-Luci cells were infected with lentiviruses carrying either the control group shLacZ or shENO1 (each also exhibiting luciferase) and transplanted into the lung parenchyma of NOD-SCID mice (1 × 10⁵ cells) (Figures 3A and 3B) or injected into the tail vein of NOD-SCID mice (1 × 10⁶ cells) (Figures 3C, 3D, 3E). Tumor formation in the lungs was monitored by bioluminescence imaging (Figure 3E). The number of tumor nodules was counted after hematoxylin and eosin staining of the excised lungs from the mice shown in (Figure 3C). **P < 0.05; ***P < 0.001.

[0035] Figures 4A to 4G: ENO1 promotes EMT by regulating SLUG stability. (Figure 4A) Morphology of CL1-5 cells infected with lentiviruses carrying control group shLacZ or shENO1. (Figure 4B) Detection of EMT markers or regulators by Western chromatograph in CL1-5 cells infected with shLacZ or shENO1 (left) or CL1-0 cells infected with vector or ENO1-FLAG (right). (Figure 4C) Detection of EMT markers or regulators by qRT-PCR in CL1-5 cells infected with shLacZ or shENO1. (Figure 4D) Analysis of protein expression of these genes in ENO1 knockdown cells by Western chromatograph after treatment with or without 10 mM MG132 (proteasome inhibitor) for 6 hours. (Figure 4E) CL1-5 cells infected with lentiviruses carrying shLacZ or shENO1. Twenty-four hours post-transfection, cells were treated with 10 mM MG132 for 6 hours prior to cell collection. Cell lysate was immunoprecipitated using anti-SLUG antibody (left panel) and the input group (right panel). Western ink dot assay was performed using the antibodies shown to detect protein interactions. (Fig. 4F) Stability of SLUG protein in CL1-5 cells infected with shLacZ or shENO1. Cells were treated with 100 μg cyclohexylamine (CHX) at the intervals shown and analyzed using Western ink dot assay. (Fig. 4G) Quantification of SLUG half-life in the groups shown. **P < 0.05; ***P < 0.001.

[0036] Figures 5A to 5G: ENO1 stabilizes SLUG by reducing GSK3β activity through activation of HGFR-Src-PI3K-AKT and Wnt-β-catenin signaling. (Figure 5A) Protein expression in ENO1 knockdown cells (shENO1-1) was analyzed by Western blotting after 24 hours of treatment with or without 2 μM GSK3β inhibitor (BIO). (Figure 5B) One group of cells was transfected with TOP reporter, CMV: Renal luciferase; the second group of cells was transfected with FOP reporter, CMV: Renal luciferase, as shown in the figure. The TOP and FOP reporters are as follows: the β-catenin-responsive "T cytokine (TCF) element" (TOP) that drives luciferase; and the non-β-catenin-responsive mutant element (FOP) that also drives luciferase. The ratio of messages from these two reporter progenitors reflects β-catenin-specific standard Wnt message delivery, with each reporter progenitor normalized to Renilla luciferase as an internal control. Wnt message delivery was measured in ENO1-knockdown CL1-5 cells or control cells after 72 hours. (Fig. 5C) β-catenin expression in CL1-5 cells was detected using confocal immunofluorescence assay. Samples were observed using a 63x objective. Green fluorescence represents β-catenin, while blue fluorescence is DAPI staining. The lower panel shows the quantification of the mean intensity of nuclear SLUG. (Fig. 5D) Matrix gel invasion assay of CL1-5 cells with the plastid assemblages shown. (Fig. 5E) TCGA data analysis (RNA-sequence data for lung adenocarcinoma) of overall survival in NSCLC patients with ENO1 low / SLUG low, ENO1 high / SLUG low, ENO1 low / SLUG high, and ENO1 high / SLUG high outcomes. Patients were grouped according to the median. (Figure 5F) Protein concentration was determined by Western ink dot assay in CL1-5 cells infected with shLacZ or ENO1 shRNA, or in ENO1 shRNA cells treated with recombinant ENO1 for 30 min (1 μg / mL). P: phosphorylated protein concentration, t: total protein concentration. (Figure 5G) Matricella invasion assay was performed using CL1-5 cells infected with shLacZ or ENO1 shRNA, or in ENO1 shRNA cells treated with recombinant ENO1 for 30 min (1 or 2 μg / mL). The number of invading cells was counted, and the results are expressed as mean ± SEM from three independent experiments. **P < 0.01; ***P < 0.001.

[0037] Figures 6A to 6G: ENO1 associates with HGFR and is essential for HGFR. (Figure 6A) CL1-5 cells infected with shLacZ or ENO1 shRNA were stimulated for 10 minutes with a vector or human HGF, followed by immunoprecipitation. (Figure 6B) GST pull-down assay. GST-agarose bead-bound GST-ENO1 or GST protein was co-cultured with purified His-HGFR. After washing, the bead-bound protein and 5% introduced His-HGFR protein were released by boiling with SDS-PAGE loading buffer. The samples were then subjected to Western ink precipitation with anti-His or ENO1 antibody. (Figure 6C) Membrane proteins were extracted from CL1-5 cells, and the IP of affinity-crosslinked ENO1 was bound to HGFR. (Figure 6D) Immunoprecipitates were prepared from CL1-5 cells after labeling ENO1 with a FLAG. Immunoprecipitates of lysate were prepared from CL1-5 cells using anti-ENO1 (IP:ENO1) or anti-HGFR (IP:HGFR) antibodies, followed by ink dot spectrometry with anti-HGFR and anti-ENO1 antibodies. (Figure 6E) Immunoprecipitates were prepared from CL1-5 cells. Lysate was immunoprecipitated using anti-ENO1 (IP:ENO1) or anti-HGFR (IP:HGFR) antibodies, followed by ink dot spectrometry with anti-HGFR and anti-ENO1 antibodies. (Figure 6F) Top: Schematic diagram of the HGFR domain. HGFR constructs or empty vectors labeled with different Myc were immunoprecipitated with anti-Myc antibodies for 72 hours and separated by SDS-polyacrylamide gel electrophoresis (PAGE). Immunomodulatory dots were detected using anti-FLAG or anti-Myc antibodies. (Figure 6G) CL1-5 cells infected with shLacZ or ENO1 shRNA were treated with an HGFR inhibitor for 24 hours, stimulated with hHGF for 10 minutes, and then subjected to immunodot spectrometry.

[0038] Figures 7A to 7I: The chENO1-22 antibody reduces the proliferation and invasion of cancer cells in vitro by inhibiting downstream signaling mediated by the ENO1-HGFR axis and promoting SLUG protein degradation. Phycoerythrin (PE) conjugated goat anti-human IgG was used to quantify the binding of anti-chENO1-22 to live CL1-5 cells infected with lentiviruses carrying LacZ or ENO1 shRNA (Figure 7A) or CL1-0 cells transfected with AS2-ENO1-FLAG or an empty vector (Figure 7B) by flow cytometry. (Figure 6C) Cell proliferation (WST-1) assay was performed on CL1-5 cells treated with two anti-ENO1 mouse mAbs. The antibody concentration was 10 μg / ml, and OD values ​​were measured at 8, 24, 48, or 72 hours after antibody treatment. (Figure 6D) Assay of matrix gel invasion was performed on CL1-5 cells treated with anti-ENO1 mouse mAbs. Invading cells were quantified, and results are expressed as the mean ± SEM of three independent experiments (*P < 0.05, Student's t-test). The following figure shows the area of ​​invading cells in the matrix gel invasion assay. (Fig. 6E) Protein concentration in CL1-5 cells treated with NHIgG or chENO1-22 was detected by Western ink dot assay. (Fig. 6F) CL1-5 cells were treated with NHIgG or chENO1-22 for 4 hours and stimulated with a carrier or hHGF for 10 minutes, followed by Western ink dot assay. (Fig. 6G) CL1-5 cells were treated with NHIgG or chENO1-22 for 4 hours and immunoprecipitated with anti-ENO1 (IP:ENO1) or anti-HGFR (IP:HGFR) antibodies, followed by Western ink dot assay. (Fig. 6H) CL1-5 cells were treated with 10 mM MG132 and chENO1-22 for 6 hours before cell collection and subsequent Western ink dot assay. (Figure 6I) Stability of Slug protein in CL1-5 cells treated with NHIgG or chENO1-22. Cells were treated with 100 μg cyclohexylamine (CHX) at the intervals shown and analyzed by Western blotting. The figure below shows the quantification of SLUG half-life in the groups shown. *P < 0.05; **P < 0.01; ***P < 0.001.

[0039] Figures 8A to 8D: chENO1-22 antibody reduces in vivo lung cancer cell metastasis. (Figure 8A) One million CL1-5-Luci cells were intravenously injected into NOD-SCID mice, and normal human IgG control or chENO1-22 antibody were intravenously injected twice weekly for a total of eight injections. Weekly bioluminescence imaging was performed to monitor the growth of intravenously injected tumor cells (median survival days: log-rank, p = 0.0019). (Figure 8B) Monitoring tumor formation in the lungs by bioluminescence imaging. (Figure 8C) Kaplan-Meier curves were generated to analyze survival. (Figure 8D) A working model describes how ENO1 promotes cell invasion by regulating the HGFR-Src-PI3K-AKT-GSK3β-SLUG and HGFR-WNT-GSK3β-SLUG axes. (a) ENO1 increases phosphorylation of HGFR and Wnt co-receptors LPR5 / 6 and decreases GSK-3β activity to enhance SLUG stability and inhibit E-cadherin expression. Ultimately, this enhances cancer cell invasion. (b) chENO1-22 mAb promotes SLUG protein ubiquitination and degradation by blocking downstream signaling mediated by the ENO1-HGFR axis, thereby inhibiting cancer cell invasion.

[0040] Figures 9A to 9D: Screening of binding activity and specificity of anti-ENO1 mAb using fusion tumor technology. The binding activity of four anti-ENO1 mAb to CL1-5 cells was analyzed on live cells using cellular ELISA (Figure 9A) and flow cytometry, with phycoerythrin (PE)-conjugated goat anti-mouse IgG as a secondary antibody (Figure 9B). X-axis: PE fluorescence intensity; Y-axis: number of events. The binding of anti-chENO1-22 to live CL1-5 cells (Figure 9C) or CL1-5 cells infected with lentiviruses carrying LacZ or ENO1 shRNA (Figure 9D) was quantitatively assessed by flow cytometry using phycoerythrin (PE)-conjugated goat anti-human IgG.

[0041] Figure 10: ENO1 manifestation is associated with more advanced cancer stages in lung cancer patients. Immunohistochemical staining data are expressed as the percentage of ENO1 positive staining intensity. Based on ENO1 staining intensity, ENO1 manifestation was divided into one of four categories: 3: strong manifestation; 2: moderate manifestation; 1: weak or indistinct manifestation; and 0: no manifestation.

[0042] Figure 11: LRP5 and LRP6 are essential for ENO1-induced stability of SLUG and β-catenin. CL1-5 cells infected with shLacZ, ENO1 shRNA, or LRP5 / 6 shRNA were stimulated with a vector or hHGF for 10 minutes, followed by Western ink dot assay.

[0043] Figure 12: ChENO1-22 antibody reduces in vivo lung cancer cell metastasis. Mice were administered 10 or 20 mg / kg of chENO1, and the body weight of the treated mice is shown.

[0044] Figure 13: The amino acid sequences of VH and VL of the chENO1-22 antibody of the present invention. [Sequence List]

[0176] Sequence List <![CDATA[ <110> Academia Sinica <![CDATA[ <120> Anti-enolase 1 (ENO1) antibodies and their applications <![CDATA[ <150> 63 / 164,137]]> <![CDATA[ <151> 2021-03-22 <![CDATA[ <160> 16 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 30]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 1]]> Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr 20 25 30 <![CDATA[ <210> 2]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 2]]> Asp Tyr Ala Met His 1 5 <![CDATA[ <210> 3]]> <![CDATA[ <211> 14]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 3]]> Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly 1 5 10 <![CDATA[ <210> 4]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 4]]> Val Ile Ser Thr Tyr Asn Gly Asn Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <![CDATA[ <210> 5]]> <![CDATA[ <211> 32]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice<![CDATA[ <400> 5]]> Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg 20 25 30 <![CDATA[ <210> 6]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 6]]> Ser Pro Leu Arg Tyr 1 5 <![CDATA[ <210> 7]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 7]]> Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <![CDATA[ <210> 8]]> <![CDATA[ <211> 23]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 8]]> Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys 20 <![CDATA[ <210> 9]]> <![CDATA[ <211> 10]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 9]]> Arg Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <![CDATA[ <210> 10]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Mice <![CDATA[ <400> 10]]> Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 1 5 10 15 <![CDATA[<210 >11]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mouse]]> <![CDATA[<400> 11]]> Ala Thr Ser Asn Leu Ala Ser 1 5 <![CDATA[<210> 12]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mouse]]> <![CDATA[<400> 12]]> Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 1 5 10 15 Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30 <![CDATA[<210> 13]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mouse]]> <![CDATA[<400> 13]]> Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 <![CDATA[<210> 14]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mouse]]> <![CDATA[<400> 14]]> Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 1 5 10 <![CDATA[<210> 15]]> <![CDATA[<211> 114]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mouse]]> <![CDATA[<400> 15]]> Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45Gly Val Ile Ser Thr Tyr Asn Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Leu Arg Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <![CDATA[<210> 16]]> <![CDATA[<211> 106]]> <![CDATA[<212> PRT]]> <![CDATA[<213> mouse]]> <![CDATA[<400> 16]]> Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to enolase 1 (ENO1), comprising: (a) a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HC CDR1) containing an amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity-determining region 2 (HC CDR2) containing an amino acid sequence of SEQ ID NO: 4, and a heavy chain complementarity-determining region 3 (HC CDR3) containing an amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LC CDR1) containing an amino acid sequence of SEQ ID NO: 9, a light chain complementarity-determining region 2 (LC CDR2) containing an amino acid sequence of SEQ ID NO: 11, and a light chain complementarity-determining region 3 (LC CDR3) containing an amino acid sequence of SEQ ID NO:

13.

2. The antibody or antigen-binding fragment of claim 1, wherein the VH contains the amino acid sequence of SEQ ID NO:

15.

3. The antibody or antigen-binding fragment of claim 1, wherein the VL contains the amino acid sequence of SEQ ID NO:

16.

4. A recombinant nucleic acid comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 3.

5. A host cell comprising the recombinant nucleic acid of claim 4.

6. A composition comprising an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 3 and a vector.

7. Use of an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 3, or a composition as defined in claim 6, for the preparation of a pharmaceutical agent for the purpose of inhibiting downstream enolase 1 (ENO1) signaling in an individual in need and / or treating a disease or condition associated with activation of downstream ENO1 signaling.

8. For the purposes of claim 7, wherein the disease or condition is cancer and cancer metastasis.

9. As requested in item 8, wherein the cancer is selected from the group consisting of: lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer.

10. As requested in claim 9, wherein the cancer is lung cancer.

11. The use of any one of claims 8 to 10, wherein the antibody or its antigen-binding fragment is effective in inhibiting tumor growth and metastasis in the individual in need.

12. The use of any of claims 7 to 10, wherein the antibody or its antigen-binding fragment is effective in prolonging the survival time of the individual in need.

13. A method for detecting enolase 1 (ENO1) in vitro, comprising: (i) contacting an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 3 with cells of an individual in need; and (ii) detecting the binding of the antibody or the antigen-binding fragment thereof to the cells.

14. The method of claim 13, wherein the cell is present in a biological sample obtained from the individual.

15. The method of claim 13, wherein the individual is at risk of developing cancer or is suspected of having cancer.

16. The method of request item 13, wherein the individual has cancer.

17. The method of claim 16, further comprising comparing the result of the test with a reference concentration and determining the prognosis of the individual based on the result of the comparison, wherein an elevated ENO1 concentration indicates a negative prognosis, wherein the reference concentration is an ENO1 concentration measured in a normal individual or a non-cancerous tissue or cell sample, or the reference concentration is the average or median number of ENO1 in a cancer patient population.

18. The method of claim 17, wherein the negative prognosis is selected from the group consisting of: reduced survival, increased tumor growth, increased risk of metastasis, increased risk of recurrence, and any combination thereof.

19. The method of any one of claims 15 to 18, wherein the cancer is selected from the group consisting of: lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer.

20. The method of claim 19, wherein the cancer is lung cancer.

21. A method for monitoring cancer progression in a patient with cancer, comprising: (i) providing a first biological sample from the patient at a first time point; (ii) providing a second biological sample from the patient at a second time point later than the first time point; (iii) detecting the concentration of enolase 1 (ENO1) in the first biological sample and the second biological sample, wherein the detection is performed by an immunoassay of an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 3; and (iv) determining cancer progression in the patient based on the ENO1 concentration in the first biological sample and the second biological sample, wherein an elevated ENO1 concentration in the second biological sample compared to the first biological sample indicates cancer progression.

22. The method of claim 21, wherein the cancer is selected from the group consisting of: lung cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and testicular cancer.

23. The method of claim 22, wherein the cancer is lung cancer.

24. Use of an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 3, or a composition as defined in claim 6, for the preparation of a kit for the detection of ENO1, diagnosis and prognosis of cancer, and monitoring of cancer progression.

25. A kit for performing the method of any one of claims 13 to 23, comprising an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 3 or a composition as described in claim 6, and instructions for using the kit to detect ENO1.

Images